CN117059166A - 用于评估总生存期和表征肿瘤免疫微环境的基因特征及其研究方法 - Google Patents
用于评估总生存期和表征肿瘤免疫微环境的基因特征及其研究方法 Download PDFInfo
- Publication number
- CN117059166A CN117059166A CN202310915518.7A CN202310915518A CN117059166A CN 117059166 A CN117059166 A CN 117059166A CN 202310915518 A CN202310915518 A CN 202310915518A CN 117059166 A CN117059166 A CN 117059166A
- Authority
- CN
- China
- Prior art keywords
- hcc
- gene
- tumor
- ascore
- genes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 106
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000011160 research Methods 0.000 title claims abstract description 11
- 230000004083 survival effect Effects 0.000 claims abstract description 30
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 27
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 16
- 238000003556 assay Methods 0.000 claims abstract description 12
- 238000010166 immunofluorescence Methods 0.000 claims abstract description 10
- 230000000869 mutational effect Effects 0.000 claims abstract description 8
- 230000004797 therapeutic response Effects 0.000 claims abstract description 5
- 230000014509 gene expression Effects 0.000 claims description 30
- 230000004048 modification Effects 0.000 claims description 15
- 238000012986 modification Methods 0.000 claims description 15
- 230000004547 gene signature Effects 0.000 claims description 13
- 230000008595 infiltration Effects 0.000 claims description 12
- 238000001764 infiltration Methods 0.000 claims description 12
- 238000010586 diagram Methods 0.000 claims description 11
- 230000002068 genetic effect Effects 0.000 claims description 11
- 239000002953 phosphate buffered saline Substances 0.000 claims description 10
- 102100034406 5'-deoxynucleotidase HDDC2 Human genes 0.000 claims description 9
- 102100027058 Bleomycin hydrolase Human genes 0.000 claims description 9
- 102100035325 Complement factor H-related protein 5 Human genes 0.000 claims description 9
- 102100039455 Cytochrome b-c1 complex subunit 6, mitochondrial Human genes 0.000 claims description 9
- 102100039264 Glycogen [starch] synthase, liver Human genes 0.000 claims description 9
- 102100039262 Glycogen [starch] synthase, muscle Human genes 0.000 claims description 9
- 102100039999 Histone deacetylase 2 Human genes 0.000 claims description 9
- 101001066900 Homo sapiens 5'-deoxynucleotidase HDDC2 Proteins 0.000 claims description 9
- 101000984541 Homo sapiens Bleomycin hydrolase Proteins 0.000 claims description 9
- 101000878134 Homo sapiens Complement factor H-related protein 5 Proteins 0.000 claims description 9
- 101000746783 Homo sapiens Cytochrome b-c1 complex subunit 6, mitochondrial Proteins 0.000 claims description 9
- 101001036117 Homo sapiens Glycogen [starch] synthase, liver Proteins 0.000 claims description 9
- 101001036130 Homo sapiens Glycogen [starch] synthase, muscle Proteins 0.000 claims description 9
- 101001035011 Homo sapiens Histone deacetylase 2 Proteins 0.000 claims description 9
- 101001044927 Homo sapiens Insulin-like growth factor-binding protein 3 Proteins 0.000 claims description 9
- 101001011746 Homo sapiens Integrator complex subunit 8 Proteins 0.000 claims description 9
- 101001056015 Homo sapiens Mannan-binding lectin serine protease 2 Proteins 0.000 claims description 9
- 101000962664 Homo sapiens Microtubule-associated protein RP/EB family member 1 Proteins 0.000 claims description 9
- 101000866795 Homo sapiens Non-histone chromosomal protein HMG-14 Proteins 0.000 claims description 9
- 101000974015 Homo sapiens Nucleosome assembly protein 1-like 1 Proteins 0.000 claims description 9
- 101000609219 Homo sapiens Polyadenylate-binding protein 4 Proteins 0.000 claims description 9
- 101000742932 Homo sapiens Retinol dehydrogenase 16 Proteins 0.000 claims description 9
- 101000697781 Homo sapiens Syntaxin-6 Proteins 0.000 claims description 9
- 101000666730 Homo sapiens T-complex protein 1 subunit alpha Proteins 0.000 claims description 9
- 101000666502 Homo sapiens Xaa-Pro aminopeptidase 1 Proteins 0.000 claims description 9
- 102100022708 Insulin-like growth factor-binding protein 3 Human genes 0.000 claims description 9
- 102100030148 Integrator complex subunit 8 Human genes 0.000 claims description 9
- 102100026046 Mannan-binding lectin serine protease 2 Human genes 0.000 claims description 9
- 102100039560 Microtubule-associated protein RP/EB family member 1 Human genes 0.000 claims description 9
- 102100031353 Non-histone chromosomal protein HMG-14 Human genes 0.000 claims description 9
- 102100022389 Nucleosome assembly protein 1-like 1 Human genes 0.000 claims description 9
- 102100039424 Polyadenylate-binding protein 4 Human genes 0.000 claims description 9
- 102100038057 Retinol dehydrogenase 16 Human genes 0.000 claims description 9
- 102100027866 Syntaxin-6 Human genes 0.000 claims description 9
- 102100038410 T-complex protein 1 subunit alpha Human genes 0.000 claims description 9
- 102100038365 Xaa-Pro aminopeptidase 1 Human genes 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 101001014572 Homo sapiens MARCKS-related protein Proteins 0.000 claims description 8
- 102100032514 MARCKS-related protein Human genes 0.000 claims description 8
- 102000033021 YBX1 Human genes 0.000 claims description 8
- 108091002437 YBX1 Proteins 0.000 claims description 8
- 238000004422 calculation algorithm Methods 0.000 claims description 8
- 238000010276 construction Methods 0.000 claims description 8
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 8
- 230000037361 pathway Effects 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 238000010201 enrichment analysis Methods 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- 239000000427 antigen Substances 0.000 claims description 6
- 108091007433 antigens Proteins 0.000 claims description 6
- 102000036639 antigens Human genes 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 6
- 238000012795 verification Methods 0.000 claims description 6
- 230000000903 blocking effect Effects 0.000 claims description 5
- 238000000513 principal component analysis Methods 0.000 claims description 5
- 239000012188 paraffin wax Substances 0.000 claims description 4
- 238000000611 regression analysis Methods 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 abstract description 97
- 238000004393 prognosis Methods 0.000 abstract description 15
- 238000011269 treatment regimen Methods 0.000 abstract description 5
- 238000013517 stratification Methods 0.000 abstract description 4
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 94
- 238000004458 analytical method Methods 0.000 description 14
- 210000002865 immune cell Anatomy 0.000 description 10
- 238000009169 immunotherapy Methods 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 8
- 201000011510 cancer Diseases 0.000 description 8
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 230000011987 methylation Effects 0.000 description 6
- 238000007069 methylation reaction Methods 0.000 description 6
- 206010064571 Gene mutation Diseases 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 210000003289 regulatory T cell Anatomy 0.000 description 5
- 108010009992 CD163 antigen Proteins 0.000 description 4
- 101001013582 Homo sapiens N6-adenosine-methyltransferase non-catalytic subunit Proteins 0.000 description 4
- 210000004322 M2 macrophage Anatomy 0.000 description 4
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 4
- 102100031578 N6-adenosine-methyltransferase non-catalytic subunit Human genes 0.000 description 4
- 102100025831 Scavenger receptor cysteine-rich type 1 protein M130 Human genes 0.000 description 4
- 238000011088 calibration curve Methods 0.000 description 4
- 230000002596 correlated effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010199 gene set enrichment analysis Methods 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102100023990 60S ribosomal protein L17 Human genes 0.000 description 3
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 description 3
- 101001068133 Homo sapiens Hepatitis A virus cellular receptor 2 Proteins 0.000 description 3
- 101001081567 Homo sapiens Insulin-like growth factor-binding protein 1 Proteins 0.000 description 3
- 102100027636 Insulin-like growth factor-binding protein 1 Human genes 0.000 description 3
- 108091054455 MAP kinase family Proteins 0.000 description 3
- 102000043136 MAP kinase family Human genes 0.000 description 3
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 229940044683 chemotherapy drug Drugs 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 3
- 229940126546 immune checkpoint molecule Drugs 0.000 description 3
- 238000003125 immunofluorescent labeling Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- SMADWRYCYBUIKH-UHFFFAOYSA-N 2-methyl-7h-purin-6-amine Chemical compound CC1=NC(N)=C2NC=NC2=N1 SMADWRYCYBUIKH-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 102100028323 ADP-ribose glycohydrolase MACROD2 Human genes 0.000 description 2
- 101710159080 Aconitate hydratase A Proteins 0.000 description 2
- 101710159078 Aconitate hydratase B Proteins 0.000 description 2
- 206010069754 Acquired gene mutation Diseases 0.000 description 2
- 102100030461 Alpha-ketoglutarate-dependent dioxygenase FTO Human genes 0.000 description 2
- 102000008096 B7-H1 Antigen Human genes 0.000 description 2
- 108010074708 B7-H1 Antigen Proteins 0.000 description 2
- 101100510617 Caenorhabditis elegans sel-8 gene Proteins 0.000 description 2
- 102100032137 Cell death activator CIDE-3 Human genes 0.000 description 2
- 102100024485 Cell division cycle-associated protein 7 Human genes 0.000 description 2
- 102100023308 Centrosomal protein of 126 kDa Human genes 0.000 description 2
- 102100024291 Cilia- and flagella-associated protein 298 Human genes 0.000 description 2
- 102100032247 Cilium assembly protein DZIP1L Human genes 0.000 description 2
- 102100035321 Complement factor H-related protein 3 Human genes 0.000 description 2
- 102100033145 Cyclin-dependent kinase 19 Human genes 0.000 description 2
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 2
- 102100037055 DCN1-like protein 5 Human genes 0.000 description 2
- 101150027068 DEGS1 gene Proteins 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 2
- 102100021558 ER lumen protein-retaining receptor 3 Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 102100031158 GAS2-like protein 3 Human genes 0.000 description 2
- 102100039272 Glycine N-acyltransferase-like protein 1 Human genes 0.000 description 2
- 102100024233 High affinity cAMP-specific 3',5'-cyclic phosphodiesterase 7A Human genes 0.000 description 2
- 108010005336 Histone H2a Dioxygenase AlkB Homolog 1 Proteins 0.000 description 2
- 101000578915 Homo sapiens ADP-ribose glycohydrolase MACROD2 Proteins 0.000 description 2
- 101001062620 Homo sapiens Alpha-ketoglutarate-dependent dioxygenase FTO Proteins 0.000 description 2
- 101000775558 Homo sapiens Cell death activator CIDE-3 Proteins 0.000 description 2
- 101000980893 Homo sapiens Cell division cycle-associated protein 7 Proteins 0.000 description 2
- 101000908170 Homo sapiens Centrosomal protein of 126 kDa Proteins 0.000 description 2
- 101000980087 Homo sapiens Cilia- and flagella-associated protein 298 Proteins 0.000 description 2
- 101001016178 Homo sapiens Cilium assembly protein DZIP1L Proteins 0.000 description 2
- 101000878136 Homo sapiens Complement factor H-related protein 3 Proteins 0.000 description 2
- 101000944345 Homo sapiens Cyclin-dependent kinase 19 Proteins 0.000 description 2
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 2
- 101000954832 Homo sapiens DCN1-like protein 5 Proteins 0.000 description 2
- 101000898776 Homo sapiens ER lumen protein-retaining receptor 3 Proteins 0.000 description 2
- 101001066167 Homo sapiens GAS2-like protein 3 Proteins 0.000 description 2
- 101000888230 Homo sapiens Glycine N-acyltransferase-like protein 1 Proteins 0.000 description 2
- 101001117267 Homo sapiens High affinity cAMP-specific 3',5'-cyclic phosphodiesterase 7A Proteins 0.000 description 2
- 101001011441 Homo sapiens Interferon regulatory factor 4 Proteins 0.000 description 2
- 101001049181 Homo sapiens Killer cell lectin-like receptor subfamily B member 1 Proteins 0.000 description 2
- 101001050886 Homo sapiens Lysine-specific histone demethylase 1A Proteins 0.000 description 2
- 101000589441 Homo sapiens Membrane progestin receptor beta Proteins 0.000 description 2
- 101000914035 Homo sapiens Pre-mRNA-splicing regulator WTAP Proteins 0.000 description 2
- 101000808521 Homo sapiens Probable U3 small nucleolar RNA-associated protein 11 Proteins 0.000 description 2
- 101001048762 Homo sapiens Protein FAM117B Proteins 0.000 description 2
- 101000873612 Homo sapiens Protein bicaudal D homolog 1 Proteins 0.000 description 2
- 101000794282 Homo sapiens Putative protein C3P1 Proteins 0.000 description 2
- 101000703608 Homo sapiens RIB43A-like with coiled-coils protein 2 Proteins 0.000 description 2
- 101000687317 Homo sapiens RNA-binding motif protein, X chromosome Proteins 0.000 description 2
- 101001062093 Homo sapiens RNA-binding protein 15 Proteins 0.000 description 2
- 101000591128 Homo sapiens RNA-binding protein Musashi homolog 2 Proteins 0.000 description 2
- 101000620798 Homo sapiens Ras-related protein Rab-11A Proteins 0.000 description 2
- 101001132643 Homo sapiens Ribonucleoprotein PTB-binding 2 Proteins 0.000 description 2
- 101000655308 Homo sapiens S-adenosylmethionine sensor upstream of mTORC1 Proteins 0.000 description 2
- 101001094146 Homo sapiens SUMO-activating enzyme subunit 2 Proteins 0.000 description 2
- 101000822448 Homo sapiens Sodium channel and clathrin linker 1 Proteins 0.000 description 2
- 101000820476 Homo sapiens Syntaxin-binding protein 4 Proteins 0.000 description 2
- 101000831807 Homo sapiens Transmembrane protein 234 Proteins 0.000 description 2
- 101000708392 Homo sapiens U5 small nuclear ribonucleoprotein 40 kDa protein Proteins 0.000 description 2
- 101000743488 Homo sapiens V-set and immunoglobulin domain-containing protein 4 Proteins 0.000 description 2
- 101000654576 Homo sapiens Vesicle transport protein SFT2A Proteins 0.000 description 2
- 101000744742 Homo sapiens YTH domain-containing family protein 1 Proteins 0.000 description 2
- 101000782060 Homo sapiens Zinc finger CCCH domain-containing protein 13 Proteins 0.000 description 2
- 102100030126 Interferon regulatory factor 4 Human genes 0.000 description 2
- 238000010824 Kaplan-Meier survival analysis Methods 0.000 description 2
- 102100023678 Killer cell lectin-like receptor subfamily B member 1 Human genes 0.000 description 2
- 102100024985 Lysine-specific histone demethylase 1A Human genes 0.000 description 2
- 102100032326 Membrane progestin receptor beta Human genes 0.000 description 2
- 102100027051 Nucleic acid dioxygenase ALKBH1 Human genes 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102100026431 Pre-mRNA-splicing regulator WTAP Human genes 0.000 description 2
- 102100038592 Probable U3 small nucleolar RNA-associated protein 11 Human genes 0.000 description 2
- 102100023780 Protein FAM117B Human genes 0.000 description 2
- 102100035898 Protein bicaudal D homolog 1 Human genes 0.000 description 2
- 102100030156 Putative protein C3P1 Human genes 0.000 description 2
- 102100033810 RAC-alpha serine/threonine-protein kinase Human genes 0.000 description 2
- 102100030683 RIB43A-like with coiled-coils protein 2 Human genes 0.000 description 2
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 2
- 102100024939 RNA-binding motif protein, X chromosome Human genes 0.000 description 2
- 101710105008 RNA-binding protein Proteins 0.000 description 2
- 102100029244 RNA-binding protein 15 Human genes 0.000 description 2
- 102100034027 RNA-binding protein Musashi homolog 2 Human genes 0.000 description 2
- 102100022873 Ras-related protein Rab-11A Human genes 0.000 description 2
- 102100033918 Ribonucleoprotein PTB-binding 2 Human genes 0.000 description 2
- 102100032896 S-adenosylmethionine sensor upstream of mTORC1 Human genes 0.000 description 2
- 102100035250 SUMO-activating enzyme subunit 2 Human genes 0.000 description 2
- 102100022483 Sodium channel and clathrin linker 1 Human genes 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 102100021678 Syntaxin-binding protein 4 Human genes 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 102100024194 Transmembrane protein 234 Human genes 0.000 description 2
- 102100031471 U5 small nuclear ribonucleoprotein 40 kDa protein Human genes 0.000 description 2
- 102100038296 V-set and immunoglobulin domain-containing protein 4 Human genes 0.000 description 2
- 102100032651 Vesicle transport protein SFT2A Human genes 0.000 description 2
- 102100039647 YTH domain-containing family protein 1 Human genes 0.000 description 2
- 102100036624 Zinc finger CCCH domain-containing protein 13 Human genes 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 2
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 2
- 229960005420 etoposide Drugs 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 230000036438 mutation frequency Effects 0.000 description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000037439 somatic mutation Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 description 1
- 102100038838 2-Hydroxyacid oxidase 2 Human genes 0.000 description 1
- 101150098072 20 gene Proteins 0.000 description 1
- AVTRLVGCVSFGCO-UHFFFAOYSA-N 6-(diethylamino)hexyl 3,4,5-trimethoxybenzoate Chemical compound CCN(CC)CCCCCCOC(=O)C1=CC(OC)=C(OC)C(OC)=C1 AVTRLVGCVSFGCO-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 102100023579 Autophagy-related protein 2 homolog A Human genes 0.000 description 1
- 102100027444 B9 domain-containing protein 2 Human genes 0.000 description 1
- 102100026324 Beclin 1-associated autophagy-related key regulator Human genes 0.000 description 1
- 102100025215 CCN family member 5 Human genes 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 101100464293 Caenorhabditis elegans plk-1 gene Proteins 0.000 description 1
- 102100021753 Cardiolipin synthase (CMP-forming) Human genes 0.000 description 1
- 102100028914 Catenin beta-1 Human genes 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 102100039497 Choline transporter-like protein 3 Human genes 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 102000003849 Cytochrome P450 Human genes 0.000 description 1
- 102100027368 Histone H1.3 Human genes 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101001031584 Homo sapiens 2-Hydroxyacid oxidase 2 Proteins 0.000 description 1
- 101000905707 Homo sapiens Autophagy-related protein 2 homolog A Proteins 0.000 description 1
- 101000936627 Homo sapiens B9 domain-containing protein 2 Proteins 0.000 description 1
- 101000766227 Homo sapiens Beclin 1-associated autophagy-related key regulator Proteins 0.000 description 1
- 101000934220 Homo sapiens CCN family member 5 Proteins 0.000 description 1
- 101000895518 Homo sapiens Cardiolipin synthase (CMP-forming) Proteins 0.000 description 1
- 101000916173 Homo sapiens Catenin beta-1 Proteins 0.000 description 1
- 101001009450 Homo sapiens Histone H1.3 Proteins 0.000 description 1
- 101001044940 Homo sapiens Insulin-like growth factor-binding protein 2 Proteins 0.000 description 1
- 101001139134 Homo sapiens Krueppel-like factor 4 Proteins 0.000 description 1
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 101000967135 Homo sapiens N6-adenosine-methyltransferase catalytic subunit Proteins 0.000 description 1
- 101000610551 Homo sapiens Prominin-1 Proteins 0.000 description 1
- 101000752221 Homo sapiens Rho guanine nucleotide exchange factor 2 Proteins 0.000 description 1
- 101000844204 Homo sapiens Thioredoxin domain-containing protein 12 Proteins 0.000 description 1
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 1
- 101000976373 Homo sapiens YTH domain-containing protein 1 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 1
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000004374 Insulin-like growth factor binding protein 3 Human genes 0.000 description 1
- 108090000965 Insulin-like growth factor binding protein 3 Proteins 0.000 description 1
- 102100022710 Insulin-like growth factor-binding protein 2 Human genes 0.000 description 1
- 102100020677 Krueppel-like factor 4 Human genes 0.000 description 1
- 102000017578 LAG3 Human genes 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 108700005084 Multigene Family Proteins 0.000 description 1
- 102100040619 N6-adenosine-methyltransferase catalytic subunit Human genes 0.000 description 1
- 239000012826 P38 inhibitor Substances 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 102100040120 Prominin-1 Human genes 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 102000012211 Retinoic Acid 4-Hydroxylase Human genes 0.000 description 1
- 108010022037 Retinoic Acid 4-Hydroxylase Proteins 0.000 description 1
- 102100021707 Rho guanine nucleotide exchange factor 2 Human genes 0.000 description 1
- 108091006587 SLC13A5 Proteins 0.000 description 1
- 108091006525 SLC27A2 Proteins 0.000 description 1
- 108091007000 SLC44A3 Proteins 0.000 description 1
- 101150043341 Socs3 gene Proteins 0.000 description 1
- 102100035210 Solute carrier family 13 member 5 Human genes 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 102000058015 Suppressor of Cytokine Signaling 3 Human genes 0.000 description 1
- 108700027337 Suppressor of Cytokine Signaling 3 Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 1
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 description 1
- 102100032032 Thioredoxin domain-containing protein 12 Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 1
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 102100023048 Very long-chain acyl-CoA synthetase Human genes 0.000 description 1
- 238000001793 Wilcoxon signed-rank test Methods 0.000 description 1
- 102100023905 YTH domain-containing protein 1 Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000005902 aminomethylation reaction Methods 0.000 description 1
- -1 and Proteins 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 230000005773 cancer-related death Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001335 demethylating effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 230000004129 fatty acid metabolism Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 238000002682 general surgery Methods 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 210000005008 immunosuppressive cell Anatomy 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 238000001325 log-rank test Methods 0.000 description 1
- 102000033952 mRNA binding proteins Human genes 0.000 description 1
- 108091000373 mRNA binding proteins Proteins 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- HPZMWTNATZPBIH-UHFFFAOYSA-N methyl adenine Natural products CN1C=NC2=NC=NC2=C1N HPZMWTNATZPBIH-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000007474 nonparametric Mann- Whitney U test Methods 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000006712 oncogenic signaling pathway Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000012514 protein characterization Methods 0.000 description 1
- 239000003197 protein kinase B inhibitor Substances 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000028710 ribosome assembly Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/50—Mutagenesis
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B30/20—Sequence assembly
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- G16H70/00—ICT specially adapted for the handling or processing of medical references
- G16H70/40—ICT specially adapted for the handling or processing of medical references relating to drugs, e.g. their side effects or intended usage
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- G16H70/00—ICT specially adapted for the handling or processing of medical references
- G16H70/60—ICT specially adapted for the handling or processing of medical references relating to pathologies
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Analytical Chemistry (AREA)
- Theoretical Computer Science (AREA)
- Evolutionary Biology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Public Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Primary Health Care (AREA)
- Oncology (AREA)
- Artificial Intelligence (AREA)
- Bioethics (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Data Mining & Analysis (AREA)
- Databases & Information Systems (AREA)
- Library & Information Science (AREA)
- Evolutionary Computation (AREA)
Abstract
本发明公开了一种用于评估总生存期和表征肿瘤免疫微环境的基因特征及其研究方法,属于生物医学技术领域,所述用于评估总生存期和表征肿瘤免疫微环境的基因特征为m6A相关基因特征,具体包括20个蛋白质编码基因。本发明综合分析了TCGA中差异表达的基因,以确定与HCC预后相关的m6A特征;而且还评估了GSEA、肿瘤突变负荷(TMB)、免疫浸润和治疗反应,并进行了质谱蛋白质组学和多重免疫荧光测定以进行验证,最终可以得出m6A特征具有显著的评估总生存期和表征HCC肿瘤免疫微环境的能力,可作为风险分层管理的有用方法,并为选择合理的治疗策略提供有价值的线索。
Description
技术领域
本发明涉及生物医学技术领域,尤其涉及用于评估总生存期和表征肿瘤免疫微环境的基因特征及其研究方法。
背景技术
肝细胞癌(HCC)是全球第六大最常见的恶性肿瘤,也是癌症相关死亡的第四大原因。包括靶向疗法、化学疗法和免疫疗法在内的多种方案已被批准用于HCC治疗。然而,HCC患者的总体预后和生存期仍然很差,每年仍有约781,000例死于HCC。因此,有必要开发有效的方法来识别预后不良的患者。对于高危HCC亚群,需要更好的临床管理和合理的治疗策略,以改善总体预后。
目前,随着新的检测技术的进步,RNA分子中的N6-甲基腺嘌呤修饰(m6A)越来越受到关注,这对RNA的稳定性、输出、剪接或翻译有重大影响。m6A修饰由组装的复合物催化,该复合物由作为“写入器”的多个甲基转移酶、作为“读取器”的m6A RNA结合蛋白和作为“擦除器”的去甲基化酶组成。成熟的m6A“写入器”包括METTL3、METTL14、WTAP、RBM15和ZC3H13。这些“写入器”负责向靶RNA添加甲基化单元。值得注意的是,m6A修饰是一个动态可逆过程,可以通过“擦除器”RNA去甲基化酶——FTO以及ALKBH5去除。m6A修饰的RNA可以被各种RNA结合蛋白“阅读器”识别以确定RNA命运,包括YTHDC1、YTHDF1/2/3、***mRNA结合蛋白家族IGFBP1/2/3和RBMX。
越来越多的证据表明,m6A修饰在癌症恶性演化中起着至关重要的作用,已成为癌症生物学研究的热点领域。有许多研究描述了m6A修饰如何调节HCC的发展。例如,“写入器”METTL14可以介导EGFR的m6A甲基化,从而抑制EGFR/PI3K/AKT活性并抑制HCC中的EMT和转移。m6A“阅读器”YTHDF1可以通过与m6A修饰的ATG2A和ATG14 mRNA结合来促进HCC细胞的自噬,从而增加它们在缺氧条件下的翻译。“擦除器”FTO也参与HCC进展,它可以通过去甲基化SOX2、KLF4和NANOG mRNA来调节癌症干细胞特性。鉴于m6A修饰的重要调节功能,m6A相关基因可能是用于患者分层的有前途的分子谱。最近,已经在包括HCC在内的各种癌症中鉴定出多个m6A相关基因特征。然而,大多数签名缺乏独立队列的验证,这些模型的可靠性和稳健性仍然难以捉摸。
发明内容
针对上述存在的问题,本发明旨在提供一种用于评估总生存期和表征肿瘤免疫微环境的基因特征及其研究方法,鉴定并验证了m6A相关蛋白编码基因特征,该特征有助于指示预后、表征肿瘤免疫微环境并预测HCC患者治疗的潜在疗效,从而有助于风险分层管理,以及为高危HCC患者选择合理的治疗策略。
为了实现上述目的,本发明所采用的技术方案如下:
用于评估总生存期和表征肿瘤免疫微环境的基因特征,其特征在于:所述基因特征为m6A相关基因特征。
进一步的,所述m6A相关基因特征包括20个蛋白质编码基因,IGFBP3、HDAC2、HMGN1、TCP1、PABPC4、RDH16、HDDC2、CFHR5、GYS1、MAPRE1、GYS2、BLMH、YBX1、NAP1L1、INTS8、MARCKSL1、STX6、MASP2、UQCRH和XPNPEP1。
进一步的,用于评估总生存期和表征肿瘤免疫微环境的基因特征的研究方法,其特征在于,包括以下步骤,
S1:获取HCC肿瘤样本和正常样本基因表达数据;
S2:构建m6A Score评分体系,并评估所有HCC肿瘤样本;
S3:确定HCC高危和低危人群,并进行GO和KEGG通路富集分析,以确定用于评估总生存期和表征肿瘤免疫微环境的基因特征;
S4:对HCC肿瘤样本的GSEA、肿瘤突变负荷、免疫浸润和治疗反应进行评估;
S5:通过质谱蛋白质组学和多重免疫荧光测定进行验证。
进一步的,步骤S2的具体操作包括以下步骤,
S201:通过一致性聚类算法分析HCC肿瘤样本中所有基因的表达,生成不同的聚类;
S202:通过维恩图获得重叠的差异表达基因DEG;
S203:使用Cox比例风险回归分析确定不同m6A修饰模式的蛋白质编码基因的预后价值,确定用于构建m6A Score评分体系的蛋白质编码基因;
S204:对步骤S203中确定的蛋白质编码基因进行主成分分析,建立m6A Score评分体系。
进一步的,步骤S5中质谱蛋白质组学的具体操作包括以下步骤,
S501a:从***固定石蜡包埋的肿瘤样本中分离蛋白质;
S502 a:对分离出来的蛋白质进行消化;
S503 a:通过质谱法进行肽测序;
S504 a:数据分析以识别蛋白质。
进一步的,步骤S5中多重免疫荧光测定的具体操作包括以下步骤,
S501b:石蜡切片脱蜡去水后,将载玻片浸入柠檬酸抗原修复缓冲液和开水中进行抗原修复;
S502 b:将样品与3%H2O2在室温下孵育,溶液在37℃下用5%FBS封闭30分钟,然后在4℃下与一抗孵育过夜;
S503 b:将样品用磷酸盐缓冲盐水PBS冲洗,与Cy5/Sp Green标记的二抗在37°C下孵育,然后再次用PBS冲洗;
S504 b:将溶液与Hoechst在室温下孵育15分钟,并通过微波处理去除先前的一抗/二抗,重复进行连续染色;
S505 b:对样本进行多重免疫荧光测定。
本发明的有益效果是:
1、本发明中提出了一种用于评估总生存期和表征肿瘤免疫微环境的m6A相关基因特征,具体包括20个蛋白质编码基因,IGFBP3、HDAC2、HMGN1、TCP1、PABPC4、RDH16、HDDC2、CFHR5、GYS1、MAPRE1、GYS2、BLMH、YBX1、NAP1L1、INTS8、MARCKSL1、STX6、MASP2、UQCRH和XPNPEP1,本发明鉴定并验证了这些蛋白编码基因特征对于评估HCC预后是可靠且稳健的。
2、本发明通过综合分析TCGA中差异表达的基因,以确定与HCC预后相关的m6A特征;而且还评估了GSEA、肿瘤突变负荷(TMB)、免疫浸润和治疗反应,并进行了质谱蛋白质组学和多重免疫荧光测定以进行验证,最终可以得出m6A特征具有显著的评估总生存期和表征HCC肿瘤免疫微环境的能力,可作为风险分层管理的有效方法,并为选择合理的治疗策略提供有价值的线索。
附图说明
图1为本发明中部分样品的D15和D17质谱基峰光谱图。
图2为本发明中组织样本的蛋白质鉴定数量结果。
图3为本发明中在HCC中建立m6A相关蛋白编码基因特征的流程图。
图4为本发明在HCC中m6A相关基因特征鉴定时,使用uniCox回归分析的15m6A相关基因与OS和基因表达的关系森林图和用于确定主成分最佳数量的碎石图。
图5为本发明中在HCC中m6A相关基因特征鉴定时,3个簇中差异表达基因(DEG)的GO和KEGG富集分析的气泡图。
图6为本发明中在HCC中m6A相关基因特征鉴定时,3个簇中的DEG数量的维恩图,以及质谱法验证基因和预后相关基因。
图7为本发明中在HCC中m6A相关基因特征鉴定时,20个基因在HCC肿瘤和癌旁标本中的表达水平以及拷贝数变异(CNV)频率。
图8为本发明中SRAMP预测的m6A相关基因序列中BLMH、HDAC2、INTS8、NAP1L1的m6A甲基化位点。
图9为本发明中SRAMP预测的m6A相关基因序列中CFHR5、HDDC2、MAPRE1、PABPC4的m6A甲基化位点。
图10为本发明中SRAMP预测的m6A相关基因序列中GYS1、HMGN1、MARCKSL1、RDH16的m6A甲基化位点。
图11为本发明中SRAMP预测的m6A相关基因序列中GYS2、IGFBP3、MASP2、STX6的m6A甲基化位点。
图12为本发明中SRAMP预测的m6A相关基因序列中TCP1、UQCRH、XPNPEP1、YBX1的m6A甲基化位点。
图13为本发明中TCGA-HCC队列中m6A相关特征的临床意义。
图14为本发明中独立HCC队列中m6A相关基因特征的蛋白质组学验证结果。
图15为本发明中m6A相关特征的功能分析和对靶向/化疗药物敏感性的预测结果。
图16为本发明中m6A相关特征的肿瘤体细胞突变和免疫治疗敏感性预测结果。
图17为本发明中m6A相关特征与免疫细胞和相关基因浸润的相关性研究结果。
图18为本发明中肝细胞癌标本中的免疫细胞浸润验证结果。
具体实施方式
为了使本领域的普通技术人员能更好的理解本发明的技术方案,下面结合附图和实施例对本发明的技术方案做进一步的描述。
实施例一:
实施例一中提供一种用于评估总生存期和表征肿瘤免疫微环境的基因特征,所述基因特征为m6A相关基因特征。
具体的,所述m6A相关基因特征包括20个蛋白质编码基因,IGFBP3、HDAC2、HMGN1、TCP1、PABPC4、RDH16、HDDC2、CFHR5、GYS1、MAPRE1、GYS2、BLMH、YBX1、NAP1L1、INTS8、MARCKSL1、STX6、MASP2、UQCRH和XPNPEP1。
实施例二:
实施例二中提供一种用于评估总生存期和表征肿瘤免疫微环境的基因特征的研究方法,包括以下步骤,
S1:获取HCC肿瘤样本和正常样本基因表达数据;
S2:构建m6A Score评分体系,并评估所有HCC肿瘤样本;
S3:确定HCC高危和低危人群,并进行GO和KEGG通路富集分析,以确定用于评估总生存期和表征肿瘤免疫微环境的基因特征;
S4:对HCC肿瘤样本的GSEA、肿瘤突变负荷、免疫浸润和治疗反应进行评估;
S5:通过质谱蛋白质组学和多重免疫荧光测定进行验证。
具体的,该研究过程具体包括以下内容:
材料和方法:
HCC样本的基因表达数据
HCC的原始基因表达数据和相应的临床信息是从癌症基因组图谱(TCGA)数据库(https://portal.gdc.cancer.gov/)中提取的。包括374个肿瘤标本和50个正常标本的转录数据。HTseq计数基于每百万转录本(TPM)方法进行了标准化。
排除标准如下:(i)组织学诊断排除了HCC,(ii)基因表达值极低,以及(iii)临床数据不完整且随访时间<30天。ID转换和RNA分类由Perl进行(The Perl ProgrammingLanguage version 5.30.1,https://www.perl.org/)。
HCC中m6A相关签名的构建
为了生成m6A相关特征并量化每位患者的m6A修饰模式,本发明中构建了一个评分***(称为m6A Score)并评估所有HCC患者。
首先,通过一致性聚类算法分析HCC肿瘤样本中所有基因的表达,以生成不同的聚类;然后通过维恩图获得重叠的差异表达基因DEG;接下来,使用Cox比例风险回归分析确定不同m6A修饰模式的蛋白质编码基因的预后价值,确定用于构建m6A Score评分体系的蛋白质编码基因(将具有显著预后价值的蛋白质编码DEGs用于构建m6A Score评分体系);鉴于HCC中的预后蛋白编码DEG,然后进行主成分分析(PCA)以建立m6A Score。PCA是一种降维方法,已广泛用于基因表达分析,本发明中添加了主成分(PC)1和2作为最后的基因特征评分。
式中,i和j是HCC中m6A相关预后蛋白编码的顺序和总数。最后,使用m6A Score的Z分数进行进一步分析。
对于不同的簇,基因本体论(Gene ontology,GO)和京都基因与基因组百科全书(KEGG)通路被识别并通过气泡图可视化。
HCC参与者注册
2015年3月至2020年6月期间,复旦大学华山医院共纳入了101名HCC患者的独立队列。所有患者在手术前均未接受放疗或化疗。每位患者都提供了知情同意书。研究方案经复旦大学华山医院人类研究伦理委员会批准。有关样品的基本信息如下表1和下表2所示。
表1华山医院独立HCC队列的临床信息
表2华山医院一个独立HCC队列的20个m6A相关基因表达
续表2(1)
续表2(2)
一般突变信息
从TCGA数据库下载拷贝数变异(CNV)数据。本发明中还从TCGA数据库下载了基因突变数据,并在高/低m6AScore组中识别出基因突变。此外,每个样本的肿瘤突变负荷(TMB)是通过Perl脚本计算的(https://www.perl.org/),TMB被定义为肿瘤组织中每兆碱基的突变总数。
基因集富集分析(GSEA)
GSEA是一种计算方法,可以对给定的基因列表执行GO和KEGG分析。GSEA软件版本4.2.3(https://www.gsea-msigdb.org/)用于预测m6A相关签名的潜在功能。结合m6A Score确定的高危和低危人群。进行了GO和KEGG通路富集分析,以可视化涉及不同通路、生物学功能及其表达模式的各种基因。数据已针对多次测试进行了校正(重复次数=1000)。
化疗反应预测
pRRhetic的R包用于预测常见化疗药物的敏感性。敏感性表示物质抑制特定生物或生化功能的有效性。组间差异通过Wilcoxon符号秩检验进行检验。
预测对免疫疗法的反应
使用肿瘤免疫功能障碍和排除(TIDE)算法进行T细胞功能障碍的基因特征分析和患者对癌症免疫治疗反应的预测。TIDE利用T细胞功能障碍和排除特征来模拟具有不同细胞毒性T淋巴细胞水平的肿瘤的免疫逃逸,并且TIDE评分与肿瘤免疫逃逸的特征一致。较高的肿瘤TIDE评分与较差的免疫检查点阻断反应相关。
免疫细胞浸润及免疫功能分析
通过进行单样本基因集富集分析(ssGSEA),计算出每位HCC患者的免疫功能评分,以进一步量化肿瘤浸润免疫细胞的组成。根据免疫评分,量化HCC组织中免疫细胞浸润的程度。下表3中列出了参与本研究的免疫细胞基因特征。
表3参与本研究的免疫细胞基因特征(部分)
质谱蛋白质组学检测
蛋白质组学检测分四个步骤进行:从***固定石蜡包埋的肿瘤样本中分离蛋白质、蛋白质消化、通过质谱法(MS)进行肽测序以及数据分析以识别蛋白质。
MS生成的原始文件的处理细节如下:使用Mascot搜索引擎(v2.3,Matrix ScienceInc.;version 04-07-2013,total 32015)在NCBI Human Refseq数据库中搜索MS原始文件条目。母离子和子离子质量偏差分别设置为20和50 ppm(QExactive HF)或0.5 Da。理论上的蛋白质切割位点是精氨酸(R)和赖氨酸(K),最多允许有两个缺失的切割位点。用于固定化的氨基甲基化(C)和用于动态修饰的乙酰基(蛋白质N端)和氧化(M)。所有鉴定的肽段均取自MS1峰计算下的面积,肽段假发现率控制在1%。在蛋白质表征层面,我们只保留那些至少包含一种独特肽和两种高质量肽(严格肽,即Mascot Ion Score>20)的蛋白质。对于蛋白质定量,我们使用基于强度的绝对量化算法(即iBAQ算法),随后将每个iBAQ值归一化为FOT-iBAQ值。这是通过将iBAQ值除以相应样品中所有检测到的蛋白质的iBAQ总和来执行的。然后,为了使这个FOT大小更易于读写,将这个FOT值乘以105,得到iFOT值。
从复旦大学华山医院普通外科获得的101个HCC标本的蛋白质组测序由国家计量研究院(中国)如上所述进行。研究涉及的样品质量控制和蛋白质谱表达如附图1和附图2所示,其中图1为本发明中部分样品的D15和D17质谱基峰光谱图,每个样品的蛋白质鉴定结果如附图2所示,在图2中,每个样本的蛋白质鉴定量超过4000,大多数样本在4500-5000之间。
多重免疫荧光(mIF)测定
石蜡切片脱蜡去水后,将载玻片浸入柠檬酸抗原修复缓冲液和开水中进行抗原修复;在用磷酸盐缓冲盐水(PBS)冲洗之前,将样品与3%H2O2在室温下孵育,溶液在37℃下用5%FBS封闭30分钟,然后在4℃下与一抗孵育过夜;样品用PBS冲洗,与Cy5/Sp Green标记的二抗在37℃下孵育,然后再次用PBS冲洗;然后将溶液与Hoechst在室温下孵育15分钟,并通过微波处理去除先前的一抗/二抗,重复进行连续染色;
统计分析
进行Kaplan-Meier分析和对数秩检验以比较高/低风险HCC组之间的生存差异。测试数据的正态分布,如果正态分布,则使用t检验,或应用非参数Mann-Whitney U检验,除非另有说明。根据参数数据的Pearson检验和非参数数据的Spearman检验评估相关性。所有统计分析均使用Prism(第8版,https://www.graphpad-prism.cn/)和R统计软件(版本4.1.3,https://www.r-project.org/)。P值<0.05表示具有统计学显着性差异(*P<0.05;**P<0.01和***P<0.001)。
实验结果
HCC中m6A相关基因特征的鉴定
TCGA数据库包括33种癌症的详细临床信息,被认为是癌症基因组学计划的里程碑。为了识别HCC中的m6A相关特征,从TCGA数据库下载了374名HCC患者的所有原始基因表达数据。在仔细核对每个样本的临床信息后,排除了31名因临床数据不完整或随访时间少于30天的患者。最终,343名具有可靠转录数据和详细临床信息的患者被纳入研究。通过文献报道,进一步对15个成熟的m6A调控基因(METTL3、METTL14、WTAP、ZC3H13、RBM15、YTHDC1、YTHDF1、YTHDF2、YTHDF3、IGFBP1、IGFBP2、IGFBP3、RBMX、FTO和ALKBH5)进行了研究分析。整个分析过程总结为附图3中的流程图。
通过使用基因表达水平的单变量Cox回归分析,15m6A调节剂中的11种与HCC预后显著相关,而METTL14、IGFBP1/2和ALKBH5未能达到统计显著,如附图4中A中所示。根据与11m6A调节器的共识算法,当横坐标为k=3时,碎石图的斜率急剧下降,表明可以将患者分为三类以获得更好的聚类呈现,如附图4中B所示。GO和KEGG分析显示每个聚类显示不同的特征,如附图5所示。簇A与RNA加工和剪接密切相关(图5中左上图)。簇B参与组蛋白修饰和核糖体组装(图5中右上图)。簇C主要富含细胞外结构、胶原基质和整合素结合(图5中左下图)。在所有三个簇中有85个差异表达基因重叠,如下表4所示。其中68个差异表达基因与HCC预后显著相关,如附图6中左图以及下表5所示。鉴于蛋白质编码基因是生物学功能的主要执行者,本发明中对101份HCC样本进行了质谱(MS)分析的蛋白质组学检测。总共有7059个蛋白质表达水平通过MS测定法成功检测到。随后,将68个预后相关基因与MS检测到的基因相交,最终将20个蛋白质编码基因定义为m6A相关特征以供进一步分析,如附图6中右图所示,它们的系数如下表6所示。
表4肝细胞癌中m6A修饰A、B和C簇的DEGs
IGFBP3 | SLC27A2 | SLC13A5 | TCP1 | CFHR5 | AL139005.1 | RPS4XP1 | KDELR3 |
FAM117B | HDAC2 | DZIP1L | FANCE | T×NDC12 | BICD1 | INTS8 | UQCRH |
KDM1A | CRLS1 | SLC44A3 | PABPC4 | GYS1 | DCUN1D5 | MARCKSL1 | MACROD2 |
AC104066.2 | RAB11A | CFHR3 | GAS2L3 | MAPRE1 | CEP126 | GLYATL1 | AC022211.1 |
CIDEC | CFAP298 | PIGS | RAVER2 | GYS2 | NAP1L1 | KLRB1 | XPNPEP1 |
H1-3 | BMT2 | PDE7A | LINC01121 | SFT2D1 | AP000892.4 | STX6 | FAM86DP |
SNRNP40 | CCN5 | CDK19 | RIBC2 | BLMH | AP002990.1 | SOCS3 | UBA2 |
C3P1 | CDCA7 | SNHG3 | TMEM234 | Y8X1 | RALGPS2-AS1 | SCLT1 | SNORA15B-1 |
PAQR8 | MSI2 | LBX2-AS1 | RDH16 | IYD | ARHGEF2-AS2 | PROM1 | |
LHFPL3-AS2 | HMGN1 | B9D2 | HDDC2 | AC114760.2 | PTMAP4 | MASP2 | |
STXBP4 | LINC01018 | UTP11 | CYP26B1 | HAO2 | AC084824.1 | ACTG1P25 |
表5 m6A修饰A、B和C簇DEG的预后分析
id | HR | HR.95L | HR.95H | pvalue | id | HR | HR.95L | HR.95H | pvalue | |
IGFBP3 | 1.202152 | 1.066211 | 1.355424 | 0.002638 | HDDC2 | 1.468087 | 1.164597 | 1.850666 | 0.001156 | |
FAM117B | 1.314641 | 1.090386 | 1.585017 | 0.004147 | CYP2681 | 1.399713 | 1.220531 | 1.6052 | 1.5E-06 | |
KDM1A | 1.956574 | 1.518727 | 2.520651 | 2.07E-07 | CFHR5 | 0.916698 | 0.858619 | 0.978706 | 0.009201 | |
AC104066 | 2.797414 | 1.85927 | 4.208924 | 7.99E-07 | TXNDC12 | 2.345491 | 1.650229 | 3.333675 | 2.01E-06 | |
CIDEC | 1.119483 | 1.01631 | 1.23313 | 0.022142 | GYS1 | 1.40372 | 1.126197 | 1.749631 | 0.002549 | |
SNRNP40 | 1.826473 | 1.397778 | 2.386646 | 1.02E-05 | MAPRE1 | 1.443094 | 1.180244 | 1.764484 | 0.00035 | |
C3P1 | 0.890395 | 0.82006 | 0.966762 | 0.005691 | GYS2 | 0.884051 | 0.816449 | 0.957251 | 0.002394 | |
PAQR8 | 1.172628 | 1.000384 | 1.374529 | 0.049449 | SFT2D1 | 1.675212 | 1.327143 | 2.11457 | 1.41E-05 | |
LHFPL3-A | 1.203727 | 1.046051 | 1.38517 | 0.00964 | BLMH | 1.20832 | 1.042625 | 1.400346 | 0.011915 | |
STXBP4 | 1.490245 | 1.126437 | 1.971554 | 0.005211 | YBX1 | 2.334344 | 1.799421 | 3.028287 | 1.73E-10 | |
HDAC2 | 1.93874 | 1.515903 | 2.479519 | 1.33E-07 | IYD | 0.793324 | 0.683902 | 0.920253 | 0.002232 | |
RAB11A | 1.363697 | 1.00219 | 1.855607 | 0.048395 | BICD1 | 1.951282 | 1.470895 | 2.588561 | 3.55E-06 | |
CFAP298 | 1.517849 | 1.106749 | 2.081652 | 0.009617 | DCUN1D5 | 1.888181 | 1.473202 | 2.420053 | 5.17E-07 | |
BMT2 | 1.649991 | 1.270008 | 2.143665 | 0.000177 | CEP126 | 2.008702 | 1.379218 | 2.925487 | 0.000277 | |
CDCA7 | 1.267975 | 1.119948 | 1.435566 | 0.000178 | NAP1L1 | 1.541312 | 1.265808 | 1.87678 | 1.66E-05 | |
MSI2 | 1.263862 | 1.017455 | 1.569945 | 0.034315 | AP002990 | 1.650051 | 1.138396 | 2.391671 | 0.008184 | |
HMGN1 | 1.298436 | 1.031847 | 1.633902 | 0.025924 | ARHGEF2 | 1.978118 | 1.312059 | 2.982296 | 0.001128 | |
LINC01018 | 0.901427 | 0.836688 | 0.971175 | 0.006349 | PTMAP4 | 2.271206 | 1.706525 | 3.022738 | 1.86E-08 | |
DZIP1L | 1.523597 | 1.237509 | 1.875823 | 7.24E-05 | AC084824 | 1.582702 | 1.122336 | 2.231903 | 0.008843 | |
CFHR3 | 0.858593 | 0.79341 | 0.929131 | 0.000154 | RPS4XP1 | 1.839692 | 1.02241 | 3.310282 | 0.041961 | |
PIGS | 1.484926 | 1.230384 | 1.792127 | 3.77E-05 | INTS8 | 1.743036 | 1.353881 | 2.244049 | 1.63E-05 | |
PDE7A | 1.413364 | 1.158933 | 1.723652 | 0.000634 | MARCKSL | 1.37243 | 1.195007 | 1.576195 | 7.38E-06 | |
CDK19 | 1.404927 | 1.147375 | 1.720292 | 0.001 | GLYATL1 | 0.844672 | 0.7687 | 0.928153 | 0.000447 | |
SNHG3 | 1.459384 | 1.24702 | 1.707913 | 2.46E-06 | KLRB1 | 0.795015 | 0.672439 | 0.939936 | 0.007253 | |
UTP11 | 3.015607 | 2.176226 | 4.178741 | 3.31E-11 | STX6 | 1.667861 | 1.306571 | 2.129055 | 4.01E-05 | |
TCP1 | 1.670928 | 1.28745 | 2.168628 | 0.000114 | SCLT1 | 1.840914 | 1.287927 | 2.631333 | 0.000813 | |
FANCE | 1.583806 | 1.307757 | 1.918124 | 2.53E-06 | MASP2 | 0.889218 | 0.822008 | 0.961923 | 0.00341 | |
PABPC4 | 1.770198 | 1.355257 | 2.312183 | 2.78E-05 | KDELR3 | 1.228993 | 1.086776 | 1.389821 | 0.001015 | |
GAS2L3 | 1.688699 | 1.410232 | 2.022152 | 1.21E-08 | UQCRH | 1.92623 | 1.493764 | 2.4839 | 4.34E-07 | |
RAVER2 | 1.215942 | 1.030797 | 1.434342 | 0.020348 | MACROD2 | 1.282311 | 1.008208 | 1.630934 | 0.042705 | |
LINC0112 | 2.441549 | 1.285963 | 4.635562 | 0.006356 | AC022211 | 3.104223 | 1.505991 | 6.398578 | 0.002144 | |
RIBC2 | 1.437506 | 1.225608 | 1.686039 | 8.19E-06 | XPNPEP1 | 1.743086 | 1.268546 | 2.395143 | 0.00061 | |
TMEM234 | 1.902631 | 1.454 | 2.489687 | 2.76E-06 | FAM86DP | 2.005571 | 1.442969 | 2.787526 | 3.43E-05 | |
RDH16 | 0.879342 | 0.816205 | 0.947363 | 0.000719 | UBA2 | 1.477286 | 1.173808 | 1.859225 | 0.000881 |
表620个m6A相关基因的系数
Gene names | PC1 | PC2 | PC |
IGFBP3 | 0.505342 | 0.016672 | 0.522013718 |
HDAC2 | 0.867325 | 0.119152 | 0.986476171 |
HMGN1 | 0.73316 | 0.056543 | 0.789703725 |
TCP1 | 0.676837 | 0.092023 | 0.768860137 |
PABPC4 | 0.728055 | -0.01474 | 0.71331339 |
RDH16 | -0.58298 | 0.522821 | -0.060163531 |
HDDC2 | 0.736668 | -0.10174 | 0.634928786 |
CFHR5 | -0.41475 | 0.519119 | 0.104369803 |
GYS1 | 0.73258 | 0.032457 | 0.765037486 |
MAPRE1 | 0.872042 | 0.062224 | 0.934266148 |
GYS2 | -0.61596 | 0.641265 | 0.025300886 |
BLMH | 0.685407 | -0.10318 | 0.58222723 |
YBX1 | 0.692319 | 0.071267 | 0.763585884 |
NAP1L1 | 0.845052 | -0.10595 | 0.739100649 |
INTS8 | 0.770094 | 0.042677 | 0.812771311 |
MARCKSL1 | 0.746138 | -0.26199 | 0.484149414 |
STX6 | 0.830609 | 0.091894 | 0.922502372 |
MASP2 | -0.67542 | 0.218138 | -0.457282071 |
UQCRH | 0.58297 | -0.1771 | 0.405866493 |
XPNPEP1 | 0.729938 | 0.135524 | 0.865462231 |
比较HCC组织和癌旁组织之间20m6A相关基因的表达水平。其中大部分被上调,其中5个被下调,如附图7中A所示,其中,红色框表示肿瘤,蓝色表示正常。丢失和增加的拷贝数变异(CNV)如附图7中B所示,其中,CNV获得和缺失分别以红色和绿色表示。此外,通过SRAMP在线分析发现这20个基因中存在大量高度密集的m6A簇,如附图8-12所示,表明这些基因可能是m6A修饰的潜在靶点。
综上所述,本发明建立了一个由20个蛋白质编码基因(IGFBP3、HDAC2、HMGN1、TCP1、PABPC4、RDH16、HDDC2、CFHR5、GYS1、MAPRE1、GYS2、BLMH、YBX1、NAP1L1、INTS8、MARCKSL1、STX6、MASP2、UQCRH和XPNPEP1)组成的m6A相关特征,与HCC预后显著相关。
m6A相关特征在HCC中的临床意义
m6A相关特征在HCC中的临床意义研究结果如附图13所示,其中,A为根据TCGA HCC样本中m6A相关特征的m6AScore分布;红色代表高m6AScore(高风险)组,蓝色代表低m6AScore(低风险)组。B为m6AScore与生存状态的关联;红色代表死亡的患者,蓝色代表幸存的患者。C中箱线图显示了m6AScore与临床病理分期之间的关系;蓝色代表I-II期患者,红色代表III-IV期患者。D为低m6AScore组(蓝色图)和高m6AScore组(红色图)的Kaplan-Meier生存曲线。E为1(红色图)、3(蓝色图)和5(绿色图)年的m6Ascore ROC下的接受者操作特征(ROC)曲线和面积。F为m6AScore 1(红色图)、3(蓝色图)和5(绿色图)年的校准曲线。
根据m6A相关特征,按照方法(HCC中m6A相关签名的构建)中的描述为每位患者计算m6AScore。根据生存曲线的最佳切点,将TCGA中的343例HCC患者分为低危组和高危组。如附图13中A和B所示。随着m6AScore的升高,死亡风险逐渐增加。同时,高m6AScore与晚期HCC分期显著相关,如附图13中C所示。因此,具有高m6AScore的患者被分层为高危HCC组。
然后,针对总生存期(OS)的Kaplan-Meier分析显示,高危人群的生存时间通常较短,如附图13中D所示。与低危患者相比,高危患者的1年、3年和5年生存率分别为62.2%对91.7%、40.1%对70.7%和21.9%对57.0%。受试者工作特征(ROC)曲线分析表明,m6AScore是HCC患者OS预测的有用预后指标,因为ROC曲线下面积(AUC)在1、3和5年分别达到0.746、0.669和0.628,如附图13中E所示。此外,m6AScore模型显示了良好的校准,1年、3年和5年生存的校准曲线都接近理想线,如附图13中F所示。
总的来说,这些发现表明m6A相关编码基因特征具有预测HCC患者总体生存的强大能力。
独立HCC队列中m6A相关基因特征的蛋白质组学验证
为了评估m6A相关蛋白编码基因标记在预后评估中的可靠性,使用101个HCC标本的独立队列进行进一步验证,结果如附图14所示,其中,A为根据独立HCC样本中m6A相关特征的m6AScore分布;红色代表高m6AScore(高风险)组,蓝色代表低m6AScore(低风险)组。B为m6AScore与生存状态的关联;红色代表死亡的患者,蓝色代表幸存的患者。C中箱线图显示了m6AScore与临床病理分期之间的关系;蓝色代表I-II期患者,红色代表III-IV期患者。D为低m6AScore组(蓝色图)和高m6AScore组(红色图)的Kaplan-Meier生存曲线。E为1(红色图)、3(蓝色图)和5(绿色图)年的m6AScore ROC下的接受者操作特征(ROC)曲线下面积。F为1(红色图)、3(蓝色图)和5(绿色图表)年的m6AScore校准曲线。
本发明中验证的HCC队列的基本信息如上表1所示。m6A相关特征的蛋白质水平通过质谱(MS)测定法测定,并计算m6AScore以如上所述分为高风险和低风险HCC组。
与附图13中的结果一致,高m6AScore组具有更高的死亡风险(如附图14中A和B所示)和更晚的临床分期(如附图14中C所示)。此外,高风险组的生存时间明显更短(如附图14中D所示)。与低危患者相比,高危患者的1年和3年生存率分别为87.5%对92.2%和44.1%对71.8%。ROC曲线分析进一步证实了m6A相关特征的准确性和敏感性,因为AUC在1年、3年分别达到0.722和0.604(如附图14中E所示)。1年和3年的校准曲线非常接近理想曲线,显示出令人满意的校准效果(如附图14中F所示)。
因此,这些经过验证的结果证实了m6A相关特征的可靠性和稳健性,可以很容易地对高风险HCC患者进行分层并评估总生存期。m6A相关特征可能有助于在早期为高风险HCC提供更好的临床管理。
m6A相关基因特征的功能表征及高危HCC人群的潜在治疗策略
为了利用m6A相关特征的潜在机制,本发明中对GO和KEGG分析进行了富集分析,以探索可能的生物学功能,结果如附图15所示,其中,A为基因本体论(GO)的基因集富集分析;下图显示了低m6AScore组的基因集富集,上图显示了高m6AScore组的基因集富集。B为KEGG通路的基因集富集分析;下图显示了低m6AScore组的基因集富集,上图显示了高m6AScore组的基因集富集。C-K为高或低m6AScore组中几种化疗药物的估计logIC50的箱线图。
如附图15中A和B所示,在高危HCC组中,异常DNA复制和重组、表观遗传失调、细胞周期紊乱以及MAPK、mTOR和VEGF等致癌信号通路的激活明显丰富。而氨基酸和脂肪酸代谢紊乱、细胞色素p450通路异常在低危HCC组明显增多。
根据富集分析结果,本发明中试图探索高风险HCC治疗的潜在有效策略。一些FDA批准的抑制剂和化疗药物可以靶向细胞周期(CGP.082996)、抑制DNA复制和合成(依托泊苷和丝裂霉素C)并使MAPK信号通路失活(PLK1、AKT和p38抑制剂)被选择用于进一步分析。然后,使用pRRophetic平台在低风险和高风险亚组中评估这些抑制剂的半数最大抑制浓度(IC50)。pRRheetic是一个独立的授权公共资源,可根据基因表达微阵列数据预测抑制剂敏感性。如附图15中C-H所示,高危HCC患者对上述抑制剂更敏感,因为低药物浓度可有效抑制细胞增殖。
这些发现表明,低风险和高风险人群可能具有不同的致癌机制来促进HCC的发展。高危HCC患者可能对CDK和MAPK抑制剂以及化疗药物依托泊苷和丝裂霉素C有更好的反应。m6A相关特征有助于选择合理的治疗策略。
m6A相关特征与肿瘤突变负荷和免疫检查点阻断疗法相关
肿瘤突变负荷(TMB)是评估癌症进展和免疫治疗疗效的重要指标,m6A相关特征的肿瘤体细胞突变和免疫治疗敏感性预测结果如附图16所示,其中,A为低m6AScore组的基因突变频率。B为高m6AScore组的基因突变频率。C为四组的Kaplan-Meier生存曲线除以肿瘤突变负荷(TMB)和m6AScore。L-TMB是低TMB的简称,H-TMB是高TMB的简称;L-m6AScore是lowm6AScore的简称,Hm6AScore是high-m6AScore的简称。D为通过TIDE评分预测四组免疫治疗反应的分析;较低的TIDE分数表明对免疫疗法更敏感。E中箱形图显示了高m6AScore组和低m6AScore组之间免疫检查点基因(PD-1、PD-L1、CTLA4、TIM3、LAG3)的差异表达。
如附图16中A和B的瀑布图所示,低风险组和高风险组的基因突变图谱明显不同。在低风险患者中,CTNNB1突变是最常见的事件(如附图16中A所示);而在高风险组中,TP53突变的频率出现在超过一半的患者中(如附图16中B所示)。然后,根据高-/低-TMB和m6AScore的组合,将HCC患者分为四个亚组,如附图16中C所示,L-TMB+L-m6AScore,L-TMB+H-m6AScore,H-TMB+L-m6AScore和H-TMB+H-m6AScore。最终发现L-TMB+H-m6AScore组的预后最差,而L-TMB+L-m6AScore组的预后最好。
使用免疫检查点抑制剂的免疫疗法在包括HCC治疗在内的各种癌症中取得了令人鼓舞的临床结果。TIDE是一种用于预测对免疫疗法的反应的算法,高TIDE分数通常表示反应不佳。本发明中还发现,无论TMB为何,具有高m6AScore评分的HCC患者都倾向于降低TIDE评分(如附图16中D所示),这表明高危人群可能受益于免疫治疗。此外,我们还分析了每个TMB联合m6AScore亚组中有据可查的免疫检查点分子的表达水平,包括CTLA4、PD-1、PD-L1、LAG3和TIM3。如附图16中E所示,大多数这些免疫检查点分子在高m6AScore亚组中表现出更高的表达水平,表明高危HCC组可能抑制T细胞活化并逃避抗肿瘤免疫。另一方面,这暗示高危HCC患者可能对免疫检查点疗法敏感,例如针对PD-1、CTLA-4、TIM3和LAG3的抗体。
综合起来,m6AScore结合TMB可以对预后最差的HCC亚组进行分层。高危HCC患者容易通过上调免疫检查点分子的表达而发生免疫逃逸,可能受益于免疫检查点治疗。
m6A特征可以表征肿瘤免疫微环境
为了***地描述肿瘤免疫微环境的状态,本发明中通过ssGSEA分析比较了各种免疫细胞的浸润百分比,结果如附图17所示,其中,A中箱线图显示了HCC中m6AScore和免疫浸润水平的ssGSEA分析;红色代表高m6AScore组,蓝色代表低m6AScore组。B中相关热图展示了m6AScore、20种基因表达和免疫细胞浸润之间的关系。C-F箱形图显示了Treg(调节性T细胞)、M2型巨噬细胞相关基因在高m6AScore组和低m6AScore组之间的表达。
值得注意的是,免疫抑制细胞如M2极化巨噬细胞和T调节细胞(Treg)在高危组中明显积累,如附图17中A所示。m6A特征中的20个基因中的每一个都对免疫细胞浸润有各自的贡献(图如附图17中B所示)。众所周知,Foxp3是Treg的重要转录因子;CD163、IRF4和VSIG4是M2巨噬细胞的标志物。因此,在TCGA数据集中分析了这些基因的表达水平。高危HCC组表现出Foxp3、CD163、IRF4和VSIG4的显着上调(如附图17中C-F所示)。
为了确认和验证这些发现,本发明进行了多重免疫荧光染色以显示部分HCC队列中的肿瘤微环境特征,结果如附图18所示,其中,A为在低m6Ascore组中,对DAPI、CD163和Foxp3进行多重免疫荧光染色结果;每个染色分别显示并合并(图中标出了比例尺)。B为在高m6Ascore组中,对DAPI、CD163和Foxp3进行多重免疫荧光染色结果;每个染色分别显示并合并(图中标出了比例尺)。从附图18中可以看出,在低风险HCC样本中,仅观察到少量M2-巨噬细胞(绿色)和Tregs(紫色)(如附图18中A所示)。一致地,在高风险HCC样本中,肿瘤微环境中有明显的M2巨噬细胞和Tregs浸润(如附图18中B所示)。
因此,这些结果表明,m6A特征可以表征肿瘤免疫微环境,高危HCC人群可能形成具有大量M2巨噬细胞和Treg浸润的免疫抑制微环境。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (6)
1.用于评估总生存期和表征肿瘤免疫微环境的基因特征,其特征在于:所述基因特征为m6A相关基因特征。
2.根据权利要求1所述的用于评估总生存期和表征肿瘤免疫微环境的基因特征,其特征在于:所述m6A相关基因特征包括20个蛋白质编码基因,IGFBP3、HDAC2、HMGN1、TCP1、PABPC4、RDH16、HDDC2、CFHR5、GYS1、MAPRE1、GYS2、BLMH、YBX1、NAP1L1、INTS8、MARCKSL1、STX6、MASP2、UQCRH和XPNPEP1。
3.如权利要求1或2所述的用于评估总生存期和表征肿瘤免疫微环境的基因特征的研究方法,其特征在于,包括以下步骤,
S1:获取HCC肿瘤样本和正常样本基因表达数据;
S2:构建m6AScore评分体系,并评估所有HCC肿瘤样本;
S3:确定HCC高危和低危人群,并进行GO和KEGG通路富集分析,以确定用于评估总生存期和表征肿瘤免疫微环境的基因特征;
S4:对HCC肿瘤样本的GSEA、肿瘤突变负荷、免疫浸润和治疗反应进行评估;
S5:通过质谱蛋白质组学和多重免疫荧光测定进行验证。
4.根据权利要求3所述的用于评估总生存期和表征肿瘤免疫微环境的基因特征的研究方法,其特征在于,步骤S2的具体操作包括以下步骤,
S201:通过一致性聚类算法分析HCC肿瘤样本中所有基因的表达,生成不同的聚类;
S202:通过维恩图获得重叠的差异表达基因DEG;
S203:使用Cox比例风险回归分析确定不同m6A修饰模式的蛋白质编码基因的预后价值,确定用于构建m6AScore评分体系的蛋白质编码基因;
S204:对步骤S203中确定的蛋白质编码基因进行主成分分析,建立m6AScore评分体系。
5.根据权利要求3所述的用于评估总生存期和表征肿瘤免疫微环境的基因特征的研究方法,其特征在于,步骤S5中质谱蛋白质组学的具体操作包括以下步骤,
S501a:从***固定石蜡包埋的肿瘤样本中分离蛋白质;
S502a:对分离出来的蛋白质进行消化;
S503a:通过质谱法进行肽测序;
S504a:数据分析以识别蛋白质。
6.根据权利要求3所述的用于评估总生存期和表征肿瘤免疫微环境的基因特征的研究方法,其特征在于,步骤S5中多重免疫荧光测定的具体操作包括以下步骤,
S501b:石蜡切片脱蜡去水后,将载玻片浸入柠檬酸抗原修复缓冲液和开水中进行抗原修复;
S502b:将样品与3%H2O2在室温下孵育,溶液在37℃下用5%FBS封闭30分钟,然后在4℃下与一抗孵育过夜;
S503b:将样品用磷酸盐缓冲盐水PBS冲洗,与Cy5/SpGreen标记的二抗在37°C下孵育,然后再次用PBS冲洗;
S504b:将溶液与Hoechst在室温下孵育15分钟,并通过微波处理去除先前的一抗/二抗,重复进行连续染色;
S505b:对样本进行多重免疫荧光测定。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310915518.7A CN117059166A (zh) | 2023-07-25 | 2023-07-25 | 用于评估总生存期和表征肿瘤免疫微环境的基因特征及其研究方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310915518.7A CN117059166A (zh) | 2023-07-25 | 2023-07-25 | 用于评估总生存期和表征肿瘤免疫微环境的基因特征及其研究方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117059166A true CN117059166A (zh) | 2023-11-14 |
Family
ID=88663603
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310915518.7A Pending CN117059166A (zh) | 2023-07-25 | 2023-07-25 | 用于评估总生存期和表征肿瘤免疫微环境的基因特征及其研究方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117059166A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117275744A (zh) * | 2023-11-22 | 2023-12-22 | 北京大学人民医院 | 一种综合基因突变特征与mIF图像特征的肺癌预后多模态预测模型构建方法 |
-
2023
- 2023-07-25 CN CN202310915518.7A patent/CN117059166A/zh active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117275744A (zh) * | 2023-11-22 | 2023-12-22 | 北京大学人民医院 | 一种综合基因突变特征与mIF图像特征的肺癌预后多模态预测模型构建方法 |
CN117275744B (zh) * | 2023-11-22 | 2024-02-13 | 北京大学人民医院 | 一种综合基因突变特征与mIF图像特征的肺癌预后多模态预测模型构建方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Wei et al. | Bioinformatics profiling utilized a nine immune‐related long noncoding RNA signature as a prognostic target for pancreatic cancer | |
Lambrechts et al. | Phenotype molding of stromal cells in the lung tumor microenvironment | |
US11079384B2 (en) | Biomarkers and methods for diagnosis of early stage pancreatic ductal adenocarcinoma | |
Ring et al. | Novel prognostic immunohistochemical biomarker panel for estrogen receptor–positive breast cancer | |
US20100099093A1 (en) | Biomarkers for the Identification Monitoring and Treatment of Head and Neck Cancer | |
Zhang et al. | Prognostic value of a stemness index-associated signature in primary lower-grade glioma | |
Hou et al. | Gene signature and identification of clinical trait-related m6 A regulators in pancreatic cancer | |
Wang et al. | Discovery of potential colorectal cancer serum biomarkers through quantitative proteomics on the colonic tissue interstitial fluids from the AOM–DSS mouse model | |
CN117059166A (zh) | 用于评估总生存期和表征肿瘤免疫微环境的基因特征及其研究方法 | |
Jiang et al. | Prognostic value of high FoxC2 expression in resectable non-small cell lung cancer, alone or in combination with E-cadherin expression | |
Wang et al. | Novel potential biomarkers associated with epithelial to mesenchymal transition and bladder cancer prognosis identified by integrated bioinformatic analysis | |
Lage-Vickers et al. | The expression of YWHAZ and NDRG1 predicts aggressive outcome in human prostate cancer | |
Xu et al. | Identification of a novel tumor microenvironment prognostic signature for bladder urothelial carcinoma | |
Feng et al. | A prognostic model based on nine DNA methylation-driven genes predicts overall survival for colorectal cancer | |
Zhang et al. | Hypoxia-related signature is a prognostic biomarker of pancreatic cancer | |
Xin et al. | A novel 9-gene signature for the prediction of postoperative recurrence in stage II/III colorectal cancer | |
Liu et al. | Dysregulation of tumor microenvironment promotes malignant progression and predicts risk of metastasis in bladder cancer | |
KR102087373B1 (ko) | 비소세포성 폐암 진단용 단백질 바이오마커 패널 및 이를 이용한 비소세포성 폐암 진단 방법 | |
Zheng et al. | Exploring potential regulatory anesthetic drugs based on RNA binding protein and constructing CESC prognosis model: A study based on TCGA database | |
Wang et al. | Heparanase is a prognostic biomarker independent of tumor purity and hypoxia based on bioinformatics and immunohistochemistry analysis of esophageal squamous cell carcinoma | |
Kim et al. | Proteomic profiling of bladder cancer for precision medicine in the clinical setting: A review for the busy urologist | |
Meng et al. | Identification and validation of a novel prognostic gene model for colorectal cancer | |
Zhang et al. | Integrated analysis of single-cell and bulk transcriptomics develops a robust neuroendocrine cell-intrinsic signature to predict prostate cancer progression | |
Charmpi et al. | Proteogenomic heterogeneity of localized human prostate cancer progression | |
Song et al. | Key genes involved with prognosis were identified in lung adenocarcinoma by integrated bioinformatics analysis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |