A kind of lncRNA related to liver cancer diagnosis and treatment
Technical field
The invention belongs to biomedicine field, it is related to a kind of lncRNA related to liver cancer diagnosis and treatment, is specifically related to
lncRNA ENSG00000271781。
Background technology
Primary carcinoma of liver (primary liver cancer, PLC) is one of malignant tumour common in the world, 90% with
Upper is hepatocellular carcinoma (hepatocelluar carcinoma, HCC), and in male, its incidence of disease accounts for Cancer Mortality
Second, in women, its incidence of disease accounts for the 6th of Cancer Mortality;According to recent statistics, the annual neopathy in the whole world
Example reaches 780,000, and because the patient numbers of PLC mortality reach 740,000, case fatality rate reaches 95%.China's onset of liver cancer number accounts for the world
The 50% of number of the infected, accounts for the 2nd in mortality of malignant tumors cis-position, is only second to be only second to stomach in lung cancer, rural area in city
Cancer, it turns into the big killer for threatening our people's health and lives.
Fast breeding is one of important Biological characteristics of liver cancer with transfer, and fast breeding and the transfer of liver cancer are to cause to be permitted
Many liver cancer patient prognosis are poor, one of low key factor of 5 years survival rates.Therefore, research causes liver cancer fast breeding and transfer
The reason for, disclose the molecular mechanism of onset of liver cancer has extremely important effect to improving liver cancer patient prognosis.
Long-chain non-coding RNA (long non-coding RNA, 1ncRNA) is the class being widely present in eucaryote
Itself not encoding proteins, transcript length exceed 200nt RNA molecule, can in a variety of aspects (epigenetic regulation, turn
Record regulation and control and post-transcriptional control etc.) controlling gene expression.Five kinds are roughly divided into by its position relationship between mRNA
Type:(1) just long-chain non-coding RNA;(2) antisense long-chain non-coding RNA;(3) two-way long-chain non-coding RNA;(4) introne
Type long-chain non-coding RNA;(5) long-chain non-coding RNA between gene (i.e. large intergenic noncoding RNA,
lincRNA).In whole gene group transcription product, the ratio of ratio shared by lncRNA considerably beyond mRNA.At present in tumour
The lncRNA of the unconventionality expression found in tissue can relate to whole body each system, be distributed it is relatively broad, for lncRNA identification
Us is understood whole functioning gene control methods again, by study lncRNA biological function and its
Regulatory mechanism in disease, can be more completely understood the genesis mechanism of disease, find new disease diagnosis marker and
Therapy target.
The content of the invention
In order to make up the deficiencies in the prior art, an object of the present invention is there is provided a kind of biomarker, for liver cancer
Early diagnosis or targeted therapy.
The second object of the present invention is there is provided a kind of method for screening treatment liver cancer potential drug, by adjusting biological marker
The expression of thing come judge candidate whether be potential treatment liver cancer medicine.
To achieve these goals, the present invention is adopted the following technical scheme that:
The invention provides the reagent of detection ENSG00000271781 expressions in the product of diagnosing liver cancer is prepared
Using, wherein, ENSG00000271781 expressions in liver cancer patient are raised.
Further, the reagent is selected from:
Specific recognition ENSG00000271781 probe;Or
Specific amplification ENSG00000271781 primer.
The invention provides a kind of product of diagnosing liver cancer, the product includes ENSG00000271781 in detection sample
The reagent of expression.As long as product of the present invention can detect ENSG00000271781 expression, without office
It is limited to common detection product such as chip, nucleic acid film bar, preparation or kit etc.." sample " include cell, tissue, dirty
Device, body fluid (blood, lymph etc.), digestive juice, expectoration, alveole bronchus cleaning fluid, urine, excrement etc..It is preferred that, the sample
For tissue, blood.
Further, the reagent includes specific recognition ENSG00000271781 probe, or specific amplification
ENSG00000271781 primer.
In the embodiment of the present invention, the reagent includes specific amplification ENSG00000271781 primer,
The primer sequence of the specific amplification ENSG00000271781 is as shown in SEQ ID NO.2~3.
The invention provides application of the ENSG00000271781 genes in screening prevention or the treatment potential material of liver cancer.
The invention provides a kind of method for the potential material for screening prevention or treatment liver cancer, methods described includes:
The system expressed or containing ENSG00000271781 genes is handled with candidate substances;With
Detect the expression of ENSG00000271781 genes in the system;
Wherein, if the candidate substances can reduce ENSG00000271781 genes expression (preferably significantly reduce,
It is such as low by more than 20%, it is preferably low by more than 50%;More preferably low more than 80%), then it is prevention or treatment to show the candidate substances
The potential material of liver cancer.The system is selected from:Cell system, subcellular fraction system, solution system, organizational framework, organ systems or
Animal system.
The candidate substances include but is not limited to:For ENSG00000271781 genes or its upstream or downstream gene
Disturbing molecule, nucleic acid inhibitor, micromolecular compound of design etc..
The invention provides applications of the ENSG00000271781 in the pharmaceutical composition for preparing treatment liver cancer.
Further, described pharmaceutical composition includes the inhibitor of ENSG00000271781 functional expressions, the inhibitor
It is selected from:Using ENSG00000271781 or its transcript as target sequence and can suppress ENSG00000271781 gene expressions or
The disturbing molecule of genetic transcription, including:ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense
Nucleic acid, or can express or be formed the construction of the shRNA, siRNA, dsRNA, Microrna, antisensenucleic acids.It is preferred that,
Described inhibitor is siRNA.
Further, described pharmaceutical composition also includes and other medicine classes of the inhibitor compatibility and pharmaceutically acceptable
Carrier and/or auxiliary material.
The invention provides a kind of pharmaceutical composition for treating liver cancer, described pharmaceutical composition includes:
The inhibitor of ENSG00000271781 functional expressions;With
Pharmaceutically acceptable carrier.
Pharmaceutically acceptable carrier includes but is not limited to buffer, emulsifying agent, suspending agent, stabilizer, preservative, life
Manage salt, excipient, filler, coagulating agent and blender, interfacial agent, diffusant, defoamer.
Brief description of the drawings
Fig. 1 is to detect expression figures of the ENSG00000271781 in liver cancer patient using QPCR;
Fig. 2 is to utilize differential expression figures of the TCGA database cross validations ENSG00000271781 in liver cancer patient;
Fig. 3 is ROC curve figures of the ENSG00000271781 in liver cancer patient;
Fig. 4 is to detect expression figures of the ENSG00000271781 in liver cancer cells using QPCR;
Fig. 5 is expression influence figures of the detection transfection siRNA on ENSG00000271781 in liver cancer cells;
Fig. 6 is the influence figure that ENSG00000271781 cell proliferations are detected using CCK8;
Fig. 7 is the influence figure for detecting ENSG00000271781 to the Clone formation colony of cell;
Fig. 8 is the influence figure for detecting ENSG00000271781 to hepatoma cell apoptosis;
Fig. 9 is the influence of the detection ENSG00000271781 Hepatocarcinoma Cells migration of Transwell cells and invasion and attack
Figure;Wherein, figure A is the influence figure of ENSG00000271781 Hepatocarcinoma Cells migration, and figure B is ENSG00000271781 bases
Because of the influence figure to fucosylation.
Specific embodiment
The present invention is most wide using current covering database by high throughput method by in-depth study extensively
LncRNA expression, finds wherein there is obvious differential expression in lncRNA chips, detection liver cancer tissue and cancer beside organism
LncRNA fragments, its relation between the generation of liver cancer is inquired into, so that early detection for liver cancer and targeted therapy are found
More preferable approaches and methods.By screening, present invention firstly discovers that ENSG00000271781 conspicuousnesses are raised in liver cancer.It is real
Checking is bright, siRNA interference silence ENSG00000271781, can effectively suppress the propagation of liver cancer cells, be the individual character of liver cancer
Change treatment and provide new way.
ENSG00000271781 genes
ENSG00000271781 genes are located on No. 5 chromosomes, a kind of representational people ENSG00000271781 genes
Nucleotide sequence as shown in SEQ ID NO.1.ENSG00000271781 in the present invention include wild type, saltant type or its
Fragment.
It would be recognized by those skilled in the art that the practicality of the present invention is not limited to appointing to the target gene of the present invention
The gene expression of what specific variants is quantified.If when nucleic acid or its fragment and other nucleic acid (or its complementary strand) optimal comparison
When (have appropriate nucleotides inserted or missing), nucleotide base at least about 60%, usually at least about 70%, more
Usually at least about 80%, it is preferably at least about 90% and more preferably at least about there is nucleosides in 95-98% nucleotide bases
The acid sequence phase same sex, then the two sequences are " substantially homologous " (or substantially similar).
Or, when nucleic acid or its fragment with another nucleic acid (or its complementary strand), a chain or its complementary series in selectivity
When hybridizing under hybridization conditions, then there is substantially homologous or (the phase same sex) therebetween.When hybridization has more than specific general loss
When selective, there is cross selection.Typically, when one section of sequence at least about 14 nucleotides is present at least about
When the 55% phase same sex, preferably at least about 65%, more preferably at least about 75% and the most preferably at least about 90% phase same sex, hair
Raw selective cross.It is as described herein, cognate pair than length can be longer sequence section, lead in certain embodiments
Often it is at least about 20 nucleotides, more frequently at least about 24 nucleotides, typically at least about 28 nucleotides, more
Typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.
Therefore, polynucleotides of the invention and SEQ ID NO.1 preferably have at least 75%, more preferably at least 85%, more
Preferably at least 90% homology.It is highly preferred that in the presence of at least 95%, more preferably at least 98% homology.
The present invention can determine gene expression using any method known in the art.Those skilled in the art should manage
Solution, the means for determining gene expression are not the importances of the present invention.The table of biomarker can be detected on transcriptional level
Up to level.
Detection technique
The present invention lncRNA detected using multiple nucleic acids technology known to persons of ordinary skill in the art, these skills
Art includes but is not limited to:Nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification technologies.
The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but is not limited to chain terminator (Sanger) sequencing and contaminated
Expect terminator sequencing.One of ordinary skill in the art it will be recognized that due to RNA in cell less stable and in an experiment
Be more vulnerable to nuclease attack, thus before sequencing generally by RNA reverse transcriptions into DNA.
The present invention can simultaneously be expanded before detection or with detection to nucleic acid (for example, ncRNA).Nucleic acid amplification technologies
Exemplary, non-limitative example include but is not limited to:PCR (PCR), reverse transcriptase polymerase chain reaction (RT-
PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleotide sequence
Expand (NASBA).One of ordinary skill in the art will be it will be recognized that some amplification techniques (for example, PCR) needs will before amplification
RNA reverse transcriptions are into DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).
Commonly referred to as PCR PCR uses annealing and the primer extend of denaturation, primer pair and opposite strand
Multiple circulations, exponentially increase target nucleic acid sequence copy number;The amplification of TMA transcriptive intermediate is (in substantial constant
Temperature, ionic strength and pH under conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, wherein target sequence is more
Individual RNA copies autocatalytically generate other copy;LCR ligase chain reaction uses miscellaneous with the adjacent area of target nucleic acid
The two groups of complementary DNA oligonucleotides handed over;Other amplification methods are included for example:The commonly referred to as NASBA expansion based on nucleotide sequence
Increase;Use rna replicon enzyme (the commonly referred to as Q β replicase) amplification of amplification probe molecule in itself;Amplification method based on transcription;
And the sequence amplification of self―sustaining.
The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.
Chip, nucleic acid film bar, kit
Chip includes in the present invention:Solid phase carrier;And the oligonucleotides being fixed in order on the solid phase carrier is visited
Pin, described oligonucleotide probe specifically corresponds to the part or all of sequence shown in ENSG00000271781.
The solid phase carrier includes inorganic carrier and organic carrier, the inorganic carrier include but is not limited to have silicon carrier,
Glass carrier, ceramic monolith etc.;The organic carrier includes polypropylene film, nylon membrane etc..
" probe " refers to the molecule that can be combined with the particular sequence or subsequence or other parts of another molecule.Unless otherwise finger
Go out, term " probe " is often referred to combine with another polynucleotides (often referred to as " target polynucleotide ") by complementary base pairing
Polynucleotide probes.Lack the complementary target of sufficient sequence according to the preciseness of hybridization conditions, probe energy and with the probe many
Nucleotides is combined.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, includes, but are not limited to:It is molten
Liquid phase, solid phase, mixed phase or in situ hybridization determination method.
Exemplary probe in the present invention includes PCR primer and gene specific DNA oligonucleotide probe, for example, fix
In the micro probe array on microarray substrate, quantitative nucleic acid enzyme protection examine probe, the probe being connected with molecular barcode and
The probe being fixed on pearl.
These probes have the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ",
As long as hybridization, can not be complete complementary.These polynucleotides, which are commonly angled relative to the specific base sequence, to be had
More than 80%, preferably more than 90%, more preferably more than 95%, particularly preferred 100% homology.These probes can be DNA,
Can also be RNA, furthermore it is possible to pass through PNA (Polyamide nucleic in one part or whole nucleotides
Acid, peptide nucleic acid), LNA (registration mark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core
Acid), ENA (registration mark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol
Nucleic acid, glycerine nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains
Polynucleotides.
In the present invention, nucleic acid film bar includes substrate and the oligonucleotide probe being fixed in the substrate;The substrate
Can be any substrate suitable for immobilized oligonucleotide probe, for example nylon membrane, nitrocellulose filter, polypropylene screen, sheet glass,
Silica gel chip, micro magnetic bead etc..
The invention provides a kind of kit, the kit can be used for detection ENSG00000271781 expression.
It is used to detect that the reagent of ENSG00000271781 expression includes specific amplification in a particular embodiment of the present invention
ENSG00000271781 primer, the primer sequence sequence is as shown in SEQ ID NO.2~3.Also contain in described kit
There are the label for labeled RNA sample, and the substrate corresponding with the label.In addition, in described kit also
It may include, for extracting the various reagents needed for RNA, PCR, hybridization, colour developing etc., to include but is not limited to:Extract, amplification liquid, miscellaneous
Hand over liquid, enzyme, comparison liquid, nitrite ion, washing lotion etc..In addition, also including the use of specification and/or chip image in described kit
Analysis software.
Gene detecting kit or genetic chip can be used for detection including ENSG00000271781 genes in the present invention
Multiple genes (for example, multiple genes related to liver cancer) expression, by multiple marks of liver cancer simultaneously examined
Survey, be greatly improved the accuracy rate of diagnosing cancer of liver.
Inhibitor and pharmaceutical composition
Discovery based on inventor, the invention provides a kind of ENSG00000271781 inhibitor, the inhibitor
Property has no importance for the present invention, as long as it suppresses the functional expression of ENSG00000271781 genes, these suppressions
Preparation is as lowering the useful materials of ENSG00000271781, available for preventing or treat liver cancer.
As a kind of preferred embodiment of the present invention, the inhibitor of the ENSG00000271781 is a kind of
The specific siRNA molecules of ENSG00000271781.As used herein, described " siRNA " refers to a kind of short-movie
Section double stranded rna molecule, can degrade specific mRNA by target of the mRNA of homologous complementary sequence, and this process is exactly RNA
(RNA interference) process of interference.SiRNA can be prepared into the form of double-strandednucleic acid, and it contains a positive-sense strand
With an antisense strand, this two chains only form double-strand under conditions of hybridization.One double-stranded RNA compound can be by being separated from each other
Positive-sense strand and antisense strand prepare.Therefore, for example, complementary positive-sense strand and antisense strand are chemical syntheses, thereafter may be used
By anneal, the double-stranded RNA compound of synthesis is produced.
When screening effective siRNA sequence, the present inventor is analyzed by substantial amounts of compare, so as to find out optimal effective
Fragment.The present inventor's design has synthesized a variety of siRNA sequences, and they are entered by transfection reagent transfecting hepatoma cells system respectively
Row checking, selects the optimal siRNA of interference effect, further tests, is as a result proved for the siRNA in energy in cellular level
The effective expression for suppressing ENSG00000271781 genes in cell, and liver cancer cells propagation.
The nucleic acid inhibitor such as siRNA of the present invention can be with chemical synthesis, can also be by a recombinant nucleic acid structure
Expression cassette is prepared after being transcribed into single stranded RNA.The nucleic acid inhibitors such as siRNA, can be by using appropriate transfection reagent quilt
It is transported to intracellular, or can be also transported to using multiple technologies known in the art intracellular.
Present invention also offers a kind of pharmaceutical composition, it contains the described ENSG00000271781 of effective dose suppression
Preparation, and pharmaceutically acceptable carrier.Described composition can be used for suppressing liver cancer.It is any foregoing
ENSG00000271781 inhibitor is used equally for the preparation of pharmaceutical composition.
In the present invention, pharmaceutically acceptable carrier includes but is not limited to buffer, emulsifying agent, suspending agent, stably
Agent, preservative, physiological saline, excipient, filler, coagulating agent and blender, interfacial agent, diffusant, defoamer.
As used herein, described " effective dose " refer to that people and/or animal can be produced function or activity and can by people and/
Or the amount that animal is received." pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various figurations
Agent and diluent.The term refers to some such medicament carriers:Themselves it is not necessary active component, and does not have after administration
Undue toxicity.Suitable carrier is well known to those of ordinary skill in the art.It is pharmaceutically acceptable in the composition to carry
Body can contain liquid, such as water, salt solution, buffer solution.In addition, complementary material is there is likely to be in these carriers, such as filler,
Lubricant, glidant, wetting agent or emulsifying agent, pH buffer substance etc..Cell can also be contained in described carrier, and (host is thin
Born of the same parents) transfection reagent.
The present invention can use with a variety of methods well known in the art by described inhibitor or its encoding gene or its
Pharmaceutical composition delivers medicine to mammal.Including but not limited to:Hypodermic injection, intramuscular injection, for percutaneous administration of, administer locally to, plant
Enter, be sustained and give;It is preferred that, the administering mode is that non-bowel is given.
It is preferred that, it can be carried out using the means of gene therapy.Such as, can be directly by ENSG00000271781 inhibitor
Subject is delivered medicine to by the method such as injecting;Or, ENSG00000271781 suppression can will be carried by certain approach
The ceneme (such as expression vector or virus etc., or siRNA or shRNA) of preparation is delivered on target spot, and is allowed to expression work
The ENSG00000271781 inhibitor of property, concrete condition need to be depending on the type of described inhibitor, and these are this area skills
Known to art personnel.
The pharmaceutical composition of the present invention can be further comprising one or more anticancers.In specific embodiments,
Compound of the pharmaceutical composition comprising at least one suppression ENSG00000271781 gene expressions and at least one chemotherapeutics.With
In the chemotherapeutics of the present invention, include but is not limited to:Micro-pipe activator, alkylating agent, anti-superfluous raw antimetabolite, platinum-like compounds,
DNA- alkylating agents, antitumor antibiotics agent, antimetabolite, microtubule stabilizing agent, tubulin destabilizing agent, hormone antagonism
Agent, topoisomerase enzyme inhibitor, kinases inhibitor, HMG-COA inhibitor, CDK inhibitor, cyclin suppresses
Agent, caspase inhibitors, metal protease inhibitors, antisensenucleic acids, triple helix DNA, aptamer, and molecular modification
Virus, bacterium and exotoxin reagent.
Pharmaceutical acceptable carrier may include but be not limited to:Virus, micro-capsule, liposome, nano particle or polymer and its any group
Close.Related delivering supporting agent may include but be not limited to:Liposome, biocompatible polymer (including natural polymer and synthesis
Polymer), lipoprotein, polypeptide, polysaccharide, lipopolysaccharides, artificial viral envelope, inorganic (including metal) particle and bacterium or disease
Malicious (such as baculoviral, adenovirus and retrovirus), bacteriophage, sticking grain or plasmid vector.
The pharmaceutical composition of the present invention can also be with other treatment liver cancer drug combination, other therapeutic compound can be with
Main active component is administered simultaneously, or even is administered simultaneously in same composition.
The pharmaceutical composition of the present invention can also be with single composition or the dosage shape different from main active component
Formula individually gives other therapeutic compounds.The Fractional of main component can be administered simultaneously with other therapeutic compounds,
And other dosage can be administered alone.Over the course for the treatment of, can be according to the order of severity of symptom, the frequency of recurrence and treatment side
The physiologic response of case, adjusts the dosage of pharmaceutical composition of the present invention.
Statistical analysis
In a particular embodiment of the present invention, experiment all completed according to being at least repeated 3 times, result data be all with
The mode of mean+SD is represented, carries out statistical analysis using SPSS18.0 statistical softwares, difference between the two
Different use t is examined, it is believed that work as P<There is statistical significance when 0.05.
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning:Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) described in condition, or according to the condition proposed by manufacturer.
Embodiment 1 screens the gene marker related to liver cancer
1st, sample collection
The cancerous tissue of each 10 liver cancer patients of collection and cancer beside organism, the equal informed consent of patient, above-mentioned all samples
Obtain the agreement by the committee of organizational ethics.
2nd, the preparation of RNA sample
Tissue RNA extraction is carried out using QIAGEN tissue RNA extracts kits, the specific steps of by specification are carried out
Operation.
3rd, reverse transcription and mark
With Low RNA Input Linear Amplification Kit by mRNA reverse transcriptions into cDNA, while using Cy3
Difference labelling experiment group and control group.
4th, hybridize
Genetic chip uses Kang Cheng biology-Human lncRNA Array, carries out the step of by chip operation instructions miscellaneous
Hand over.
5th, data processing
Chip Agilent scanner scannings after hybridization, resolution ratio is 5 μm, and scanner is automatically with 100% and 10%PMT
Each to scan 1 time, 2 times result Agilent softwares merge automatically.Scan image data is using at FeatureExtraction
Reason analysis, obtained initial data application Bioconductor program bags carry out follow-up data processing.Differential gene screening criteria:
FDR<0.01, abs (log2FC)>1.5。
6th, result
Compared with cancer beside organism, expressions of the ENSG00000271781 in liver cancer tissue is significantly higher than cancer beside organism.
The differential expression of the QPCR sequence verification ENSG00000271781 genes of embodiment 2
1st, large sample QPCR checkings are carried out to ENSG00000271781 gene differential expressions.According to the sample in embodiment 1
Collection mode collects liver cancer tissue and each 60 of cancer beside organism's sample.
2nd, RNA extraction steps be the same as Example 1.
3rd, reverse transcription:
Using 25 μ l reaction systems, each sample takes 1 μ g total serum IgEs as template ribonucleic acid, is separately added into PCR pipe following
Component:DEPC water, 5 × RT Buffer, 10mM dNTP, 0.1mM DTT, 30 μM of Oligo dT, 200U/ μ l M-MLV,
Template ribonucleic acid.42 DEG C are incubated 1h, 72 DEG C of 10min, of short duration centrifugation.
(3) QPCR amplifications are examined
Design of primers:
The primer sequence of ENSG00000271781 genes is:
Forward primer:5’-GATGAAAGAGGTGATGAG-3’(SEQ ID NO.2)
Reverse primer:5’-CTTTATTTCGGTGGTTTG-3’(SEQ ID NO.3)
House-keeping gene GAPDH primer sequence is:
Forward primer:5’-CCGGGAAACTGTGGCGTGATGG-3’(SEQ ID NO.4)
Reverse primer:5’-AGGTGGAGGAGTGGGTGTCGCTGTT-3’(SEQ ID NO.5)
Prepare 25 μ l reaction systems:The μ l of SYBR Green PCRs system 12.5, forward and reverse primer (5 μM)
Each 1 μ l, template cDNA 2.0 μ l, no μ l of enzyme water 8.5.Operations are carried out on ice.Each sample sets 3 parallel pipes,
All amplified reactions in triplicate more than to ensure the reliability of result.
Amplification program is:95 DEG C of 60s, (95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 45s) × 35 circulations.
It is anti-in the enterprising performing PCR of Light Cycler fluorescence real-time quantitative PCR instrument using SYBR Green as fluorescent marker
Should, purpose band is determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out relative quantification.
3rd, result
As a result as shown in figure 1, compared with cancer beside organism, ENSG00000271781 genes are in Expression In Hepatocellular Carcinoma level
Up-regulation, difference has statistical significance (P<0.05) it is, consistent with RNA-sep results.
Embodiment 3 analyzes expressions of the ENSG00000271781 in TCGA databases
1st, Data Collection
The lncRNA expression modal datas of 200 liver cancer tissues and 50 cancer beside organisms, analysis are collected from TCGA databases
Expressions of the ENSG00000271781 in liver cancer tissue and cancer beside organism;Draw box-shaped figure.
2nd, ROC curve is analyzed
ENSG00000271781 Receiver Operating Characteristics are analyzed using the pROC bags in R language, binomial is calculated and accurately puts
Believe space, draw ROC curve.
3rd, result
ENSG00000271781 expression is as shown in Fig. 2 compared to control group, ENSG00000271781 is in liver cancer group
Knit middle expression significantly up-regulation.
ENSG00000271781 ROC curve as shown in figure 3, ENSG00000271781 AUC is up to 0.8897, and
With higher specificity and sensitiveness, illustrate that ENSG00000271781 is applied to the diagnosis of liver cancer with higher accuracy.
Differential expression of the ENSG00000271781 genes of embodiment 4 in liver cancer cell lines
1st, cell culture
HepG2 cell lines, Huh7 and normal liver cell system HL-7702, with containing 10% hyclone and 1%P/S
Culture medium DMEM in 37 DEG C, 5%CO2, relative humidity for 90% incubator in cultivate.Change within 2-3 days liquid 1 time, use
The 0.25% trypsase conventional digestion passage containing EDTA.
2nd, RNA extraction
1) pancreatin digestion attached cell, blows and beats the cell obtained after centrifugation, resuspension, cleaning, with the DMEM containing 10%FBS
Culture medium is resuspended;
2) cell of resuspension is transferred to 6 orifice plates, addition culture medium is to 2m1/ holes, and the orifice plate of jog 6 makes cell uniformly be resuspended;
3) cell attachment growth 48h, removes culture medium;
4) with 1mlTrizol reagent cell lysis, 6 orifice plate walls are blown and beaten repeatedly, cell is cracked completely as far as possible;
5) in the EP pipes that transfer cell pyrolysis liquid is treated to 1.5ml DEPC, it is placed on ice.0.2m1 chloroforms are added, are remained
Remaining operating procedure is with RNA extraction process in blood.
3rd, reverse transcription
Specific steps be the same as Example 2.
4th, result
As a result as shown in figure 4, compared with normal liver cell system, ENSG00000271781 genes hepatocellular carcinoma H22,
Express and raise in Huh7, difference has statistical significance (P<0.05) it is, consistent with RNA-sep results.
The silence of the ENSG00000271781 genes of embodiment 5
1st, cell culture
HepG2 cell lines, with the culture medium DMEM containing 10% hyclone and 1%P/S in 37 DEG C, 5%CO2、
Relative humidity is culture in 90% incubator.Change within 2-3 days liquid 1 time, use the 0.25% trypsase conventional digestion containing EDTA
Passage.
2nd, siRNA is designed
For the siRNA sequence of ENSG00000271781 genes:
Negative control siRNA sequence (siRNA-NC):
Positive-sense strand:5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.6),
Antisense strand:5’-ACGUGACACGUUCGGAGAA-3’(SEQ ID NO.7);
siRNA1:
Positive-sense strand:5 '-AGAAAAGCCAUGAAACUGCUC-3 ' (SEQ ID NO.8),
Antisense strand:5’-GCAGUUUCAUGGCUUUUCUGG-3’(SEQ ID NO.9);
siRNA2:
Positive-sense strand:5 '-AAGUUAUGCCAGAAAAGCCAU-3 ' (SEQ ID NO.10),
Antisense strand:5’-GGCUUUUCUGGCAUAACUUUC-3’(SEQ ID NO.11);
siRNA3:
Positive-sense strand is 5 '-UAUUUCAUUCCUACAUUGCCG-3 ' (SEQ ID NO.12),
Antisense strand is 5 '-GCAAUGUAGGAAUGAAAUACA-3 ' (SEQ ID NO.13)
Cell is pressed 2 × 105/ hole is inoculated into six porocyte culture plates, in 37 DEG C, 5%CO2Cell culture in incubator
24h;
In without DMEM culture mediums dual anti-, containing 10%FBS, transfection (is purchased from according to lipofectamine 2000
Invitrogen companies) specification transfection.
Experiment be divided into blank control group (HepG2), negative control group (siRNA-NC) and experimental group (20nM) (siRNA1,
SiRNA2, siRNA3), the sequence of wherein negative control group siRNA and ENSG00000271781 genes is without homology, and concentration is
20nM/ holes, while being transfected respectively.
3rd, QPCR detects the expression of ENSG00000271781 genes
The extraction of 3.1 cell total rnas
Specific steps be the same as Example 4.
3.2 reverse transcription step be the same as Examples 2.
3.3 QPCR amplification steps be the same as Example 2.
4th, result
As a result such as Fig. 5 is shown, compared to HepG2, transfection zero load siRNA-NC, siRNA2, siRNA3 group, siRNA1 groups can
ENSG00000271781 expression is significantly reduced, difference has statistical significance (P<0.05).
The CCK8 of embodiment 6 detects cell proliferation experiment
1st, cell culture and transfection procedure be the same as Example 4
2nd, CCK8 detects cell propagation
1) the HepG2 cells of logarithmic proliferation phase are inoculated in 96 orifice plates, per hole 2 × 103Individual cell;
2) three groups of experiment point, is blank control group, transfection siRNA-NC groups and transfection siRNA1 respectively, every group sets 6 again
Hole;
3) 10 μ l/ holes CCK8 reagents are added after transfection 0h, 24h, 48h, 72h respectively;
4) A450 light absorption value is detected after 2h using ELIASA.
3rd, result
Result shown in Fig. 6 is shown:Blank control group transfects the cell life of siRNA1 groups with unloaded group no significant difference
The vitro growth rates of the obvious low control group of long speed, difference has statistical significance (P<0.05), the above results show
ENSG00000271781 expression can promote the growth of liver cancer cells.
The formation experiment of the soft-agar cloning of embodiment 7
1st, the cell of exponential phase is in 0.25% Trypsin Induced, gently piping and druming makes unicellular outstanding
Liquid, is collected by centrifugation cell precipitation.
2nd, it is resuspended, is counted after appropriate dilution with the DMEM complete mediums containing 20% hyclone, adjustment cell concentration is 5
×103Individual/ml.
3rd, prepare after the low melting point agar liquid glucose that two concentration are respectively 1.2% and 0.7%, autoclaving, maintain 40
In DEG C water-bath.
4th, 1.2% agarose and 2 × DMEM culture mediums 1:1 mixing, adds 2 × antibiotic and 20% calf serum,
Take 3ml mixed liquors to inject and 5min cooled and solidifieds are placed in diameter 6cm plates, CO is placed in as bottom-layer agar2It is standby in incubator.
5th, 1 in sterile test tube:The agarose and 2 × DMEM culture mediums of 1 mixing 0.7%, then addition 0.2ml is dense into pipe
Spend for 5 × 103Individual/ml stable infection cell suspension, fully mixes, injects in above-mentioned plate, gradually form double agar layers, often
Individual experimental group repeats 4 samples.
6th, after after top-layer agar solidification, 37 DEG C of 5%CO are inserted2Cultivated in incubator, every 3 days plus culture medium 1.5ml.7th, cultivate
Culture dish is taken out after 14 days, 90min is dyed for 0.005% gentian violet with 1ml concentration.Plate is placed under inverted microscope
Observation, every group of cell randomly selects the number of cell clones of technology formation under 10 low-power fields, mirror.
8th, result
As a result as shown in fig. 7, compared with control group, transfecting siRNA2-ENSG00000271781 unicellular gram of groups of cells
Grand Colony forming number is significantly reduced.
The influence of the ENSG00000271781 Hepatocarcinoma Cells apoptosis of embodiment 8
Use the influence of flow cytomery ENSG00000271781 gene pairs Apoptosis.
1st, cell culture step be the same as Example 4.
2nd, cell transfecting step be the same as Example 5.
3rd, step
1) by 10 × sample-loading buffers of 3m1 27ml distilled water dilutings.
2) collection of cellular samples and with the PBS of precooling.
3) cell is added into 1 × sample-loading buffers of lml, 300g centrifugation 10min suction out buffer solution.
4) 1 × sample-loading buffers of lml are added again, and cell concentration in cell suspension is adjusted to 1 × 106Individual/ml.
5) cell suspension is taken out into 100 μ 1, added in EP pipes.
6) 5 μ l Annexin V FITC are added in EP pipes, mixes the liquid in EP pipes, lucifuge is incubated at room temperature
10min。
7) 5 μ 1PI dye liquors are added into EP pipes, at room temperature lucifuge 5min.
8) 500 μ l PBS solution is added in EP pipes, is gently mixed, flow cytometer is detected in 1h.
4th, result:
As a result as shown in figure 8, experimental group is compared with control group, apoptosis rate rise (P<0.05), the result illustrates,
The apoptosis of ENSG00000271781 expression inhibiting liver cancer cells.
The cell migration of embodiment 9 and Matrigel
1st, prepared by Transwell cells
Matrigel is ice bath melted under aseptic condition, and 20 times of dilutions are carried out with PBS, are layered on the volume in 50 μ l/ holes
On the polycarbonate membrane of Transwell cells.37 DEG C of placement 4h, take out after Matrigel glue aggregates into gel, gently suction out
Upper strata separates out liquid.The serum-free medium containing BSA that 50 μ l are added in per hole carries out hydration process, 37 DEG C of placements to basilar memebrane
30min。
2nd, cell suspension is configured
Cell removes serum starvation processing 12-24h, carries out digestion process to cell, is centrifuged after terminating digestion, in removal
Layer nutrient solution.Sedimentation cell is cleaned with PBS, the serum free medium containing BSA is added and it is resuspended.Adjustment is thin
The density of born of the same parents is to 5 × l05Individual/ml.
3rd, cell is inoculated with
The μ 1 (migration experiment is 100 μ 1, and Matrigel is 200 μ 1) of cell suspension 200 is taken to be added to Transwell cells
In.Room adds 1640 culture mediums of 500 μ 1 containing FBS under 24 orifice plates.Cell is put into cell culture incubator and cultivates 24h.
4th, dye
Cell is dyed after culture terminates using DAPI.Small ventricular cell is first rinsed 2 times with PBS, DAPI working solutions are put into
Middle room temperature dyes 5-20min.Rinsed 2 times with PBS, be put into fluorescence microscopy Microscopic observation and count.
5th, result
As a result as shown in figure 9, after liver cancer cells transfection RNA interfering, compared with control group, the migration of experimental group and invading
Attack ability to be decreased obviously, as a result illustrate that ENSG00000271781 can promote the migration and invasion and attack of liver cancer cells.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improve can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>A kind of lncRNA related to liver cancer diagnosis and treatment
<160> 13
<170> PatentIn version 3.5
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ccctggttgc ctctgccgtc ccctagagct gggggagcct gcaaggcagt cagctggaga 300
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