CN113234824A - Application of RP11-379k17.4 overexpression in preparation of endometrial cancer treatment drug - Google Patents

Application of RP11-379k17.4 overexpression in preparation of endometrial cancer treatment drug Download PDF

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CN113234824A
CN113234824A CN202110495750.0A CN202110495750A CN113234824A CN 113234824 A CN113234824 A CN 113234824A CN 202110495750 A CN202110495750 A CN 202110495750A CN 113234824 A CN113234824 A CN 113234824A
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overexpression
endometrial cancer
preparation
cells
hec
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辛卫娟
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Obstetrics and Gynecology Hospital of Fudan University
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Obstetrics and Gynecology Hospital of Fudan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

The invention discloses application of RP11-379k17.4 overexpression in preparation of a drug for treating endometrial cancer. The RP11-379k17.4 overexpression can reduce the activity of HEC-1B and Ishikawa cells. Meanwhile, overexpression of RP11-379k17.4 induced apoptosis in HEC-1B and Ishikawa cells. The RP11-379k17.4 overexpression can reduce the activity of HEC-1B and Ishikawa cells and promote the apoptosis of the HEC-1B and Ishikawa cells, and the imbalance of RP11-379k17.4 can be involved in the occurrence and development of endometrial cancer to play a role in inhibiting cancer, so that the application has great clinical significance.

Description

Application of RP11-379k17.4 overexpression in preparation of endometrial cancer treatment drug
Technical Field
The invention relates to the technical field of biology, in particular to application of RP11-379k17.4 overexpression in preparation of a drug for treating endometrial cancer.
Background
Endometrial Cancer (EC) is a group of epithelial malignant tumors occurring in endometrium, takes adenocarcinoma derived from endometrial gland as one of three major malignant tumors of female genital tract, accounts for 7 percent of female systemic malignant tumors, has different incidence rates in different regions worldwide, is the most common gynecological malignant tumor in western industrialized countries, has the highest incidence rate in North America and northern Europe regions, and has lower incidence rates in Asia Japan, India, China, south America and other regions. China lacks detailed epidemiological investigation data of endometrial cancer, and the disease is estimated to be similar to the Japanese disease. The developed countries in North America and Europe are higher than those in developing countries, the former is 10 times of the latter, the incidence rate of the former is behind breast cancer, colorectal cancer and lung cancer, the former is the fourth most common malignant tumor of the whole body of a female, and the former is the first malignant tumor of the genital tract of the female.
Disclosure of Invention
The invention provides application of RP11-379k17.4 overexpression in preparation of a medicament for treating endometrial cancer, aiming at enriching treatment methods of endometrial cancer.
The invention adopts the following technical scheme.
Use of RP11-379k17.4 overexpression in preparation of a medicament for treating endometrial cancer.
Further, the sequence of the RP11-379k17.4 is shown as SEQ ID No.1 or SEQ ID No. 2.
Furthermore, the RP11-379k17.4 overexpression vector is a eukaryotic expression vector or a viral expression vector.
Further, the virus expression vector is an adenovirus vector.
Further, overexpression of RP11-379k17.4 decreased the activity of HEC-1B and Ishikawa cells.
Further, overexpression of RP11-379k17.4 induced apoptosis in HEC-1B and Ishikawa cells.
The present invention obtains the following advantageous effects.
The overexpression of RP11-379k17.4 can reduce the activity of HEC-1B and Ishikawa cells and promote the apoptosis of the HEC-1B and Ishikawa cells, and the dysregulation of RP11-379k17.4 can be involved in the occurrence and development of endometrial cancer to play a cancer inhibiting role.
Drawings
FIG. 1 is a graph showing the effect of the over-expression plasmid (6 kb) for RP11-379K17.4 of the present invention on the apoptotic activity of cells;
FIG. 2 is a graph showing the effect of the over-expression plasmid (6 kb) for RP11-379K17.4 of the present invention on the proliferative capacity of cells;
FIG. 3 is a graph showing the effect of the over-expression plasmid (1.7 kb) for RP11-379K17.4 of the present invention on the apoptotic activity of cells;
FIG. 4 is a graph showing the effect of the over-expression plasmid (1.7 kb) for RP11-379K17.4 of the present invention on the proliferation potency of cells.
Detailed Description
The present invention will be further described with reference to examples.
1. Construction of RP11-379k17.4 overexpression plasmid
Gene ID: 100128164
LOC100128164[Human]:
NR_027622.1 [Full:6057bp] [Protein:0aa]
NR_024409.2 [Full:1752bp] [Protein:0aa]
The gene is lncRNA, and has 2 transcripts; the first 758bp of these 2 transcripts are identical, with the remaining sequences being different.
Construction of adenovirus plasmid from transcript NR _027622.1 (6 kb);
the transcript NR-024409.2 (1.7 kb) was used to construct a general plasmid (i.e., a general eukaryotic expression plasmid).
1. Construction of an overexpression plasmid for the transcript NR-027622.1 (6 kb):
>NR_027622.1|LOC100128164[Human]| 6031bp(SEQ ID No.1)
aatccgccggctggcgcccctggaatggggaggtcggccgcctggcggggccagcggtccttagcggtgccttctctcccctcttccctccgctcctgctgtgcgggaactggagaatcgttaggacgtggaggaagcagagaagtggatcagagaacaggagggctgggaagagtcctggtttctcaatcctgactgagcatctgaatcacctgagaagctactaaaatgcagattccccgaccccatccccttagagtcagagtctgtgcatttagggtgcggcctttctcagagggatcgactgagacctactgaaccctagaagtctttacctgaggcagttcctgcaatgtactgtatatttcatgacattccatgtaaaagatgacaagaccgagaccgttgagtgacagagataagaactgacttatctaaggctccacaggtggtagatggcagagccgagacctgagccaattatcttttctcctgcactgtgcggggtaatacagctcgtttaaaacaagctggagtctccattccgttttccgcgttgcttctcacaactcactgttagaatccagaagtaagagaacccggggaagcgtaaggaagatgaataggacaaagattctaagaaggtgggaaggaagaaggctgcagtctatttcaagtctggtgaatttcaatcactgtagcctttattgttattttagagccacctagaccaaaagagaaggagctcaaattgctggtgaaagaagactgcagctgagttctattatcaaagcaaagtgctgtactgtacttgaaataactctagcaaatactgtgcttcctgcacagaacctgttggattactcacacaaatgtcaaagaacaggttgacattttagcaagttatcagcctggcccatataaaaaaagacatttctagtttttacactttgcagggttcagagagcagttgaatgttatttttagtctatgatccaaagcagctaatgacagagtctaccttagaagataaacaggcttctgatttataacacaaggctaatggtagatattgtgtgatgtgtccagttaaattaacgaaatagctttgcaacagtgtggagtaaagacttaggcttaaaacagagaatttgaggattgagataagtccagagaagcagcaggatggagtgcagatgcaagagaaaggctatgtcaatgccatattataaatatagctcatagacagcatatcctagtgtgtgtctgaagaggcctgtgcccttgaggaaaggggagattatgaccttgtcccccagtgtcatttgcataaattcttagagaccttggggcagtgagtaggagactggttgagatgattactataggaaaattgaataacttaaagtatggatatcttactgttacaggaaatgcttaggaggtgaggtactctgaatttagaatatgatggcctaagggattcggagtcaagaagcaggacatgggatgaagatacatccactgtcaagctggtcattacgacagggagaaatagccctccacagagtgagactagtgagacttgcaagaattgaatgatggtcccgttggacgaaactctttagggtgagtttgcttgtctgggaaagacagaactcagagccctaattgagatgaactgcaagatgggttcagcaggagaaagtggctggaaagggggagggaccaggagccaaatgggtgactatctgccaggtggtctcctgagggcacgcatcttccctggtggtgtgactagaaagagggagtccaaagcacagtcccttctcctccacttggcacacatcaacactttcctcttttcctgtgagccaaggtagaaggtctggtccagaatggacgatatgggtggaggggtgagtcagtattgtttttcatgttggtggttggactcagtttaaaagatggatgtcttagggtagtaggtagcatagttttcctagctttgttgtcacaggtgatagaagctggaccataagttagggtcacagctcccttgattctccttcttctttgttctccttcctctttcacaactctctctcctaggtacttttagtcaggctgcagatggaattctgggcgtttgctccagagagaattttcctaggtagcttttgcgttaaacagacttcattatacattttgctttgcatggggcctctctctcctgacacagatacatgggccccttgtagcccacagcccccagtgtcacacattagtggcacaggcttggttttgacattggttcaaggtacccagtgaagagcactttctgtaagaacagttcaatggcaggacccataatggttatgagcacggaggcctccagtgcaggagcatccgcagtggttgctgatccccagaagggagacgtactctctttctttttgtgtttccaaaggcaaaggcatactcttaggaggcagacagaaagatgtacagttaagaagtggggtttttaggttctgttaacagctggtttattaccttttttggaactttctcaccctttctgcagtgaaataggctatttctctatgtaggagtacacctgtggggaaaaaaatgtgtggaaacctagctattccccaaaaaggagcacaaaaagaaggacatctgagattgctgaatacttttcagatgacaaagaacttagaatgttgagaggggacttacagggaatgggctgataaaagacttgagtgtcatctagtatttacccctttctcctccaacttgagttagctaaatacacacacacacacacacacacacacacacacacacacgaatatacctgcttataataaacgctctaatcaacaaatctactggatcataagctctgccaaggtagcgcctgtggtgttccctttttatccttagcctgtagcaccaggcctggcacatagattcttggtaaatatctactgagtgaatgaattgttgaacagctcgtaatgaagaactgattctgagaatctgtggaatacttcaaaagagaatgaatacaatgcatatattaaaagaggaggcagtctgcagccactgccacctccgccaccgccagctgtgcgggtgagcatccccaggcagcacgttggaagttgttttcaaccatatccagcccttgccaaatacatcctgtttgtcacacatccaaagtgaggtccctccagctataaggtgggcaccatggcagagaagtttttgactgtctctactgcagggaccccttgcagaggaaaaagtatgtgcagaagggtgaccaccactgctgcctgaaatgctttgacaggttctgtgccaacacctgcgtggaatgctgcaagcccatccatgtggactccaaggaggtgcactataaaaactgctcctggcatgacccctgcttccactgtgccaagtgccttcaccccttgccaatgagacctttgtggccaaggacaacaagatcctgtgcaacaagtttgccactcaggaggactctcccaagtgtaaggggtgcttcaaggcctttgtggcaggagatcaaaaatggagtacaaggggaccatctcgcacaaagaccgcttcacctgtagcaactgcaagcaagtcatggggactggaagcttcttctctaaagggaaggacttctactgtgtgacttgccatgagaccaagtttgctaagcattgtgtgaagtgcaacaaggccatcgcatcttggggagtcacttactaggatgagccctggcatgccgagggctttgtatgtgttacctgctctaagaagctggctgtgcagcatttcaccactgtggaggaccagtaatactgtgtggattgctacaagaactttgtggccaagaagtgtgctggatgcaagcaccccatcactgggtttggtaaaggctccagtgtggtggcctatgaaggacaatcctggcacgactgctgcttccactgcaaaaaatgctctgtgaatctggccaacaagcactttgttttccacgaggagcaagtatattgccctgactgtgccaaaaagttgtaaactgacaggggctcctgccctgtaaaatggaatttgaattttgctctttgtgtccttactctctgccctataccatcaatggggaagagtggtctttcccctctttaaaggtctcctgtcttttctcccgttttacagtattactcaaataagggcacacagtgatcatattaggatttagcaaaaagcaaccctgcagcaaagtgaatttctgtccagctgcaactgaaaaacgaaaatttaagtagattgactcttctgcatgtttctcacagagcagaaaagtgctagtcatttagccacttagtgatgtaagcaagaagcatatgagataaagcccccactgagatgcctcttgcggctcagctgggacctaccgtgtagtcacgcgacatgcaagagttgtagtggctgcttcaactcgctgctcaccctcttctgtgagcaggaaaagaacttactgacgtgcatgatttaacttcctcatcaaaactctgaccttccttctgttattttgtgctttcaaatgactaatatgcacttccagaaaattaacatttgaacttagctgtaattctaaactgacctttccccatactaatgtttgatttccccatgtggcatgttttctgagtgttcctaccttaaagcatggaacatgcaggtgatttgggaagtgtaggcagatctgagaaaacgagcctgtttcagaggaacattgtcacagcaaatacttctggaaacttaacaaaaccaaccctgatgtcctttttattgtttttaattaggaatatttttgttttaattgatagcaaaatagtttataggtttggaaacatccatgaaaatattctatttattttattttattattattattattttttgagacggagccttgctctgtcgcccaggctgcagtgcagtggcgcattctcagctcactgcaaccttcgcctcccaggttcaagcaatactcctgtctcagcctcctgagtagctgggactacaggcatgcgccaccacacccagttaatttttgaatttttagtagagatggggtttcaacatattggtcaggctggtcttgaactcctgacctcaggtgatccacccacctaggcctcccaaaatgttggcattacaggcgtgagccgccatgcctggcctgcatgaaaatattctagccctttcagatgttcctgtagtgttgaaattcatcctatggaagtaactgcaaaactctatagggggagttgagcaggtgccaggactgtcatcaacatggatatgacagtacagagcagtggtgagttgaatcccttgtaacgcagttgtctgctctttgtccatgtgttaatgagggctgcaaagtccctcctatagcgattcctaggaattttcctcaagaggaaatgcggatttccacctaccacttacctgaaatacaggatcacctacttactgtattctacattattttatgatataatgagacaatatcaaaagtaaacatgtaatgactatgtgtactaacattcttgtaggagtggttagagaagctgatgtctcatttctacattctgtcattagctgttatcgtctaatgtttcagtgtatccttatggaaataaagcagcatatgaatttaaaaaaagaggaggattagtccaggaactacagaatgattaacttacttttatactgtaaaaaagttagtatccccatgatgtttctccaaaatagtttgaaaataattagaagaattataaattatagtgtgtctctcctctcttaccaaggactaagggctaaagtatcgacatacctcattttactgtgctttgcagatatggcatgttttacaaatggaaggtttgtgggaaccctgagttgagtaaatctattggcgttgttttcccaa
the plasmid construction scheme is as follows: (adenovirus vector, with independent expression of Green fluorescence)
Carrier: h12588 pAdeno-EF1A (S) -mNeonGreen-CMV-MCS-3FLAG
The construction method comprises the following steps: extraction of carrier and target gene enzyme digestion-connection-transformation colibacillus-plasmid
After construction: pAdeno-EF1A (S) -mNeon Green-CMV-RP11-379k17.4(LOC100128164-6kb)
Comparison: h12588
2. Construction of an overexpression plasmid for the transcript NR-024409.2 (1.7 kb):
>NR_024409.2|LOC100128164[Human]| 1738bp(SEQ ID No.2)
aatccgccggctggcgcccctggaatggggaggtcggccgcctggcggggccagcggtccttagcggtgccttctctcccctcttccctccgctcctgctgtgcgggaactggagaatcgttaggacgtggaggaagcagagaagtggatcagagaacaggagggctgggaagagtcctggtttctcaatcctgactgagcatctgaatcacctgagaagctactaaaatgcagattccccgaccccatccccttagagtcagagtctgtgcatttagggtgcggcctttctcagagggatcgactgagacctactgaaccctagaagtctttacctgaggcagttcctgcaatgtactgtatatttcatgacattccatgtaaaagatgacaagaccgagaccgttgagtgacagagataagaactgacttatctaaggctccacaggtggtagatggcagagccgagacctgagccaattatcttttctcctgcactgtgcggggtaatacagctcgtttaaaacaagctggagtctccattccgttttccgcgttgcttctcacaactcactgttagaatccagaagtaagagaacccggggaagcgtaaggaagatgaataggacaaagattctaagaaggtgggaaggaagaaggctgcagtctatttcaagtctggtgaatttcaatcactgtagcctttattgttattttagagccacctagaccaaaagagaaggagctcaaattgctggaggggatttagtggcaggaggacaaagtcagaagagaaatttttcacagtatatcttcttgagccatgtgaccatatgctctaatttttttaactataaaatttaaattaaaaaaaccacaaaaattcgacgcaagagcagcagccacagtggctgaagatgagaggactcctgtgccagccaaggtggaagcagaaattactccccagtctccactgactacaggaacaggagggccacacacccatttggtggccataacattgaaagctgggacagcaggactggtctggacctgaggcgtaaaagacaacacttgcagccagccctggtacttgcatagtggcaaaatgtcccaagctggaggaaatggataagaattgggtacacatcacccgtactgggaataaacaaatgtgttctgattatttggaaaaagcagacattctaggggtagggagttctaaagccttggtgcacttgaatggataaacttcccaggagcagagttttccaagcatggctccctgtggttcacctctaccaagcttaacctaccttctgcttaggcagagcgaagaggcacctcagcataactcagatcaaaatggaagcatctcatttcttaaaagctgtcaaagaattggtgtgaggtttaaaaacgtctctgagcttgatggaaggaaggtaggcggtgatagttcatgctccctttcctccagacacttctcattttcctttcttaacctgctttgatattatgatttcttttgcttcatttgttaagttctcttacaacacttcttgttcacaagtgagtgaagagctgttctctgcttgtaacgttggaaatgggggagtgtttcagaaatgacacaacaaagcattaagccatgaaacaagaaggggaacttcagacagaattacaacagtcatttgagaatctcactgagagctaataaaagccacacacacacaga
the plasmid construction scheme is as follows: (general eukaryotic expression vectors, non-fluorescent)
Carrier: h302 pcDNA3.1(+)
The construction method comprises the following steps: extraction of carrier and target gene enzyme digestion-connection-transformation colibacillus-plasmid
After construction: pcDNA3.1(+) -RP11-379k17.4(LOC100128164-1.7kb)
Comparison: h302
2. Cell culture
Figure 181706DEST_PATH_IMAGE001
Fresh 100mm plates of 1 containing 12mL of CM1-1 medium (90% DMEM-H +10% FBS);
Figure 747817DEST_PATH_IMAGE002
the frozen cell strains (HEC-1B and Ishikawa) taken out from the liquid nitrogen tank are quickly placed in a water bath at 37 ℃ for 1-2min, and are moved into a safety cabinet for recovery as soon as possible after being completely dissolved;
Figure 190430DEST_PATH_IMAGE003
sucking the dissolved solution by using a sterile suction pipe, pumping into a new plate, and shaking up clockwise;
Figure 754267DEST_PATH_IMAGE004
placing at 37 deg.C with 5% CO2The culture was performed in a humidified incubator (Takara Shuzo), and the culture was changed overnight.
3. Cell passage
When the cell density was close to 80% or more, the culture medium was washed twice with PBS by aspiration, 6 mL/(100 mm dish) of trypsin solution was added, the dish was not shaken during observation under a microscope, and when the cells were just shed, most of the trypsin was aspirated, about 0.5mL was left, transferred to an incubator, digested, and taken out for about 2 min. Stopping digestion with 12mL of CM1-1 culture solution, gently blowing and beating uniform cells, and then continuously culturing by separating 3-6 dishes.
4. Cell transfection
The two plasmids prepared by the method transfect cells respectively, and the specific cell transfection process is as follows:
Figure 141386DEST_PATH_IMAGE001
1X 10 per hole5 Performing cotransfection on the cells when the cell density reaches more than 90% on the next day;
Figure 42346DEST_PATH_IMAGE002
plasmid dilution: 0.25ug of the over-expression plasmid and 0.5ul lipo3000 were dissolved in 25ul Opti-mem serum-free medium;
Figure 932941DEST_PATH_IMAGE003
diluting lncRNA: 0.5ul of the over-expression plasmid was dissolved in 25ul of Opti-mem serum-free medium;
Figure 870942DEST_PATH_IMAGE004
dilution lipo 3000: dissolving 1ul lipo3000 in 50ul Opti-mem serum-free medium, mixing well and standing at room temperature for 5 min;
Figure 10936DEST_PATH_IMAGE005
will be provided with
Figure 184428DEST_PATH_IMAGE002
Figure 398372DEST_PATH_IMAGE003
Figure 772853DEST_PATH_IMAGE004
Mixing the three pipes, and standing for 20 min;
Figure 868985DEST_PATH_IMAGE006
adding the plasmid-lipo 3000 mixed solution into a culture hole containing 400 mu L of culture medium of cells, and gently mixing;
Figure 846168DEST_PATH_IMAGE007
place the plates in 37 ℃ CO2Culturing for 48h in an incubator;
Figure 117880DEST_PATH_IMAGE008
culturing for 48h, and collecting cells for subsequent experiments.
5. Apoptosis detection
The study used flow cytometry experiments to detect apoptosis:
Figure 256738DEST_PATH_IMAGE001
digesting with pancreatin without EDTA, centrifuging at 4 deg.C for 5min, and collecting cells;
Figure 40498DEST_PATH_IMAGE002
preparing 1 XBinding Buffer, namely diluting 4 XBinding Buffer (4 mL of Binding Buffer solution and 12mL of deionized water) by deionized water according to a ratio of 1: 4;
Figure 555793DEST_PATH_IMAGE003
the cells were washed 2 times with pre-chilled PBS and centrifuged for 5min at 4 ℃;
Figure 541067DEST_PATH_IMAGE004
250. mu.L of 1 XBinding Buffer was added to resuspend the cells and the concentration was adjusted to 1X 106/mL；
Figure 257350DEST_PATH_IMAGE005
Taking 100 mu L of cell suspension to put in a 5mL flow tube, adding 5 mu L of Annexin V-Alexa Fluor647 and 10 mu L of PI, and gently mixing uniformly;
Figure 531337DEST_PATH_IMAGE006
reacting for 15 min at room temperature in a dark place;
Figure 584743DEST_PATH_IMAGE007
adding 400 mu L of 1 × Binding Buffer, mixing uniformly, and detecting a sample by using a flow cytometer within 1 hour;
Figure 955682DEST_PATH_IMAGE008
a double dispersion dot diagram (two-color dot plot) was drawn by Flow Jo, with Alexa Fluor647 on the abscissa and PI on the ordinate.
Experimental results referring to fig. 1 and 3, the above experimental results show that the overexpression of RP11-379k17.4 significantly induces the apoptosis of endometrial cancer cells compared to the control group (empty plasmid group).
6. Cell proliferation assay
CCK-8 was used to detect the proliferation of cells. The general experimental procedure is as follows:
Figure 577287DEST_PATH_IMAGE001
collecting well-grown endometrial cancer cells, adding 100ul (2000 cells) of cell suspension to each well, 5% CO at 37 deg.C2Culturing for 12 hours in the cell culture box;
Figure 869728DEST_PATH_IMAGE002
after the cells are attached to the wall sufficiently, 10ul of CCK8 reaction solution is added into each hole, and after uniform mixing, the cells are placed into an incubator for culture hours;
Figure 257984DEST_PATH_IMAGE009
and after 24h, 48h and 72h after cell inoculation, reading the absorbance of each hole sample under the condition of 450nm by using an enzyme-labeling instrument, and further calculating the cell proliferation capacity of the sample.
Experimental results referring to fig. 2 and 4, the above experimental results indicate that RP11-379k17.4 can regulate the activity of endometrial cancer cells, and that RP11-379k17.4 overexpression significantly reduces the activity of HEC-1B and Ishikawa cells.
In conclusion, the dysregulation of RP11-379k17.4 can be involved in the occurrence and development of endometrial cancer and play a role in inhibiting cancer.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Sequence listing
<110> affiliated obstetrical and gynecological hospital of double-denier university
<120> application of RP11-379k17.4 overexpression in preparation of endometrial cancer treatment drug
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6031
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
aatccgccgg ctggcgcccc tggaatgggg aggtcggccg cctggcgggg ccagcggtcc 60
ttagcggtgc cttctctccc ctcttccctc cgctcctgct gtgcgggaac tggagaatcg 120
ttaggacgtg gaggaagcag agaagtggat cagagaacag gagggctggg aagagtcctg 180
gtttctcaat cctgactgag catctgaatc acctgagaag ctactaaaat gcagattccc 240
cgaccccatc cccttagagt cagagtctgt gcatttaggg tgcggccttt ctcagaggga 300
tcgactgaga cctactgaac cctagaagtc tttacctgag gcagttcctg caatgtactg 360
tatatttcat gacattccat gtaaaagatg acaagaccga gaccgttgag tgacagagat 420
aagaactgac ttatctaagg ctccacaggt ggtagatggc agagccgaga cctgagccaa 480
ttatcttttc tcctgcactg tgcggggtaa tacagctcgt ttaaaacaag ctggagtctc 540
cattccgttt tccgcgttgc ttctcacaac tcactgttag aatccagaag taagagaacc 600
cggggaagcg taaggaagat gaataggaca aagattctaa gaaggtggga aggaagaagg 660
ctgcagtcta tttcaagtct ggtgaatttc aatcactgta gcctttattg ttattttaga 720
gccacctaga ccaaaagaga aggagctcaa attgctggtg aaagaagact gcagctgagt 780
tctattatca aagcaaagtg ctgtactgta cttgaaataa ctctagcaaa tactgtgctt 840
cctgcacaga acctgttgga ttactcacac aaatgtcaaa gaacaggttg acattttagc 900
aagttatcag cctggcccat ataaaaaaag acatttctag tttttacact ttgcagggtt 960
cagagagcag ttgaatgtta tttttagtct atgatccaaa gcagctaatg acagagtcta 1020
ccttagaaga taaacaggct tctgatttat aacacaaggc taatggtaga tattgtgtga 1080
tgtgtccagt taaattaacg aaatagcttt gcaacagtgt ggagtaaaga cttaggctta 1140
aaacagagaa tttgaggatt gagataagtc cagagaagca gcaggatgga gtgcagatgc 1200
aagagaaagg ctatgtcaat gccatattat aaatatagct catagacagc atatcctagt 1260
gtgtgtctga agaggcctgt gcccttgagg aaaggggaga ttatgacctt gtcccccagt 1320
gtcatttgca taaattctta gagaccttgg ggcagtgagt aggagactgg ttgagatgat 1380
tactatagga aaattgaata acttaaagta tggatatctt actgttacag gaaatgctta 1440
ggaggtgagg tactctgaat ttagaatatg atggcctaag ggattcggag tcaagaagca 1500
ggacatggga tgaagataca tccactgtca agctggtcat tacgacaggg agaaatagcc 1560
ctccacagag tgagactagt gagacttgca agaattgaat gatggtcccg ttggacgaaa 1620
ctctttaggg tgagtttgct tgtctgggaa agacagaact cagagcccta attgagatga 1680
actgcaagat gggttcagca ggagaaagtg gctggaaagg gggagggacc aggagccaaa 1740
tgggtgacta tctgccaggt ggtctcctga gggcacgcat cttccctggt ggtgtgacta 1800
gaaagaggga gtccaaagca cagtcccttc tcctccactt ggcacacatc aacactttcc 1860
tcttttcctg tgagccaagg tagaaggtct ggtccagaat ggacgatatg ggtggagggg 1920
tgagtcagta ttgtttttca tgttggtggt tggactcagt ttaaaagatg gatgtcttag 1980
ggtagtaggt agcatagttt tcctagcttt gttgtcacag gtgatagaag ctggaccata 2040
agttagggtc acagctccct tgattctcct tcttctttgt tctccttcct ctttcacaac 2100
tctctctcct aggtactttt agtcaggctg cagatggaat tctgggcgtt tgctccagag 2160
agaattttcc taggtagctt ttgcgttaaa cagacttcat tatacatttt gctttgcatg 2220
gggcctctct ctcctgacac agatacatgg gccccttgta gcccacagcc cccagtgtca 2280
cacattagtg gcacaggctt ggttttgaca ttggttcaag gtacccagtg aagagcactt 2340
tctgtaagaa cagttcaatg gcaggaccca taatggttat gagcacggag gcctccagtg 2400
caggagcatc cgcagtggtt gctgatcccc agaagggaga cgtactctct ttctttttgt 2460
gtttccaaag gcaaaggcat actcttagga ggcagacaga aagatgtaca gttaagaagt 2520
ggggttttta ggttctgtta acagctggtt tattaccttt tttggaactt tctcaccctt 2580
tctgcagtga aataggctat ttctctatgt aggagtacac ctgtggggaa aaaaatgtgt 2640
ggaaacctag ctattcccca aaaaggagca caaaaagaag gacatctgag attgctgaat 2700
acttttcaga tgacaaagaa cttagaatgt tgagagggga cttacaggga atgggctgat 2760
aaaagacttg agtgtcatct agtatttacc cctttctcct ccaacttgag ttagctaaat 2820
acacacacac acacacacac acacacacac acacacacga atatacctgc ttataataaa 2880
cgctctaatc aacaaatcta ctggatcata agctctgcca aggtagcgcc tgtggtgttc 2940
cctttttatc cttagcctgt agcaccaggc ctggcacata gattcttggt aaatatctac 3000
tgagtgaatg aattgttgaa cagctcgtaa tgaagaactg attctgagaa tctgtggaat 3060
acttcaaaag agaatgaata caatgcatat attaaaagag gaggcagtct gcagccactg 3120
ccacctccgc caccgccagc tgtgcgggtg agcatcccca ggcagcacgt tggaagttgt 3180
tttcaaccat atccagccct tgccaaatac atcctgtttg tcacacatcc aaagtgaggt 3240
ccctccagct ataaggtggg caccatggca gagaagtttt tgactgtctc tactgcaggg 3300
accccttgca gaggaaaaag tatgtgcaga agggtgacca ccactgctgc ctgaaatgct 3360
ttgacaggtt ctgtgccaac acctgcgtgg aatgctgcaa gcccatccat gtggactcca 3420
aggaggtgca ctataaaaac tgctcctggc atgacccctg cttccactgt gccaagtgcc 3480
ttcacccctt gccaatgaga cctttgtggc caaggacaac aagatcctgt gcaacaagtt 3540
tgccactcag gaggactctc ccaagtgtaa ggggtgcttc aaggcctttg tggcaggaga 3600
tcaaaaatgg agtacaaggg gaccatctcg cacaaagacc gcttcacctg tagcaactgc 3660
aagcaagtca tggggactgg aagcttcttc tctaaaggga aggacttcta ctgtgtgact 3720
tgccatgaga ccaagtttgc taagcattgt gtgaagtgca acaaggccat cgcatcttgg 3780
ggagtcactt actaggatga gccctggcat gccgagggct ttgtatgtgt tacctgctct 3840
aagaagctgg ctgtgcagca tttcaccact gtggaggacc agtaatactg tgtggattgc 3900
tacaagaact ttgtggccaa gaagtgtgct ggatgcaagc accccatcac tgggtttggt 3960
aaaggctcca gtgtggtggc ctatgaagga caatcctggc acgactgctg cttccactgc 4020
aaaaaatgct ctgtgaatct ggccaacaag cactttgttt tccacgagga gcaagtatat 4080
tgccctgact gtgccaaaaa gttgtaaact gacaggggct cctgccctgt aaaatggaat 4140
ttgaattttg ctctttgtgt ccttactctc tgccctatac catcaatggg gaagagtggt 4200
ctttcccctc tttaaaggtc tcctgtcttt tctcccgttt tacagtatta ctcaaataag 4260
ggcacacagt gatcatatta ggatttagca aaaagcaacc ctgcagcaaa gtgaatttct 4320
gtccagctgc aactgaaaaa cgaaaattta agtagattga ctcttctgca tgtttctcac 4380
agagcagaaa agtgctagtc atttagccac ttagtgatgt aagcaagaag catatgagat 4440
aaagccccca ctgagatgcc tcttgcggct cagctgggac ctaccgtgta gtcacgcgac 4500
atgcaagagt tgtagtggct gcttcaactc gctgctcacc ctcttctgtg agcaggaaaa 4560
gaacttactg acgtgcatga tttaacttcc tcatcaaaac tctgaccttc cttctgttat 4620
tttgtgcttt caaatgacta atatgcactt ccagaaaatt aacatttgaa cttagctgta 4680
attctaaact gacctttccc catactaatg tttgatttcc ccatgtggca tgttttctga 4740
gtgttcctac cttaaagcat ggaacatgca ggtgatttgg gaagtgtagg cagatctgag 4800
aaaacgagcc tgtttcagag gaacattgtc acagcaaata cttctggaaa cttaacaaaa 4860
ccaaccctga tgtccttttt attgttttta attaggaata tttttgtttt aattgatagc 4920
aaaatagttt ataggtttgg aaacatccat gaaaatattc tatttatttt attttattat 4980
tattattatt ttttgagacg gagccttgct ctgtcgccca ggctgcagtg cagtggcgca 5040
ttctcagctc actgcaacct tcgcctccca ggttcaagca atactcctgt ctcagcctcc 5100
tgagtagctg ggactacagg catgcgccac cacacccagt taatttttga atttttagta 5160
gagatggggt ttcaacatat tggtcaggct ggtcttgaac tcctgacctc aggtgatcca 5220
cccacctagg cctcccaaaa tgttggcatt acaggcgtga gccgccatgc ctggcctgca 5280
tgaaaatatt ctagcccttt cagatgttcc tgtagtgttg aaattcatcc tatggaagta 5340
actgcaaaac tctatagggg gagttgagca ggtgccagga ctgtcatcaa catggatatg 5400
acagtacaga gcagtggtga gttgaatccc ttgtaacgca gttgtctgct ctttgtccat 5460
gtgttaatga gggctgcaaa gtccctccta tagcgattcc taggaatttt cctcaagagg 5520
aaatgcggat ttccacctac cacttacctg aaatacagga tcacctactt actgtattct 5580
acattatttt atgatataat gagacaatat caaaagtaaa catgtaatga ctatgtgtac 5640
taacattctt gtaggagtgg ttagagaagc tgatgtctca tttctacatt ctgtcattag 5700
ctgttatcgt ctaatgtttc agtgtatcct tatggaaata aagcagcata tgaatttaaa 5760
aaaagaggag gattagtcca ggaactacag aatgattaac ttacttttat actgtaaaaa 5820
agttagtatc cccatgatgt ttctccaaaa tagtttgaaa ataattagaa gaattataaa 5880
ttatagtgtg tctctcctct cttaccaagg actaagggct aaagtatcga catacctcat 5940
tttactgtgc tttgcagata tggcatgttt tacaaatgga aggtttgtgg gaaccctgag 6000
ttgagtaaat ctattggcgt tgttttccca a 6031
<210> 2
<211> 1738
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
aatccgccgg ctggcgcccc tggaatgggg aggtcggccg cctggcgggg ccagcggtcc 60
ttagcggtgc cttctctccc ctcttccctc cgctcctgct gtgcgggaac tggagaatcg 120
ttaggacgtg gaggaagcag agaagtggat cagagaacag gagggctggg aagagtcctg 180
gtttctcaat cctgactgag catctgaatc acctgagaag ctactaaaat gcagattccc 240
cgaccccatc cccttagagt cagagtctgt gcatttaggg tgcggccttt ctcagaggga 300
tcgactgaga cctactgaac cctagaagtc tttacctgag gcagttcctg caatgtactg 360
tatatttcat gacattccat gtaaaagatg acaagaccga gaccgttgag tgacagagat 420
aagaactgac ttatctaagg ctccacaggt ggtagatggc agagccgaga cctgagccaa 480
ttatcttttc tcctgcactg tgcggggtaa tacagctcgt ttaaaacaag ctggagtctc 540
cattccgttt tccgcgttgc ttctcacaac tcactgttag aatccagaag taagagaacc 600
cggggaagcg taaggaagat gaataggaca aagattctaa gaaggtggga aggaagaagg 660
ctgcagtcta tttcaagtct ggtgaatttc aatcactgta gcctttattg ttattttaga 720
gccacctaga ccaaaagaga aggagctcaa attgctggag gggatttagt ggcaggagga 780
caaagtcaga agagaaattt ttcacagtat atcttcttga gccatgtgac catatgctct 840
aattttttta actataaaat ttaaattaaa aaaaccacaa aaattcgacg caagagcagc 900
agccacagtg gctgaagatg agaggactcc tgtgccagcc aaggtggaag cagaaattac 960
tccccagtct ccactgacta caggaacagg agggccacac acccatttgg tggccataac 1020
attgaaagct gggacagcag gactggtctg gacctgaggc gtaaaagaca acacttgcag 1080
ccagccctgg tacttgcata gtggcaaaat gtcccaagct ggaggaaatg gataagaatt 1140
gggtacacat cacccgtact gggaataaac aaatgtgttc tgattatttg gaaaaagcag 1200
acattctagg ggtagggagt tctaaagcct tggtgcactt gaatggataa acttcccagg 1260
agcagagttt tccaagcatg gctccctgtg gttcacctct accaagctta acctaccttc 1320
tgcttaggca gagcgaagag gcacctcagc ataactcaga tcaaaatgga agcatctcat 1380
ttcttaaaag ctgtcaaaga attggtgtga ggtttaaaaa cgtctctgag cttgatggaa 1440
ggaaggtagg cggtgatagt tcatgctccc tttcctccag acacttctca ttttcctttc 1500
ttaacctgct ttgatattat gatttctttt gcttcatttg ttaagttctc ttacaacact 1560
tcttgttcac aagtgagtga agagctgttc tctgcttgta acgttggaaa tgggggagtg 1620
tttcagaaat gacacaacaa agcattaagc catgaaacaa gaaggggaac ttcagacaga 1680
attacaacag tcatttgaga atctcactga gagctaataa aagccacaca cacacaga 1738

Claims (6)

1. Use of RP11-379k17.4 overexpression in preparation of a medicament for treating endometrial cancer.
2. The use of RP11-379k17.4 overexpression in the preparation of a medicament for the treatment of endometrial cancer, according to claim 1, wherein: the sequence of the RP11-379k17.4 is shown as SEQ ID No.1 or SEQ ID No. 2.
3. The use of RP11-379k17.4 overexpression in the preparation of a medicament for the treatment of endometrial cancer, according to claim 1, wherein: the RP11-379k17.4 overexpression vector is a eukaryotic expression vector or a viral expression vector.
4. The use of RP11-379k17.4 overexpression in the preparation of a medicament for the treatment of endometrial cancer, according to claim 3, wherein: the virus expression vector is an adenovirus vector.
5. The use of RP11-379k17.4 overexpression in the preparation of a medicament for the treatment of endometrial cancer, according to claim 1, wherein: overexpression of RP11-379k17.4 can reduce the activity of HEC-1B and Ishikawa cells.
6. The use of RP11-379k17.4 overexpression in the preparation of a medicament for the treatment of endometrial cancer, according to claim 1, wherein: overexpression of RP11-379k17.4 induced apoptosis in HEC-1B and Ishikawa cells.
CN202110495750.0A 2021-05-07 2021-05-07 Application of RP11-379k17.4 overexpression in preparation of endometrial cancer treatment drug Pending CN113234824A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106479981A (en) * 2016-12-20 2017-03-08 中国医科大学附属第医院 A kind of people's Endometrial carcinoma cell line and its method for building up
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WO2019104579A1 (en) * 2017-11-30 2019-06-06 SHIEH, Darbin Method for predicting and modulating susceptibility of cancer cell to programmed cell death
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106479981A (en) * 2016-12-20 2017-03-08 中国医科大学附属第医院 A kind of people's Endometrial carcinoma cell line and its method for building up
CN106676110A (en) * 2017-02-20 2017-05-17 中国医科大学附属第医院 Small interfering ribonucleic acid (siRNA) of long noncoding RNA (lncRNA) ABHD11-AS1 related to ovarian cancer and endometrial cancer, and application of siRNA
WO2019104579A1 (en) * 2017-11-30 2019-06-06 SHIEH, Darbin Method for predicting and modulating susceptibility of cancer cell to programmed cell death
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