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Epithelial ovarian cancer target RET and application thereof in diagnosis and treatment

A technology for ovarian cancer and diagnostic reagents, applied in the field of molecular biology, can solve the problems of lack of typical symptom screening methods, low response rate, chemotherapy resistance and other problems

Active Publication Date: 2021-09-14
THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the lack of typical symptoms and effective screening methods, more than 70% of patients have reached the advanced stage when they are first diagnosed. The standard treatment for patients with advanced ovarian cancer is tumor cytoreductive surgery combined with postoperative platinum-based combined chemotherapy , although most patients are sensitive to initial chemotherapy, most patients will relapse within 18 months after the end of initial treatment, and will eventually develop chemotherapy resistance after experiencing multiple relapses. The response rate is very low, and the median survival time of patients is only about 1 year

Method used

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  • Epithelial ovarian cancer target RET and application thereof in diagnosis and treatment
  • Epithelial ovarian cancer target RET and application thereof in diagnosis and treatment
  • Epithelial ovarian cancer target RET and application thereof in diagnosis and treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Example 1 Analysis of RET gene mutations associated with ovarian cancer

[0141] By analyzing the mutation frequency of 100 tyrosine kinase receptors in TCGA database in epithelial ovarian cancer, the top 5 genes with mutation frequency are: RON, PTK7, ABL1, RET, PDGFRB. Among the five genes, although the RET gene has been marketed as a targeted therapy drug vandetanib in clinical practice, its role in ovarian cancer has not been reported. Therefore, the present inventors conducted intensive studies on RET gene mutations associated with ovarian cancer.

[0142] In TCGA (The Cancer Genome Atlas), COSMIC (Catalogue of Somatic Mutations in Cancer), ICGC (International Cancer Genome Consortium), CCLE (Broad Institute Cancer Cell Line Encyclopedia) databases, potential activating mutations of the RET gene in epithelial ovarian cancer were screened. First, the mutation site of RET gene in epithelial ovarian cancer was selected. Then, the mutations predicted as "possibly dam...

Embodiment 2

[0146] Example 2 Screening for activating mutations of the RET gene

[0147] WT plasmid (available from Queen’s University, Canada), use Not1-HF (NEB#R3189S) and Nhel-HF (NEB#R3131V) to digest the RET-WT sequence in the plasmid, and digest pCDH-CMV-MCS-EF1- Puro lentiviral expression plasmid (System biosciences#CD510B-1), use T4 ligase (NEB#M0202V) to insert the RET-WT fragment into the digested pCDH-CMV-MCS-EF1-Puro lentiviral expression plasmid (System biosciences# CD510B-1), construct the RET-WT plasmid with pCDH backbone. The point mutation kit QuikChange used the point mutation kit (QuikChangeLightning Site-Directed Mutagenesis Kit (#210518)) to successfully construct plasmids containing 14 potential activating mutations of the RET gene with pCDH as the backbone, and plasmids containing the medullary thyroid carcinoma C634R Pathogenic mutation plasmid.

[0148] Using the liposome transient method, 14 kinds of plasmids were used to transfect 293T cells, and the phosphory...

Embodiment 3

[0151] Example 3 Verification of the carcinogenesis of RET-R693H and RET-A750T in ovarian cancer

[0152] Digest the NIH3T3 cells with trypsin, neutralize the culture medium, resuspend and count, press 0.25×10 6 pCDHRET-WT, pCDHRET-R693H, pCDHRET-A750T, pCDHRET-C634R virus infection after the cell confluency reaches 50%, stop the infection with fresh medium after 16 hours, add puromycin containing screening media. On the fourth day, the protein was collected for Western Blot.

[0153] The screened cells were used for Soft Agar experiments. Specifically, the selected cells were diluted to 5×10 with 2×RPMI1640 medium (Hyclone#SH30197.03) and 0.7% supernatant 4 Add 1 cell / well to the solidified lower layer gel, put it back into the cell culture incubator after solidified at room temperature, replenish 200ul fresh medium twice a week, stain with thiazolium blue (Sigma#M5655-1G) after 3 weeks and take pictures , to count the number of clones.

[0154] Result: if Figure 4 As ...

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Abstract

The invention provides application of RET in diagnosis and treatment of ovarian cancer. Specifically, the invention provides application of RET mutant protein or a coding sequence thereof or a corresponding detection reagent, the RET mutant protein or the coding sequence thereof or the corresponding detection reagent is used for preparing a diagnostic reagent or a kit, and the diagnostic reagent or the kit is (a) used for detecting ovarian cancer and / or susceptibility thereof, or (b) used for prognosis. The invention also provides a corresponding diagnostic kit for diagnosing the ovarian cancer and a pharmaceutical composition for treating the ovarian cancer. The research shows that a plurality of RET gene mutations which are most closely related to the epithelial ovarian cancer exist in an RET epithelial ovarian cancer patient. In addition, medicines such as Vandetanib and the like can effectively inhibit ovarian cancer by inhibiting activation of RET and downstream MAPK signal channels thereof.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of RET in the diagnosis and treatment of ovarian cancer. Background technique [0002] Ovarian cancer is the disease with the highest female gynecological cancer mortality in developed countries, and it is also the fourth leading cause of female cancer death; it ranks second only to cervical cancer in female gynecological cancer mortality in developing countries. Gynecological cancer is the disease with the highest mortality rate. [0003] Due to the lack of typical symptoms and effective screening methods, more than 70% of patients have reached the advanced stage when they are first diagnosed. The standard treatment for patients with advanced ovarian cancer is tumor cytoreductive surgery combined with postoperative platinum-based combined chemotherapy , although most patients are sensitive to initial chemotherapy, most patients will relapse within 18 months afte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886G01N33/574A61K45/00A61P35/00
CPCC12Q1/6886G01N33/57449G01N33/57492A61K45/00A61P35/00C12Q2600/156C12Q2600/118C12Q2600/112G01N2333/72
Inventor 卢媛王跃祥管路遥
Owner THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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