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Method for studying METTL3 or 14 mediated m6A modification regulation of EC metastasis

A mediation, m6a technology, applied in the field of biomedicine, can solve the problems of reduced affinity between HNRNPG and specific RNA sequences, affecting RNA variable splicing and mRNA expression, and reducing the level of m6A modification

Inactive Publication Date: 2021-12-07
无锡准因生物科技有限公司
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Problems solved by technology

[0007] In summary, the inactivating mutation of METTL14 and the downregulation of METTL3 expression in EC patients can lead to a decrease in the level of m6A modification on the overall mRNA, resulting in a decrease in the affinity of HNRNPG to specific RNA sequences, thereby affecting RNA alternative splicing and mRNA expression

Method used

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  • Method for studying METTL3 or 14 mediated m6A modification regulation of EC metastasis
  • Method for studying METTL3 or 14 mediated m6A modification regulation of EC metastasis
  • Method for studying METTL3 or 14 mediated m6A modification regulation of EC metastasis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Such as figure 1 As indicated, the expression polymorphisms of METTL3, METTL14, HNRNPG, m6A, and ERaD7 in EC patients were studied at the clinical level (in the figure, WB: western blot; IHC: immunohistochemistry; IF: immunofluorescence immunofluorescence)

[0071] 1.1 Entry conditions

[0072] Peripheral blood samples:

[0073] Experimental group: Female EC patients aged 18-70 years, with no primary tumor metastasis (n=20), distant primary tumor metastasis (n=20), recurrence and metastasis (n=20), no treatment (at the time of diagnosis) or 7 days or more after receiving radiotherapy or chemotherapy (based on the latest treatment).

[0074] Control group: healthy females aged 18-70, n=60, age matched with the experimental group.

[0075] Both the experimental group and the control group need to meet the conditions: no other diseases (normal function of important organs: heart, lung, liver, kidney, blood), no HIV, HBV, HCV infection, confirmed HPV infection type, no a...

Embodiment 2

[0112] Such as figure 2 As shown, the role of METTL3 and METTL14 in regulating m6A modification and HNRNPG function in tumor metastasis was studied in cell models

[0113] 2.1 Experimental cell grouping and gene knockdown and overexpression

[0114] Experimental group 1: CD133 isolated from fresh tumor tissue + CXCR4 + Cancer stem cells, CD133 in the control group - CXCR4 - tumor cells. Tissues from n=3 patients were taken, wherein n=1 primary cancer without metastasis, n=1 primary metastatic cancer, and n=1 recurrent cancer.

[0115] Experimental group 2: EC model cells, 3 cell lines: 1. Ishikawa cells (Department of Pathology, Peking University); 2. HEC-1A (Shanghai Institute of Cellular Sciences, Chinese Academy of Sciences); 3. RL95-2 cells (ATCC, Manassas, VA, USA ).

[0116] Gene knockdown treatment:

[0117] Group 1: CD133 + CXCR4 + / CD133 - CXCR4 - +siControl,CD133 + CXCR4 + / CD133 - CXCR4 - +siMETTL3,CD133 + CXCR4 + / CD133 - CXCR4 - +siMETTL14, CD...

Embodiment 3

[0142] Such as image 3 As shown, in the PDX mouse model, the regulation of METTL3 / METTL14 affects the ability of EC tumor stem cells to form tumors and metastasize

[0143] 3.1 Experimental grouping and cell processing

[0144] Experimental group 1:

[0145] The primary isolated EC tumor stem cells (CD133 + CXCR4 + ) for METTL3 / METT14 knockdown: a. CD133 + CXCR4 + +pLenti-shRNAcontrol; b. CD133 + CXCR4 + +pLenti-shMETTL3; c.CD133 + CXCR4 + +pLenti-shMETTL14

[0146] Overexpression:

[0147] a. CD133 + CXCR4 + +pLenti-oeGFP; b.CD133 + CXCR4 + +pLenti-oeMETTL3; c.CD133 + CXCR4 + +pLenti-oeMETTL14; d.CD133 + CXCR4 + +pLenti-oeMETTL3+14

[0148] Control group 1:

[0149] Knockdown: a.CD133 - CXCR4 - +pLenti-shRNAcontrol; b. CD133 - CXCR4 - +pLenti-shMETTL3; c.CD133 - CXCR4 - +pLenti-shMETTL14

[0150] Overexpression:

[0151] a. CD133 - CXCR4 - +pLenti-oeGFP; b.CD133 - CXCR4 - +pLenti-oeMETTL3; c.CD133 - CXCR4 - +pLenti-oeMETTL14; d.CD133 - CXCR...

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Abstract

The invention relates to a method for studying METTL3 or 14 mediated m6A modification regulation of EC metastasis. The method comprises the following steps: 1) studying expression polymorphism of METTL3 / 14, HNRNPG, m6A and ERaD7 in an EC patient in a clinical level experiment; (2) studying the use of METTL3 and METTL14 for regulating m6A modification and HNRNPG function in tumor metastasis in a cell model; and (3) studying regulation of METTL3 / 14 on the tumor formation and metastasis capability of EC tumor stem cells in a PDX mouse model. The method provided by the invention has the advantages that the molecular mechanism that the METTL3 / 14 regulates the m6A modification level in EC stem cells so as to influence combination of HNRNPG and target RNA and influence ERa variable shear regulation and ERaD7 specific transcription product expression level can be studied, and a new thought is provided for improving the m6A modification level in EC and exploring potential treatment means.

Description

technical field [0001] The invention relates to a method for studying the mechanism of m6A modification mediated by METTL3 or METTL14 to regulate the metastasis of endometrial cancer, and belongs to the technical field of biomedicine. Background technique [0002] Endometrial cancer (EC) accounts for up to 6% of female tumors and is the most common gynecological malignancy in the Western world. The annual incidence of endometrial cancer is 15-20 / 100,000 people, 89% of which occur in the age group of 65-59 years. Although 75% of patients can be treated at an early stage, about 20% of patients relapse after primary tumor resection. EC is mainly divided into two types: estrogen-dependent type I, which refers to excessive exposure to estrogen that loses progesterone regulation, manifested as non-ovulatory uterine bleeding, infertility, delayed menopause, obesity, endometrial hyperplasia, etc.; Hormone-dependent type II has no obvious symptoms and no metabolic disorders, but ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886G01N33/574C12Q1/02
CPCC12Q1/6886G01N33/57442G01N33/57484G01N33/5073G01N33/5017G01N33/5026C12Q2600/158C12Q2600/156G01N2800/7028
Inventor 俞珏华程禹
Owner 无锡准因生物科技有限公司
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