CN-122012705-A - Experimental method for researching TRPC5 to promote glioma TMZ drug resistance by regulating MUL1 induced mitochondrial autophagy

Abstract

The invention discloses an experimental method for researching TRPC5 to promote TMZ drug resistance of glioma by adjusting MUL1 to induce mitochondrial autophagy, which comprises the following steps of 1, analyzing changes of expression amounts of TRPC5 in recurrent glioma and primary glioma after TMZ chemotherapy. Constructing a TMZ-resistant U87 cell strain, analyzing the variation of the expression quantity of TRPC5 in the TMZ-resistant U87 and normal U87 cells so as to determine the relation between TPRC5 and glioma TMZ resistance, analyzing the influence of exogenous over-expression TRPC5 on the proliferation, invasion capacity and TMZ resistance of the U87 glioma cells, constructing a nude glioma brain model, verifying whether the same conclusion exists in an in vivo environment, and exploring the expression of the TMZ resistance in brain glioma under other paths.

Inventors

• ZHAO XUDONG
• ZOU YAN
• ZHANG YUANHAI
• Tao zhenxing
• LIU ZIXIANG
• MIAO ZENGLI

Assignees

• 无锡市第二人民医院

Dates

Publication Date

20260512

Application Date

20251231

Claims (3)

1. 1. An experimental method for studying TRPC5 to promote glioma TMZ resistance by modulating MUL 1-induced mitochondrial autophagy, comprising the steps of: Step 1, analyzing the change of the expression amount of TRPC5 in recurrent glioma and primary glioma after TMZ chemotherapy. Constructing a TMZ-resistant U87 cell strain, and analyzing the change of the expression quantity of TRPC5 in the TMZ-resistant U87 and normal U87 cells so as to determine the relation between TPRC5 and glioma TMZ resistance; Step 2, analyzing the influence of exogenous overexpression TRPC5 on the proliferation and invasion capacity of the U87 glioma cells and the drug resistance of TMZ, constructing a nude glioma brain model, and verifying whether the same conclusion exists in the in-vivo environment; step 3, analyzing the relation between TPRC5 and a mitochondrial autophagy related protein MUL 1; step 4, analyzing the effect of exogenous over-expressed TRPC5 on the mitochondrial autophagy level of the cells.
2. 2. The experimental method for studying TRPC5 to promote glioma TMZ resistance by modulating MUL 1-induced mitochondrial autophagy according to claim 1, wherein step1 comprises: (1) Analyzing the expression level of TRPC5 mRNA and protein in glioma tissues of a glioma patient and glioma tissue specimens of an initial glioma patient who relapse after a standard TMZ chemotherapy regimen; (2) Analyzing the expression level of mRNA and protein of TRPC5 in the constructed TMZ-resistant U87 cell line U87-MT and normal U87-M; The step 2 comprises the following steps: (1) Constructing a U87 cell line U87-C5 for stably transferring TRPC5 and a control group U87-CON thereof, and analyzing the influence of over-expression TRPC5 on proliferation and invasion capacities of the U87 cells and the drug resistance of TMZ; (2) Constructing a U87 cell line U87-C5-LUC which stably converts TRPC5 and Luciferase and a control group U87-CON-LUC thereof, establishing a corresponding brain glioma cell nude mouse in-situ transplantation model, and analyzing the influence of over-expressed TRPC5 on the TMZ drug resistance of U87 cells in an in-vivo environment; the step 3 comprises the following steps: (1) Analyzing changes in expression levels of mitochondrial autophagy-related proteins in HELA cells overexpressing TPRC5 by sequencing the HELA, and TRPC5 overexpressing cervical cancer cells; (2) Analyzing the expression level of mRNA and protein of MUL1 in glioma tissue of glioma patients and glioma tissue of primary glioma patients relapsed after standard TMZ chemotherapy regimen, and analyzing the expression and distribution of TRPC5 and MUL1 in the tissue; (3) Constructing U87-siC and a control group U87-siCON, analyzing the MUL1 expression quantity in U87-siC and U87-siCON, U87-C5 and U87-CON, and verifying the relation between TRPC5 and MUL1 expression quantity; (4) CO-immunoprecipitation CO-immunoprecipitation experiments were performed on TRPC5 and MUL1 in U87 cells to analyze the relationship of TRPC5 to MUL 1; the step4 comprises the following steps: (1) Analyzing the effect of over-expression of TRPC5 on mitochondrial related protein degradation under normal conditions and TMZ treatment conditions; (2) Analyzing the effect of over-expressing TRPC5 on HELA autophagosome formation under normal conditions and TMZ treatment conditions; (3) Analysis of the effect of over-expression of TRPC5 on HELA cell mitochondrial membrane potential under normal conditions and TMZ treatment.
3. 3. The experimental method for studying TRPC5 to promote glioma TMZ resistance by modulating MUL 1-induced mitochondrial autophagy according to claim 2, wherein step 1 further comprises: 1) The pathological tissue frozen section specimens of 27 glioma cases are collected, the case selection standard is ①, the imaging clear diagnosis comprises CT and nuclear magnetic resonance, ②, the postoperative pathological clear diagnosis is carried out, ③ tissue typing and pathological grading refer to the WHO central nervous system tumor classification standard in 2007, III-IV grade tumor tissues are selected, 12 glioma tissues of patients subjected to one TMZ standard chemotherapy are collected to be an experimental group, and glioma tissues of 15 primary glioma patients are collected to be a control group; 2) Analyzing the expression level of mRNA and protein of TRPC5 in the recurrent group and the initial group by Western Blot and RT-PCR; 3) TMZ is administrated and screened after subcutaneous tumor formation of mice, a TMZ-resistant U87-MT cell line is constructed by primary cell culture, the control group is U87-M, and IC50 verification is carried out on TMZ resistance; 4) Analysis of mRNA and protein expression levels of TRPC5 in U87-MT and U87-M by Western Blot and RT-PCR; the step2 further includes: 1) Constructing an exogenous U87-C5 stable transgenic cell line over-expressing TRPC5, verifying the expression quantity of the TRPC5 by using Western Blot, and analyzing whether construction is successful; 2) The influence of over-expression TRPC5 on proliferation and invasion capacity of U87 cells and TMZ drug resistance is analyzed by carrying out a plate cloning experiment, a transwell experiment and a CCK8 experiment on U87-C5 and a control group U87-CON thereof; 3) Constructing a U87 cell line U87-C5-LUC which stably converts TRPC5 and Luciferase and a control group U87-CON-LUC thereof, establishing a corresponding brain glioma cell nude mice in-situ transplantation model, performing gastric lavage with 20ug/kg TMZ 7 days after operation, performing treatment once for 3 days, performing total treatment for 15 days, measuring the tumor size once every 5 days, and analyzing the influence of over-expressed TRPC5 on the TMZ drug resistance of U87 cells in an in-vivo environment; The step 3 further includes: Sequencing HELA cells which over-express TRPC5 by QIAGEN company, and analyzing the change of the expression level of the mitochondrial autophagy related protein in HELA cells which over-express TPRC 5; 2) RT-PCR and Western Blot analysis of the expression level of MUL1 in glioma tissues of patients with primary glioma and recurrent glioma; 3) RT-PCR and Western Blot analysis of MUL1 expression levels in U87-CON and U87-C5; 4) Constructing a U87 cell line U87-siC knocked down TRPC5, verifying the expression level of the TRPC5 by Western Blot, and analyzing whether construction is successful; 5) Detecting the expression level of MUL1 in U87-C5, U87-siC and U87-CON by Western Blot, and analyzing the regulation and control effect of TPRC5 on the MUL1 expression level; 6) Analyzing the expression condition and distribution of TRPC5 and MUL1 in glioma tissues of patients with primary glioma and recurrent glioma by immunofluorescence staining and fluorescence microscopy; 7) The protein binding relationship between TRPC5 and MUL1 is analyzed through autophagy related protein LC3B by combining immunoprecipitation with Western Blot; The step4 further includes: 1) After TMZ treatment, detecting the levels of mitochondrial related proteins in U87-C5 cells and U87-CON cells by Western Blot, and analyzing the influence of TRPC5 on mitochondrial autophagy; 2) Observing HELA-C5 cells and HELA-CON cells transfected with LC3-GFP and Mitochondria-RFP under a fluorescence electron microscope, transiently turning, subsequently adding TMZ treatment and observing again, and analyzing the influence of over-expressed TRPC5 on HELA cell autophagosome formation under normal conditions and TMZ treatment; 3) The effect of over-expressing TRPC5 on the mitochondrial membrane potential of HELA cells under normal conditions and TMZ treatment was analyzed by detecting HELA-C5 cells and HELA-CON cells using JC-1 membrane potential detection kit, transiently turning, followed by treatment with TMZ and re-detection.

Description

Experimental method for researching TRPC5 to promote glioma TMZ drug resistance by regulating MUL1 induced mitochondrial autophagy Technical Field The invention relates to an experimental method for researching TRPC5 to promote glioma TMZ drug resistance by regulating MUL1 to induce mitochondrial autophagy. Background Gliomas are the most common malignancy in the Central Nervous System (CNS), with Temozolomide (TMZ) being the first anti-glioma chemotherapeutic drug. However, tumor resistance to TMZ is one of the main causes of clinical treatment failure. Some studies indicate that abnormal drug transport is associated with resistance development. At the same time, there is evidence that several mechanisms contribute to the development of drug resistance, including activation of DNA repair systems, impairment of apoptotic signals, and reduced uptake of drugs by cells, but the exact mechanism is still under investigation. Thus, understanding the exact mechanism of glioma cell resistance is critical to the study of new therapeutic strategies to overcome TMZ resistance. In cancer chemotherapy, most studies have tended to use autophagy as a protective pathway to delay or reverse apoptosis. However, the exact mechanism and potential initiation factors for chemo-induced protective autophagy remain unknown. Based on previous studies, we postulate TRPC5 to act as a promoter of autophagy during chemotherapy. We found that TRPC5 enhances TMZ resistance of glioma cells by mediating autophagy, promoting tumor cell survival. Several papers have pointed out several pathways by which TRPC5 modulates TMZ resistance of glioma cells. However, in order to achieve/explore the optimization of the therapeutic regimen, it is still necessary to design experiments to explore the expression of TMZ resistance in gliomas under other pathways/paths. Disclosure of Invention In order to solve the problems, the technical scheme is that an experimental method for researching TRPC5 to promote TMZ resistance of glioma by regulating MUL1 to induce mitochondrial autophagy is provided, which is characterized by comprising the following steps: Step 1, analyzing the change of the expression amount of TRPC5 in recurrent glioma and primary glioma after TMZ chemotherapy. Constructing a TMZ-resistant U87 cell strain, and analyzing the change of the expression quantity of TRPC5 in the TMZ-resistant U87 and normal U87 cells so as to determine the relation between TPRC5 and glioma TMZ resistance; Step 2, analyzing the influence of exogenous overexpression TRPC5 on the proliferation and invasion capacity of the U87 glioma cells and the drug resistance of TMZ, constructing a nude glioma brain model, and verifying whether the same conclusion exists in the in-vivo environment; step 3, analyzing the relation between TPRC5 and a mitochondrial autophagy related protein MUL 1; step 4, analyzing the effect of exogenous over-expressed TRPC5 on the mitochondrial autophagy level of the cells. Further, the step 1 includes: (1) The expression levels of TRPC5 mRNA and protein in glioma tissues of glioma patients and glioma tissue specimens of primary glioma patients, relapsing following standard TMZ chemotherapy regimens, were analyzed. (2) The constructed TMZ-resistant U87 cell line U87-MT and normal U87-M were analyzed for the expression levels of mRNA and protein of TRPC 5. The step 2 comprises the following steps: (1) The method comprises the steps of constructing a U87 cell line U87-C5 for stably transferring TRPC5 and a control group U87-CON thereof, and analyzing the influence of over-expression TRPC5 on proliferation and invasion capacity of the U87 cells and TMZ drug resistance. (2) Constructing a U87 cell line U87-C5-LUC which stably converts TRPC5 and Luciferase and a control group U87-CON-LUC thereof, establishing a corresponding brain glioma cell nude mouse in-situ transplantation model, and analyzing the influence of over-expressed TRPC5 on the TMZ drug resistance of the U87 cells in an in-vivo environment. The step 3 comprises the following steps: (1) Changes in the expression level of mitochondrial autophagy-related proteins in TRPC 5-overexpressing HELA cells were analyzed by sequencing TRPC 5-overexpressing cervical cancer cells (HELA) cells. (2) The levels of mRNA and protein expression of MUL1 in glioma tissues of glioma patients and glioma tissues of primary glioma patients, relapsing following standard TMZ chemotherapy regimen, and the expression and distribution of TRPC5 and MUL1 in tissues were analyzed. (3) Constructing U87-siC and a control group U87-siCON, analyzing the MUL1 expression quantity in U87-siC and U87-siCON, U87-C5 and U87-CON, and verifying the relation between TRPC5 and MUL1 expression quantity. (4) CO-immunoprecipitation (CO-immunoprecipitation experiment) was performed on TRPC5 and MUL1 in U87 cells to analyze the relationship of TRPC5 and MUL 1. The step4 comprises the following steps: (1) Analysis of the effect of over-expre