Experimental method for researching TRPC5 for promoting glioma TMZ drug resistance by regulating MUL1 induced mitochondrial autophagy

Abstract

The invention discloses an experimental method for researching TRPC5 for promoting glioma TMZ drug resistance by regulating MUL1 induced mitochondrial autophagy. The experimental method comprises the following steps: step 1, analyzing the change of the expression quantity of TRPC5 in recurrent glioma and primary glioma after TMZ chemotherapy; a TMZ-resistant U87 cell strain is constructed, and the change of the expression quantity of TRPC5 in TMZ-resistant U87 and normal U87 cells is analyzed, so that the relationship between TPRC5 and glioma TMZ drug resistance is determined. 2, analyzing the influence of exogenous overexpression TRPC5 on U87 glioma cell proliferation, invasion ability and TMZ drug resistance, constructing a nude mouse glioma intracerebral model, and verifying whether the nude mouse glioma intracerebral model has the same conclusion in the in-vivo environment; and exploring the expression of drug resistance of TMZ in brain glioma under other pathways / paths.

Description

Technical Field

[0001] This invention relates to an experimental method for studying how TRPC5 promotes TMZ resistance in gliomas by regulating MUL1-induced mitophagy. Background Technology

[0002] Gliomas are the most common malignant tumors of the central nervous system (CNS), and temozolomide (TMZ) is the first-line chemotherapy drug for gliomas. However, tumor resistance to TMZ is one of the main reasons for clinical treatment failure. Some studies have indicated that aberrant drug transport is associated with the development of resistance. Meanwhile, there is evidence that several mechanisms contribute to the development of resistance, including activation of the DNA repair system, impairment of apoptosis signaling, and reduced cellular drug uptake, but the exact mechanisms are still under investigation. Therefore, understanding the precise mechanisms of glioma cell resistance is crucial for developing new therapeutic strategies to overcome TMZ resistance.

[0003] In cancer chemotherapy, most studies tend to view autophagy as a protective pathway that delays or reverses apoptosis. However, the exact mechanisms and potential initiating factors of chemotherapy-induced protective autophagy remain unknown.

[0004] Based on previous research, we hypothesized that TRPC5 acts as an initiator of autophagy during chemotherapy. We found that TRPC5 enhances TMZ resistance in glioma cells by mediating autophagy, thus promoting tumor cell survival. Several papers have identified several pathways by which TRPC5 regulates TMZ resistance in glioma cells. However, to achieve / explore the optimal treatment regimen, it is still necessary to design experiments to explore the expression of TMZ resistance in gliomas under other pathways / pathways. Summary of the Invention

[0005] To address the above problems, the following technical solution is provided: an experimental method for studying how TRPC5 promotes TMZ resistance in gliomas by regulating MUL1-induced mitophagy, characterized by the following steps: Step 1: Analyze the changes in TRPC5 expression levels in recurrent and newly diagnosed gliomas after TMZ chemotherapy. A TMZ-resistant U87 cell line was constructed, and the changes in TRPC5 expression levels in TMZ-resistant and normal U87 cells were analyzed to clarify the relationship between TRPC5 and TMZ resistance in gliomas. Step 2: Analyze the effects of exogenous overexpression of TRPC5 on the proliferation, invasion ability and TMZ resistance of U87 glioma cells, and construct a nude mouse glioma brain model to verify whether the same conclusions are reached in vivo. Step 3: Analyze the relationship between TPRC5 and the mitophagy-associated protein MUL1; Step 4: Analyze the effect of exogenous overexpression of TRPC5 on the level of mitochondrial autophagy in cells.

[0006] Furthermore, step 1 includes: (1) The expression levels of TRPC5 mRNA and protein in glioma tissues of patients with recurrent glioma after standard TMZ chemotherapy and glioma tissues of patients with newly diagnosed glioma were analyzed.

[0007] (2) The expression levels of TRPC5 mRNA and protein in the constructed TMZ-resistant U87 cell line U87-MT and the normal U87-M were analyzed.

[0008] Step 2 includes: (1) The U87 cell line U87-C5 with stable TRPC5 expression and its control group U87-CON were constructed, and the effects of TRPC5 overexpression on the proliferation, invasion ability and TMZ resistance of U87 cells were analyzed.

[0009] (2) Construct the U87 cell line U87-C5-LUC with stable transduction of TRPC5 and luciferase and its control group U87-CON-LUC, and establish the corresponding nude mouse orthotopic transplantation model of glioma cells to analyze the effect of TRPC5 overexpression on TMZ resistance of U87 cells in vivo.

[0010] Step 3 includes: (1) By sequencing cervical cancer cells (HELA) that overexpress TRPC5, the changes in the expression levels of mitochondrial autophagy-related proteins in HELA cells that overexpress TRPC5 were analyzed.

[0011] (2) The expression levels of MUL1 mRNA and protein in glioma tissues of patients with recurrent glioma after standard TMZ chemotherapy and patients with newly diagnosed glioma were analyzed, and the expression and distribution of TRPC5 and MUL1 in tissues were also analyzed.

[0012] (3) Construct U87-siC5 and control group U87-siCON, analyze the expression level of MUL1 in U87-siC5 and U87-siCON, and verify the relationship between TRPC5 and MUL1 expression level.

[0013] (4) Perform CO-immunoprecipitation on TRPC5 and MUL1 in U87 cells to analyze the relationship between TRPC5 and MUL1.

[0014] Step 4 includes: (1) The effect of TRPC5 overexpression on the degradation of mitochondrial-related proteins was analyzed under normal conditions and TMZ treatment.

[0015] (2) The effect of TRPC5 overexpression on autophagosome formation in HELA cells under normal conditions and TMZ treatment was analyzed.

[0016] (3) The effects of TRPC5 overexpression on mitochondrial membrane potential in HELA cells under normal conditions and TMZ treatment were analyzed.

[0017] Furthermore, step 1 also includes: 1) Frozen section specimens of pathological tissue were collected from 27 glioma cases. The case selection criteria were: ① clear imaging diagnosis including CT and MRI; ② clear postoperative pathological diagnosis; ③ histological classification and pathological grading were in accordance with the "2007 WHO Classification of Tumors of the Central Nervous System", and grade III-IV tumor tissues were selected. Glioma tissues from 12 patients who had undergone one TMZ standard chemotherapy regimen were collected as the experimental group, and glioma tissues from 15 patients with newly diagnosed gliomas were collected as the control group. 2) The expression levels of TRPC5 mRNA and protein in the relapse group and the initial relapse group were analyzed by Western Blot and RT-PCR. 3) After subcutaneous tumor formation in mice, TMZ was administered and screened. Primary cell cultures were used to construct the TMZ-resistant U87-MT cell line, with the control group being U87-M. IC50 was used to verify its TMZ resistance. 4) The expression levels of TRPC5 mRNA and protein in U87-MT and U87-M were analyzed by Western Blot and RT-PCR; Step 2 also includes: 1) Construct a stable U87-C5 cell line that overexpresses exogenous TRPC5, verify TRPC5 expression level by Western blotting, and analyze whether the construction was successful.

[0018] 2) The effects of TRPC5 overexpression on the proliferation, invasion ability and TMZ resistance of U87 cells were analyzed by plate colony formation assay, transwell assay and CCK8 assay of U87-C5 and its control group U87-CON.

[0019] 3) Construct the U87 cell line U87-C5-LUC, which is stably transfected with TRPC5 and luciferase, and its control group U87-CON-LUC. Establish the corresponding glioma cell orthotopic transplantation model in nude mice. Seven days after the operation, the cells were administered 20 ug / kg TMZ by gavage once every three days for a total of 15 days. The tumor size was measured every five days to analyze the effect of TRPC5 overexpression on TMZ resistance in U87 cells under in vivo conditions.

[0020] Step 3 also includes: 1) QIAGEN sequenced HELA cells overexpressing TRPC5 and analyzed the changes in the expression levels of mitochondrial autophagy-related proteins in HELA cells overexpressing TRPC5.

[0021] 2) RT-PCR and Western Blot analysis were used to analyze the expression level of MUL1 in glioma tissues of patients with primary and recurrent gliomas.

[0022] 3) RT-PCR and Western Blot analysis were used to analyze the expression level of MUL1 in U87-CON and U87-C5.

[0023] 4) Construct the U87-siC5 cell line with TRPC5 knockdown, verify TRPC5 expression level by Western blotting, and analyze whether the construction was successful.

[0024] 5) The expression levels of MUL1 in U87-C5, U87-siC5 and U87-CON were detected by Western Blot, and the regulatory effect of TPRC5 on the expression level of MUL1 was analyzed.

[0025] 6) The expression and distribution of TRPC5 and MUL1 in glioma tissues of patients with primary and recurrent gliomas were analyzed by immunofluorescence staining and fluorescence microscopy.

[0026] 7) Immunoprecipitation combined with Western blotting was used to analyze the protein binding relationship between TRPC5 and MUL1 using autophagy-related protein LC3B.

[0027] Step 4 also includes: 1) After TMZ treatment, the levels of mitochondrial-related proteins in U87-C5 and U87-CON cells were detected by Western Blot to analyze the effect of TRPC5 on mitophagy.

[0028] 2) HELA-C5 cells and HELA-CON cells (transfected transiently) transfected with LC3-GFP and Mitochondria-RFP were observed under fluorescence electron microscopy. TMZ treatment was then applied, and the cells were observed again to analyze the effect of TRPC5 overexpression on autophagosome formation in HELA cells under normal conditions and TMZ treatment.

[0029] 3) HELA-C5 cells and HELA-CON cells (transient transduction) were detected using the JC-1 membrane potential detection kit. TMZ treatment was then applied, and the results were detected again to analyze the effect of TRPC5 overexpression on mitochondrial membrane potential in HELA cells under normal conditions and TMZ treatment.

[0030] This invention, through extensive and in-depth research, has discovered a novel pathway, Mul1, by which TRPC5 regulates resistance to the chemotherapeutic drug temozolomide (TMZ) in tumors. This new pathway is closely related to the development of resistance to TMZ. Compared with primary cells U87 and U251, the expression level of TRPC5 in temozolomide-resistant cells U87R and U251R was significantly increased, and the mitophagy pathway was activated. Reducing TRPC5 gene expression can enhance the sensitivity of cell lines to temozolomide and inhibit the proliferation of tumor cells. This invention not only provides new data on the drug resistance mechanism of malignant gliomas but also provides new targets for the design of anti-tumor drug resistance agents. Attached Figure Description

[0031] Figure 1 TRPC5 expression was elevated in TMZ-resistant gliomas. (AC) RT-PCR and Western Blot analysis were used to analyze the mRNA and protein expression levels of TRPC5 in tissues from patients with primary gliomas (n=15) and recurrent gliomas (n=12). (D) CCK8 analysis was used to analyze the TMZ resistance of the constructed U87-MT and U87-M. (EG) RT-PCR and Western Blot analysis were used to analyze the mRNA and protein expression levels of TRPC5 in U87-M and U87-MT. P < 0.05 P < 0.01, P < 0.001.

[0032] Figure 2Exogenous overexpression of TRPC5 enhanced the proliferation, invasion, and TMZ resistance of U87 glioma cells. (AB) Western blotting verified the expression level of TRPC5 protein in U87-C5 cells stably transfected with TRPC5. (CD) Plate colony assay: 800 cells were seeded per well, and after 2 weeks of growth, crystal violet staining was performed to count and statistically analyze the number of colonies. (EF) Transwell assay: 2000 cells were seeded per well, and after 36 hours of culture, crystal violet staining was performed to count and statistically analyze the cells at the bottom of the chamber. (GH) CCK8 assay: The tolerance of U87-C5 and U87-CON cells to different concentrations of TMZ was investigated, as well as the growth of U87-C5 and U87-CON cells under 100 µM TMZ treatment.

[0033] Figure 3 (I) Mouse in vivo imaging monitors intracranial tumor formation in mice in the U87-C5-LUC and U87-CON-LUC groups under 100µM TMZ treatment. P < 0.05 P < 0.01, P < 0.001.

[0034] Figure 4 Altering TPRC5 expression levels synchronously alters MUL1 expression levels, and TPRC5 and MUL1 can bind at the protein level. (A) QIAGEN sequenced HELA cells overexpressing TRPC5 and analyzed changes in the expression levels of mitophagy-related proteins in HELA cells overexpressing TPRC5, showing a significant increase in MUL1 expression. (BD) RT-PCR and Western Blot analysis were used to analyze the expression levels of MUL1 in glioma tissues from patients with primary and recurrent gliomas.

[0035] Figure 5 (EG) RT-PCR and Western Blot analysis were used to analyze the expression levels of MUL1 in U87-CON and U87-C5. (HI) Western Blot analysis was used to analyze the effect of TRPC5 overexpression on MUL1 expression in U87. (JL) Western Blot analysis was used to analyze the effect of TRPC5 knockdown on MUL1 expression in U87.

[0036] Figure 6 (M) Immunofluorescence assay revealed the expression and distribution of TRPC5 and MUL1 in glioma tissues from patients with primary and recurrent gliomas. (N) CO-IP assay analyzed the protein binding relationship between TRPC5 and MUL1 using the autophagy-related protein LC3B. P < 0.05 P < 0.01, P < 0.001.

[0037] Figure 7 Exogenous overexpression of TRPC5 can increase the level of mitochondrial autophagy in cells. (AC) Western blot analysis was conducted to investigate the effect of TRPC5 overexpression on the expression levels of mitochondrial-associated proteins TOMM20 and TIMM23 in U87 cell mitochondria.

[0038] Figure 8 (DE) Immunofluorescence assay was used to analyze the effects of TRPC5 overexpression and 100µM TMZ treatment on the distribution of autophagosome LC3 and mitochondria in HELA cells, as well as the formation of autophagosomes. (FG) JC-1 mitochondrial membrane potential assay was used to analyze the changes in mitochondrial autophagy levels in HELA cells caused by TRPC5 overexpression and 100µM TMZ treatment. P < 0.05 P < 0.01, P < 0.001. Specific Implementation

[0039] The following is an example; please refer to the relevant appendix for details. Figure 1-8 . Example 1

[0040] Step 1: Analyze the changes in TRPC5 expression levels in recurrent and newly diagnosed gliomas after TMZ chemotherapy. A TMZ-resistant U87 cell line was constructed, and the changes in TRPC5 expression levels in TMZ-resistant and normal U87 cells were analyzed to clarify the relationship between TRPC5 and TMZ resistance in gliomas. Step 2: Analyze the effects of exogenous overexpression of TRPC5 on the proliferation, invasion ability and TMZ resistance of U87 glioma cells, and construct a nude mouse glioma brain model to verify whether the same conclusions are reached in vivo. Step 3: Analyze the relationship between TPRC5 and the mitophagy-associated protein MUL1; Step 4: Analyze the effect of exogenous overexpression of TRPC5 on the level of mitochondrial autophagy in cells. Example 2

[0041] Step 1 includes: (1) The expression levels of TRPC5 mRNA and protein in glioma tissues of patients with recurrent glioma after standard TMZ chemotherapy and glioma tissues of patients with newly diagnosed glioma were analyzed.

[0042] (2) The expression levels of TRPC5 mRNA and protein in the constructed TMZ-resistant U87 cell line U87-MT and the normal U87-M were analyzed.

[0043] Step 2 includes: (1) The U87 cell line U87-C5 with stable TRPC5 expression and its control group U87-CON were constructed, and the effects of TRPC5 overexpression on the proliferation, invasion ability and TMZ resistance of U87 cells were analyzed.

[0044] (2) Construct the U87 cell line U87-C5-LUC with stable transduction of TRPC5 and luciferase and its control group U87-CON-LUC, and establish the corresponding nude mouse orthotopic transplantation model of glioma cells to analyze the effect of TRPC5 overexpression on TMZ resistance of U87 cells in vivo.

[0045] Step 3 includes: (1) By sequencing cervical cancer cells (HELA) that overexpress TRPC5, the changes in the expression levels of mitochondrial autophagy-related proteins in HELA cells that overexpress TRPC5 were analyzed.

[0046] (2) The expression levels of MUL1 mRNA and protein in glioma tissues of patients with recurrent glioma after standard TMZ chemotherapy and patients with newly diagnosed glioma were analyzed, and the expression and distribution of TRPC5 and MUL1 in tissues were also analyzed.

[0047] (3) Construct U87-siC5 and control group U87-siCON, analyze the expression level of MUL1 in U87-siC5 and U87-siCON, and verify the relationship between TRPC5 and MUL1 expression level.

[0048] (4) Perform CO-immunoprecipitation on TRPC5 and MUL1 in U87 cells to analyze the relationship between TRPC5 and MUL1.

[0049] Step 4 includes: (1) The effect of TRPC5 overexpression on the degradation of mitochondrial-related proteins was analyzed under normal conditions and TMZ treatment.

[0050] (2) The effect of TRPC5 overexpression on autophagosome formation in HELA cells under normal conditions and TMZ treatment was analyzed.

[0051] (3) The effects of TRPC5 overexpression on mitochondrial membrane potential in HELA cells under normal conditions and TMZ treatment were analyzed. Example 3

[0052] Step 1 further includes: 1) Frozen section specimens of pathological tissue were collected from 27 glioma cases. The case selection criteria were: ① clear imaging diagnosis including CT and MRI; ② clear postoperative pathological diagnosis; ③ histological classification and pathological grading were in accordance with the "2007 WHO Classification of Tumors of the Central Nervous System", and grade III-IV tumor tissues were selected. Glioma tissues from 12 patients who had undergone one TMZ standard chemotherapy regimen were collected as the experimental group, and glioma tissues from 15 patients with newly diagnosed gliomas were collected as the control group. 2) The expression levels of TRPC5 mRNA and protein in the relapse group and the initial relapse group were analyzed by Western Blot and RT-PCR. 3) After subcutaneous tumor formation in mice, TMZ was administered and screened. Primary cell cultures were used to construct the TMZ-resistant U87-MT cell line, with the control group being U87-M. IC50 was used to verify its TMZ resistance. 4) The expression levels of TRPC5 mRNA and protein in U87-MT and U87-M were analyzed by Western Blot and RT-PCR; Step 2 also includes: 1) Construct a stable U87-C5 cell line that overexpresses exogenous TRPC5, verify TRPC5 expression level by Western blotting, and analyze whether the construction was successful.

[0053] 2) The effects of TRPC5 overexpression on the proliferation, invasion ability and TMZ resistance of U87 cells were analyzed by plate colony formation assay, transwell assay and CCK8 assay of U87-C5 and its control group U87-CON.

[0054] 3) Construct the U87 cell line U87-C5-LUC, which is stably transfected with TRPC5 and luciferase, and its control group U87-CON-LUC. Establish the corresponding glioma cell orthotopic transplantation model in nude mice. Seven days after the operation, the cells were administered 20 ug / kg TMZ by gavage once every three days for a total of 15 days. The tumor size was measured every five days to analyze the effect of TRPC5 overexpression on TMZ resistance in U87 cells under in vivo conditions.

[0055] Step 3 also includes: 1) QIAGEN sequenced HELA cells overexpressing TRPC5 and analyzed the changes in the expression levels of mitochondrial autophagy-related proteins in HELA cells overexpressing TRPC5.

[0056] 2) RT-PCR and Western Blot analysis were used to analyze the expression level of MUL1 in glioma tissues of patients with primary and recurrent gliomas.

[0057] 3) RT-PCR and Western Blot analysis were used to analyze the expression level of MUL1 in U87-CON and U87-C5.

[0058] 4) Construct the U87-siC5 cell line with TRPC5 knockdown, verify TRPC5 expression level by Western blotting, and analyze whether the construction was successful.

[0059] 5) The expression levels of MUL1 in U87-C5, U87-siC5 and U87-CON were detected by Western Blot, and the regulatory effect of TPRC5 on the expression level of MUL1 was analyzed.

[0060] 6) The expression and distribution of TRPC5 and MUL1 in glioma tissues of patients with primary and recurrent gliomas were analyzed by immunofluorescence staining and fluorescence microscopy.

[0061] 7) Immunoprecipitation combined with Western blotting was used to analyze the protein binding relationship between TRPC5 and MUL1 using autophagy-related protein LC3B.

[0062] Step 4 also includes: 1) After TMZ treatment, the levels of mitochondrial-related proteins in U87-C5 and U87-CON cells were detected by Western Blot to analyze the effect of TRPC5 on mitophagy.

[0063] 2) HELA-C5 cells and HELA-CON cells (transfected transiently) transfected with LC3-GFP and Mitochondria-RFP were observed under fluorescence electron microscopy. TMZ treatment was then applied, and the cells were observed again to analyze the effect of TRPC5 overexpression on autophagosome formation in HELA cells under normal conditions and TMZ treatment.

[0064] 3) HELA-C5 cells and HELA-CON cells (transient transduction) were detected using the JC-1 membrane potential detection kit. TMZ treatment was then applied, and the results were detected again to analyze the effect of TRPC5 overexpression on mitochondrial membrane potential in HELA cells under normal conditions and TMZ treatment.

Claims

1. An experimental method for investigating how TRPC5 promotes TMZ resistance in gliomas by regulating MUL1-induced mitophagy, characterized by the following: Includes the following steps: Step 1: Analyze the changes in TRPC5 expression levels in recurrent and newly diagnosed gliomas after TMZ chemotherapy. A TMZ-resistant U87 cell line was constructed, and the changes in TRPC5 expression levels in TMZ-resistant and normal U87 cells were analyzed to clarify the relationship between TRPC5 and TMZ resistance in gliomas. Step 2: Analyze the effects of exogenous overexpression of TRPC5 on the proliferation, invasion ability and TMZ resistance of U87 glioma cells, and construct a nude mouse glioma brain model to verify whether the same conclusions are reached in vivo. Step 3: Analyze the relationship between TPRC5 and the mitophagy-associated protein MUL1; Step 4: Analyze the effect of exogenous overexpression of TRPC5 on the level of mitochondrial autophagy in cells.

2. The experimental method described in claim 1 for studying how TRPC5 promotes TMZ resistance in gliomas by regulating MUL1-induced mitophagy, characterized in that: Step 1 includes: (1) The expression levels of TRPC5 mRNA and protein in glioma tissues of patients with recurrent glioma after standard TMZ chemotherapy and glioma tissues of patients with newly diagnosed glioma were analyzed. (2) The expression levels of TRPC5 mRNA and protein in the constructed TMZ-resistant U87 cell line U87-MT and the normal U87-M were analyzed. Step 2 includes: (1) Construct a stable TRPC5-transfected U87 cell line U87-C5 and its control group U87-CON, and analyze the effects of TRPC5 overexpression on the proliferation, invasion ability and TMZ resistance of U87 cells; (2) Construct the U87 cell line U87-C5-LUC with stable transduction of TRPC5 and luciferase and its control group U87-CON-LUC, and establish the corresponding nude mouse orthotopic transplantation model of glioma cells to analyze the effect of TRPC5 overexpression on TMZ resistance of U87 cells in vivo. Step 3 includes: (1) By sequencing cervical cancer cells, HELA cells overexpressing TRPC5, the changes in the expression level of mitochondrial autophagy-related proteins in HELA cells overexpressing TRPC5 were analyzed. (2) The expression levels of MUL1 mRNA and protein in glioma tissues of patients with recurrent glioma after standard TMZ chemotherapy and patients with newly diagnosed glioma were analyzed, and the expression and distribution of TRPC5 and MUL1 in tissues were also analyzed. (3) Construct U87-siC5 and control group U87-siCON, analyze the MUL1 expression level in U87-siC5 and U87-siCON, and U87-C5 and U87-CON, and verify the relationship between TRPC5 and MUL1 expression level; (4) CO-immunoprecipitation was performed on TRPC5 and MUL1 in U87 cells to analyze the relationship between TRPC5 and MUL1. Step 4 includes: (1) The effect of TRPC5 overexpression on the degradation of mitochondrial-related proteins was analyzed under normal and TMZ treatment conditions. (2) The effect of TRPC5 overexpression on autophagosome formation in HELA cells under normal conditions and TMZ treatment was analyzed. (3) The effects of TRPC5 overexpression on mitochondrial membrane potential in HELA cells under normal conditions and TMZ treatment were analyzed.

3. The experimental method described in claim 2 for studying how TRPC5 promotes TMZ resistance in gliomas by regulating MUL1-induced mitophagy, characterized in that: Step 1 further includes: 1) Frozen section specimens of pathological tissue were collected from 27 glioma cases. The case selection criteria were: ① clear imaging diagnosis including CT and MRI; ② clear postoperative pathological diagnosis; ③ histological classification and pathological grading were in accordance with the "2007 WHO Classification of Tumors of the Central Nervous System", and grade III-IV tumor tissues were selected. Glioma tissues from 12 patients who had undergone one TMZ standard chemotherapy regimen were collected as the experimental group, and glioma tissues from 15 patients with newly diagnosed gliomas were collected as the control group. 2) The expression levels of TRPC5 mRNA and protein in the relapse group and the initial relapse group were analyzed by Western Blot and RT-PCR. 3) After subcutaneous tumor formation in mice, TMZ was administered and screened. Primary cell cultures were used to construct the TMZ-resistant U87-MT cell line, with the control group being U87-M. IC50 was used to verify its TMZ resistance. 4) The expression levels of TRPC5 mRNA and protein in U87-MT and U87-M were analyzed by Western Blot and RT-PCR; Step 2 also includes: 1) Construct a stable U87-C5 cell line that overexpresses exogenous TRPC5, verify TRPC5 expression level by Western Blot, and analyze whether the construction was successful; 2) The effects of TRPC5 overexpression on the proliferation, invasion ability and TMZ resistance of U87 cells were analyzed by plate colony formation assay, transwell assay and CCK8 assay of U87-C5 and its control group U87-CON. 3) Construct the U87 cell line U87-C5-LUC, which is stably transfected with TRPC5 and luciferase, and its control group U87-CON-LUC. Establish the corresponding glioma cell orthotopic transplantation model in nude mice. Seven days after the operation, the cells were administered 20 ug / kg TMZ by gavage once every 3 days for a total of 15 days. The tumor size was measured every 5 days to analyze the effect of TRPC5 overexpression on TMZ resistance in U87 cells under in vivo environment. Step 3 also includes: QIAGEN sequenced HELA cells overexpressing TRPC5 and analyzed the changes in the expression levels of mitochondrial autophagy-related proteins in HELA cells overexpressing TRPC5. 2) RT-PCR and Western Blot analysis were used to analyze the expression level of MUL1 in glioma tissues of patients with primary and recurrent gliomas; 3) RT-PCR and Western Blot analysis were used to analyze the expression level of MUL1 in U87-CON and U87-C5; 4) Construct the U87-siC5 cell line with TRPC5 knockdown, verify TRPC5 expression level by Western Blot, and analyze whether the construction was successful; 5) The expression levels of MUL1 in U87-C5, U87-siC5 and U87-CON were detected by Western Blot, and the regulatory effect of TPRC5 on the expression level of MUL1 was analyzed. 6) The expression and distribution of TRPC5 and MUL1 in glioma tissues of patients with primary and recurrent gliomas were analyzed by immunofluorescence staining and fluorescence microscopy. 7) Immunoprecipitation combined with Western blotting was used to analyze the protein binding relationship between TRPC5 and MUL1 using the autophagy-related protein LC3B. Step 4 also includes: 1) After TMZ treatment, the levels of mitochondrial-related proteins in U87-C5 and U87-CON cells were detected by Western Blot to analyze the effect of TRPC5 on mitophagy; 2) HELA-C5 and HELA-CON cells transfected with LC3-GFP and Mitochondria-RFP were observed under fluorescence electron microscopy. The cells were transiently transfected and then treated with TMZ and observed again. The effect of TRPC5 overexpression on autophagosome formation in HELA cells under normal conditions and TMZ treatment was analyzed. 3) HELA-C5 and HELA-CON cells were detected by using the JC-1 membrane potential detection kit. After transient transfection, TMZ treatment was applied and the cells were detected again to analyze the effect of TRPC5 overexpression on mitochondrial membrane potential of HELA cells under normal conditions and TMZ treatment.

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