| Objective: Renal cell carcinoma(RCC)is one of the most common malignant tumors in the urinary system.In recent years,the latest epidemiological data show that its morbidity and mortality are increasing year by year.Clear cell renal cell carcinoma(ccRCC)is the most common type of all renal cell carcinomas,accounting for more than70% of all renal cell carcinomas.The main treatment for early clear cell renal cell carcinoma is surgical treatment.Nephron-sparing surgery and partial nephrectomy are the two most used surgical methods.However,for patients with advanced and metastatic renal cell carcinoma,because the patients are not suitable for surgical treatment,a comprehensive treatment strategy based on internal medicine is often adopted.Although targeted therapy can significantly improve the prognosis of patients,there are still some patients who cannot obtain clinical effects after long-term treatment.Therefore,it is necessary to clarify the molecular mechanism of the occurrence and development of renal clear cell carcinoma in order to discover new prognostic markers and develop new targeted therapeutic drugs.Previous studies have shown that the lack of succinate dehydrogenase(SDH)function in cells can promote tumor growth.SDH is an important part of the mitochondrial respiratory chain and Krebs cycle.Many studies have shown that genetic defects of SDH are related to a variety of tumors,including familial head and neck paraganglioma,pheochromocytoma,gastrointestinal stroma Cell tumor,kidney cancer,etc.The lack of succinate dehydrogenase(SDH)function has been shown to play an important role in the regulation of tumor cell biological functions.Previous studies have found that succinate dehydrogenase subunit A(SDHA)is under-expressed in renal cell carcinoma tissues,and SDH-deficient renal cell carcinoma has the biological characteristics of high invasiveness and high degree of deterioration.However,there are few reports about the intact SDHA’s regulatory effect in renal cell carcinoma.Taking this as an opportunity,we detected the expression level of SDHA in renal cell carcinoma tissues and renal cancer cell lines,discussed the detailed mechanism of SDHA in renal cell carcinoma,and provided new ideas and potential targets for the diagnosis and treatment of renal cell carcinoma.Methods:1.Sample collection1.1 Collection of RCC samplesThe collection of renal cell carcinoma tissue samples in this study was approved and supported by the Ethics Committee of the First Affiliated Hospital of China Medical University.We collected a total of 84 pairs of kidney cancer tissues and their corresponding adjacent normal kidney tissues.The adjacent normal kidney tissues are more than 2 cm away from the edge of the tumor.After the specimens are cut,the tissues are labeled and stored in a-80 ℃ refrigerator for subsequent follow-up experimental use.All specimens were taken from patients with renal cell carcinoma who underwent radical nephrectomy at the Department of Urology,First Affiliated Hospital of China Medical University from September 2015 to December 2017.The patients were diagnosed by abdominal enhanced CT,abdominal magnetic resonance and urinary tract ultrasound examinations before the operation,and all the patients signed the scientific research informed consent form before the operation.All patients did not receive any treatment before surgery,and all specimens were diagnosed as clear cell renal cell carcinoma after surgery.1.2 Cell cultureIn this study,OSRC,769-P,ACHN renal carcinoma cell lines and human renal proximal tubular epithelial cell lines(HK-2)were cultured.The cell lines were all from the Institute of Urology,The First Affiliated Hospital of China Medical University.2.Analysis process2.1 The expression level of SDHA in renal cell carcinoma tissues and renal cancer cell lines was detectedRetrieve and extract renal cancer related data through the public online database of TCGA,and analyze the m RNA expression level of SDHA in renal cell carcinoma.Extract total RNA and protein from kidney cancer tissues and normal kidney tissues adjacent to the cancer,and detect the expression levels of SDHA m RNA and protein in the tissues using q PCR and western blot techniques.Culture each renal cancer cell line and immortalized normal renal tubular epithelial cell line,extract their total RNA and protein,and detect the difference in expression of SDHA protein and m RNA.2.2 The clinical significance of SDHA protein in RCC was analyzedBy searching TCGA database,the influence of SDHA expression difference on the prognosis of renal cell carcinoma patients was analyzed.The in-situ expression of SDHA in renal clear cell carcinoma tissues was detected by immunohistochemistry,and the correlation between SDHA protein expression level and pathological grade and stage of renal cell carcinoma was analyzed.2.3 Detection of the SDHA’s effect on biological behavior in RCC cell linesThe SDHA overexpression plasmid was constructed,transfected and screened for cell lines stably overexpressing SDHA,the changes in cell proliferation were detected by CCK8 cell proliferation experiments and Brd U experiments,and the changes in cell invasion and migration were detected by Transwell experiments and scratch healing experiments.2.4 Verification of the ability of SDHA to regulate the proliferation,invasion and migration of renal cancer cells by regulating the Wnt/β-catenin signaling pathwayThe influence of overexpressed SDHA on the expression levels of key proteins in the Wnt /β-catenin signaling pathway in renal carcinoma cells was detected by Western blot assay.The regulation effect of SDHA on Wnt /β-catenin signaling pathway in renal carcinoma cells was demonstrated by Western blot rescue assay.The proliferation,Transwell and scratch healing rescue experiments were designed to further verify the ability of SDHA to regulate the proliferation,invasion and migration of renal cancer cells by regulating the Wnt/β-catenin signaling pathway.2.5 To further verify the biological effects of SDHA on kidney cancer cells in vivo through nude mouse transplantation tumor experimentCarry out nude mouse transplantation tumor experiment in nude mice,inject 786-O cells overexpressing SDHA subcutaneously into nude mice,observe the growth of transplanted tumors,and compare the transplanted tumors with the control group for statistical analysis.The total protein of transplanted tumor tissue was extracted,and the protein expression levels of PCNA,E-cadherin,N-cadherin,and MMP-9 in the transplanted tumor tissue were detected by western blot to further verify the effects of SDHA on the proliferation,migration and invasion of renal cancer cells in vivo.Results:1.Differential expression analysis of SDHA in renal cell carcinoma tissues and cell linesThe m RNA and protein expression levels of SDHA in renal carcinoma tissues and adjacent tissues were detected by real-time PCR and Western-blot analysis.Meanwhile,the expression levels of SDHA in renal cell lines and renal tubular epithelial cell lines were also detected.The results showed that the m RNA and protein levels of SDHA were lower in renal carcinoma tissues and renal tubular epithelial cells than in paracancer tissues and normal renal tubular epithelial cells,with statistically significant differences(P<0.05).Immunohistochemical results also indicated that SDHA expression was decreased in renal cell carcinoma tissues compared with the normal adjacent tissues,and the difference was statistically significant(P<0.05).2.The expression difference of SDHA in renal clear cell carcinoma and its influence on the prognosis of patientsAccording to the data of the expression level of SDHA gene in renal clear cell carcinoma tissues extracted from the TCGA database,we found that compared with normal renal tissue,SDHA m RNA showed a lower expression level in renal clear cell carcinoma tissue;the results of survival analysis showed that the survival time of renal cancer patients with low SDHA expression group was lower than that of patients with high SDHA expression group.The results of immunohistochemical experiments showed that SDHA protein was under-expressed in clear cell renal cell carcinoma tissues compared with normal kidney tissues adjacent to cancer.3.SDHA can inhibit the proliferation,migration and invasion of kidney cancer cellsWe constructed an SDHA overexpression plasmid,transfected endogenous low-expressing SDHA renal cancer cell lines 786-O and ACHN,and used G418 to screen cell clones stably expressing SDHA.The results of CCK8 cell proliferation experiment and Brd U cell proliferation experiment showed that after transfection of SDHA overexpression plasmid,the proliferation ability of renal cancer cells was significantly reduced compared with control cells.The results of Transwell experiment and scratch healing experiment showed that after transfection with SDHA overexpression plasmid,the cell migration and invasion ability of 786-O and ACHN cells were also significantly inhibited compared with control cells.4.The effect of overexpression of SDHA on Wnt/β-catenin signaling pathway and phenotypic rescue experimentOverexpression of SDHA can down-regulate the protein expression levels of key proteins p-GSK-3β,β-catenin,and c-Myc in the wnt/β-catenin signaling pathway.Western blot rescue experiment results show that Li Ci reagent(Wnt/β-catenin signal Pathway activator)can antagonize the inhibitory effect of SDHA on Wnt/β-catenin signaling pathway.The results of phenotypic rescue experiments further prove that SDHA inhibits the proliferation,migration and invasion of renal cancer cells by inhibiting the Wnt/β-catenin signaling pathway..5.The effect of SDHA on the proliferation of renal carcinoma cells in vivo was detected by nude mouse transplantation tumor experimentThe results of nude mouse transplantation tumor experiments showed that the transplanted tumors of SDHA overexpression group mice grew slower and smaller than those of the control group.Western blot detection of protein expression in transplanted tumor tissues showed that transplanted tumors in SDHA overexpression group had higher E-cadherin protein expression than negative control group mouse tumors,while N-cadherin,MMP-9 and PCNA protein expression level is lower.6.Statistical analysisThe statistical analysis in this study was carried out using SPSS 19.0 software.According to the design of the experiment and the data type of the experimental data,statistical analysis methods such as t-test,paired sample t-test,analysis of variance,chi-square test,etc.were reasonably selected to perform statistics on the experimental data.analysis.P<0.05 was judged to be statistically significant.Conclusion: 1.Bioinformatics analysis and immunohistochemical results show that SDHA is lower expressed in renal clear cell carcinoma.Survival analysis results show that the survival time of patients with renal clear cell carcinoma in the SDHA low expression group is lower than that of the SDHA high expression group.2.Overexpression of SDHA at the cellular level can significantly inhibit the proliferation,migration,and invasion of renal cancer cells.3.SDHA inhibits the proliferation,migration and invasion of renal cancer cells by inhibiting the Wnt/β-catenin signaling pathway. |
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