The Effect And Mechanism Of TMEM97 In Inhibiting The Progression Of Renal Cancer

Objective: Renal cancer is one of the most common malignant tumors in the urinary system.On account of being not sensitive to radiotherapy and chemotherapy,surgery is still the main treatment method.At present,the prognosis of early stage renal cancer is significantly good after radical resections.For the treatment of advanced renal cancer,especially cases with distant metastasis,is still limited.Therefore,it is urgent to clarify the pathogenesis and find effective treatment methods for renal cancer,especially the advanced renal cancer.The sigma receptor has been divided into two subtypes,sigma-1 and sigma-2.The sigma-1 receptor plays an important role in regulating cell survival,cell differentiation,and tumorigenesis and progression.The research of sigma-2 receptor has been limited because the gene encoding it has not been discovered.Therefore,the study of sigma-2 receptor generally relies on its agonist,inhibitor or radionuclide tracers.Until 2017,American scientists confirmed that the sigma-2 receptor was encoded by TMEM97.A large number of studies have shown that TMEM97 is involved in the development of a variety of tumors,but there are few reports in renal cancer.Therefore,we started with this point to evaluate the expression of TMEM97 in clear cell renal cell carcinoma(ccRCC)tissues and its relationship with the prognosis through database analysis and tissue sample detection.The sigma-2 receptor specific agonists and inhibitors,knock down plasmid and overexpression lentiviruses were selected to knock down or over-express TMEM97.Then in vitro and in vivo experiments were performed to verify the biological roles of TMEM97 in renal cancer.Finally,stable transfection of the human renal cancer cell line OSRC-2 with TMEM97 was performed transcriptome sequencing to screen out the key difference factors and signal pathways.All mentioned above can provide theoretical basics for revealing the biological mechanism of renal cancer progression.Methods: 1.The expression of TMEM97 in renal cancerous&normal tissues.Renal cancerous&adjacent normal tissue specimens were selected from the specimen library randomly to extract total proteins or make paraffin sections,then western blot or immunohistochemistry assay was used to clarify the expression of TMEM97.Analysis of ONCOMINE database,THE HUMAN PROTEIN ALTAS database and other online public databases demonstrated the impact of TMEM97 on the prognosis of renal cancer.Bioinformatics analysis(GESA)was used to find out signal pathways which are significantly related to TMEM97 in renal cancer.2.The effect of sigma-2 receptor specific agonist or inhibitor on the biological functions of renal cancer cells.Sigma-2 receptor specific agonist PB28 and specific inhibitor sigma-2 receptor antagonist 1 were selected to culture with renal cancer cells at various concentrations.Then clone formation experiment,transwell experiment and scratch experiment were performed to verify the effect of PB28 or sigma-2 receptor antagonist 1 on renal cancer cells(proliferation,invasion and migration).3.siRNA interference technology,plasmid transfection and lentiviral transfection technology were used to verify the biological function of TMEM97 in renal cancer.Renal cancer cells were transfected with the si-RNA,sh-RNA and overexpressed-lentivirus of TMEM97,and the transfection efficiency of TMEM97 was confirmed by western blot experiment.RTCA(Real Time Cellular Analysis),clone formation experiment,transwell experiment,scratch experiment were selected to detect the proliferation,invasion and migration ability of renal cancer cells after transfection.4.In vivo experiments to verify the biological function of TMEM97 in renal cancer.Plasmid or lentiviral were used to construct stable knockout or overexpressing TMEM97 renal cancer cell lines.4-6 weeks Balb/c nude mice were selected to inject the renal cancer cells mentioned above into the right axilla.Then we observed the growth of transplanted tumors.After 3-4 weeks,all mice were sacrificed,we extracted and weighed the tumors of different groups.The subcutaneous xenograft tumors were taken for immunohistochemical experiment to detect the expression of Ki-67 and TMEM97.In addition,we observed the lung metastasis by injecting renal cancer cells into the tail vein of nude mice,the renal cancer cells overexpressing TMEM97 and the control cells were injected into the tail vein,and the mice were sacrificed after 60 days of observation.Then lung tissues of the nude mice were taken to observe the number of metastasis.5.The effect of knockdown or overexpression of TMEM97 on the expression of EMT-related protein and PI3K-AKT-mTOR pathway related proteins.Western blot experiment was used to detect the expression of EMT-related proteins after the treatment of PB28 or sigma-2 receptor antagonist 1 and TMEM97’s knockdown or overexpression.PI3K-AKT-mTOR pathway related proteins were also needed the detections above.6.Transcriptome changes after TMEM97’s overexpression.Lentiviral transfection technology was used to construct OSRC-2 cells stably overexpressing TMEM97,then the experimental group and the control group were subjected to transcriptomics sequencing.To identify the differential genes and signal pathways after TMEM97’s overexpression.Results: 1.Western blot results indicated that the expression of TMEM97 showed no significant differences in the tumorous&normal tissues(63 pairs).However,TMEM97 presented a higher expression on the clear cell renal cell carcinoma with earlier T stage.Immunohistochemical results showed that the higher the Fuhrman grade,the lower the expression of TMEM97.By analyzing the online public databases such as ONCOMINE and THE HUMAN PROTEIN ALTAS,we found that the lower the expression of TMEM97,the worse the prognosis of clear cell renal cell carcinoma.Moreover,bioinformatics analysis results suggested that TMEM97 is positively correlated with PI3K-AKT-mTOR pathway in clear cell renal cell carcinoma.2.Western blot results suggested that TMEM97 expressed in all the four renal cancer cell lines 786-O,ACHN,Caki-1 and OSRC-2.TMEM97 was expressed at a higher level in 786-O and ACHN cells,and expressed lower in Caki-1 and OSRC-2 cells.The results of clone formation experiment indicated that the sigma-2 receptor agonist PB28 inhibited the proliferation of renal cancer cells,while the sigma-2 receptor inhibitor sigma-2 receptor antagonist 1 induced it.The results of Transwell experiment and scratch experiment showed that the sigma-2 receptor agonist PB28 inhibited the invasive and migratory abilities of renal cancer cells;while the sigma-2receptor inhibitor sigma-2 receptor antagonist 1 promoted the invasion and migration of renal cancer cells.3.Small interference RNA,sh-RNA or overexpressed lentiviral of TMEM97 were used to tranfect renal cancer cells.After knockdown of TMEM97,the proliferative,invasive and migratory abilities of renal cancer cells was significantly enhanced.However,overexpression of TMEM97 could suppress the proliferation,invasion and migration of renal cancer cells.In mice models,TMEM97’s knockdown increased the ability of subcutaneous tumor formation in nude mice;TMEM97’s overexpression inhibited the ability of it.The Ki-67 staining of subcutaneous tumors in the TMEM97-knockdown group was stronger than that of the control group;and the Ki-67 staining of subcutaneous tumors in the TMEM97-overexpression group was weaker than that of the control group.And overexpression of TMEM97 resulted in a smaller number of lung metastasis in nude mice.4.Western blot results suggested that the expression of EMT-related proteins(N-cadherin and Vimentin)increased after sigma-2 receptor antagonist 1 added to renal cancer cells or TMEM97 was knocked down;the expression of EMT-related proteins(N-cadherin and Vimentin)decreased after PB28 added to renal cancer cells or TMEM97 was overexpressed.5.PB28 or TMEM97’s overexpression inhibited phosphorylation of constituents of the PI3K-AKT-mTOR signaling pathway;while TMEM97’s knockdown activated the PI3K-AKT-mTOR signaling pathway.6.Renal cancer cells stably overexpressing TMEM97 by lentiviral transfection.This experimental group and the control group were tested by transcriptomics sequencing.KEGG analysis found that PI3K-AKT-mTOR pathway was inhibited after the overexpression of TMEM97.GO analysis suggested that overexpression of TMEM97 mainly affects regulation of lipid metabolic process,cell migration,lipid localization,membrane region,sterol binding,growth factor binding,cholesterol transporter activity.The obvious difference in gene changes is SCD.By bioinformatics and database analysis,thirteen transcription factors were screened out to be related to SCD.According to TCGA and other databases,ETS1,TFAP2 A,NR3C1 were the most related to SCD.Western blot results confirmed that the expression of SCD and ETS1 decreased significantly after TMEM97 overexpressed.Conclusion: TMEM97 was highly expressed in early T stage clear cell renal cell carcinoma.Sigma-2 receptor agonist PB28 or overexpression of TMEM97 inhibited the proliferation,invasion and migration of renal cancer cells;sigma-2 receptor inhibitor sigma-2 receptor antagonist 1 or knockdown of TMEM97 increased the proliferative,invasive and migratory abilities of renal cancer cells.Sigma-2 receptor agonist PB28 or overexpression of TMEM97 suppressed the PI3K-AKT-mTOR pathway,while knockdown of TMEM97 activated PI3K-AKT-mTOR pathway.In vitro and in vivo results proved that TMEM97 inhibited the progression of renal cancer.Transcriptome sequencing results showed that the lipid metabolism pathway was mainly affected by overexpression of TMEM97,the expression of SCD and ETS1 were significantly reduced.The inhibitory effect of TMEM97 in renal cancer is achieved by inhibiting the expression of SCD and EST1,thereby inhibiting the lipid metabolism of renal cancer cells.