Regulatory Role Of TRPM7 In Renal Cell Carcinoma And Its Mechanisms Research

?Background?Renal cell carcinoma(RCC)is the most common adult kidney cancer,with a 5-year disease-specific survival rate of 50–69%.However,a previous study has demonstrated that the median survival of patients with metastatic RCC is ~13 months.Transient receptor potential melastatin 7(TRPM7)is a ubiquitously expressed ion channel with intrinsic kinase activity.Review studies have indicated the important roles of TRPM7 channel-kinase in fundamental cellular processes,physiological responses,and embryonic development.Accumulating evidence has shown that TRPM7 is aberrantly expressed and/or activated in human diseases including cancer,and plays a variety of functional roles in cancer cells including survival,cell cycle progression,proliferation,growth,migration,invasion,and epithelial-mesenchymal transition(EMT).In recent studies,TRPM7 was identified as a regulators of PI3K/AKT,MAPK,ERK1/2 pathways,regulating several oncogenes regulators and cell cycle genes and its function was potentially associated with several human cancers,such as human colon cancer,ovarian cancer,prostate cancer,breast cancer,bladder cancer,pancreatic cancer,nasopharyngeal carcinoma,and esophageal squamous cell carcinoma.Up to now,no association of TRPM7 and RCC was reported.Here,we aimed to investigate the regulatory role and mechanisms of TRPM7 in RCC and discuss the signal pathway of TRPM7 in RCC,using a series of in vitro assays and in vivo mouse model.?Purpose?To investigate the regulatory role and mechanisms of TRPM7 in RCC and discuss the signal pathway of TRPM7 in RCC.?Methods? 1)The expression of TRPM7 in HK-2 cells and RCC cells in vitro was detected byWestern blot and q-PCR,and the hypothesis was put forward.2)Functional research:(1).TRPM7 was silenced or overexpressed in human RCCcells,and the expression of TRPM7 in RCC cells and its effect on the viabilityand proliferation of RCC cells were observed in vitro.(2).To investigate theimpact of TRPM7 on viability and proliferation of RCC cells in vivo,OSRC-2shTRPM7-1/scramble cells were injected subcutaneously into the dorsal flank ofeach nude mice,respectively,to establish xenograft tumor model.3)Mechanism research: To explore the relationship between TRPM7,PI3K/AKTsignaling pathway and FOXO1 in the pathogenesis and progression of RCC.TRPM7 and/or FOXO1 were silenced or overexpressed in RCC cells,and AKTinhibitor was added,then the expression of p-akt,akt,p-foxo1 and foxo1 in RCCcells after transfection were tested,and the effects of the activity andproliferation of RCC cells were tested,respectively.?Results? 1)TRPM7 was overexpressed in RCC cellsThe TRPM7 expression level of the four RCC cells was significantly higher thanthat of the normal renal epithelial HK-2 cells.2)TRPM7 promotes cell proliferation and clonogenicity of RCC cell lines invitro.(1).A subsequent MTS analysis and colony-forming assay showed that bothACHN and OSRC-2 cells transfected with TRPM7 shRNA displayed asubstantial decrease in cell proliferation and clone capacity compared withcontrol cells,while overexpression of TRPM7 in 786-O can increase theability of cell proliferation and clone formation.(2).To further confirm the effect of TRPM7 required for RCC tumor growth invivo,xenograft tumor model assays were conducted by injectingOSRC-2-shTRPM7/scramble cells into the dorsal flank of nude micesubcutaneously.The OSRC-2-shTRPM7 cells grew at a much slower ratethan OSRC-2/scramble cells.Furthermore,the average weight of tumor wassignificantly lower in the TRPM7 depletion group compared with thescramble group.3)TRPM7 suppresses FOXO1 transcript activity via activating AKT signalingpathways.The expression level of phosphor-FOXO1 was significantly decreased inTRPM7-silenced cells.Furthermore,immunofluorescence analysis showed thecellular localization of FOXO1 from the cytoplasm into the nucleus afterknocking down the TRPM7,which also suggested that silencing the TRPM7 canactivate the FOXO1 transcript activity.AKT kinases is known to have key rolesin phosphorylating and repressing FOXO1 transcriptional activity.As predicted,phospho-AKT(p-AKT)levels were decreased by TRPM7 silencing,suggestingthat TRPM7 suppresses FOXO1 activity via activating the AKT signalingpathway.To confirm these results,we overexpressed the TRPM7 in RCC cellsand treated with an AKT inhibitor(LY294002).LY294002 led a significantlyreduced expression levels of p-AKT and p-FOXO1 but increasing expressionlevels of FOXO1 in overexpression of TRPM7 RCC cells.Next,we alsoexamined the growth and clonogenicity ability of TRPM7-silenced RCC cellsusing LY294002.MTS and colony formation assays showed that the growth ofTRPM7-overexpressed cells was significantly compromised by treatment withthe AKT inhibitors compared to control cells.Moreover,colony formation andMTT assays showed that knocking down of FOXO1 blocked the growth rate ofTRPM7-silenced RCC cells,suggesting that FOXO1 plays an importantdownstream involved the regulation of TRPM7 on proliferation in RCC cells.Taken together,these data indicate that TRPM7 may promote proliferationpartly via activation of the AKT to suppress the FOXO1 activity.?Conclusions?Our results suggest that TRPM7 plays an important regulatory role in the genesis and progression of RCC.In addition,the function of TRPM7 in cell proliferation is via PI3K/AKT pathways.These findings suggest that TRPM7 has role in the development and progression of human RCC,which render TRPM7 may serve as a novel therapeutic target in RCC.