Effects Of Knockdown Of LMNA On Clear Cell Renal Cell Carcinoma Cells And Its Molecular Mechanism Study

Objectives:Kidney cancer is a common urinary system tumor.The most common histologic type of kidney cancer is clear cell renal cell carcinoma(ccRCC).Our data showed that the copy number variation of LMNA family genes in ccRCC can reach 13.8%.Its role in the development of ccRCC and if LMNA interacts with other tumor suppression genes,such as PBRM1,are still unclear.We aim to investigate the role of LMNA(encoding lamin A/C)in ccRCC cells and its molecular mechanisms,in an attempt to provide valuable information for better diagnosis and treatment for ccRCC patients.Methods:Renal carcinoma cells 786-O and SN12C were cultured in RPMI 1640 medium.(1)SiRNA was transfected into 786-O/SN12C cells with siRNA-mate to knock down the expression level of lamin A/C;The experiment was divided into three groups:siControl group,siLMNA-1 and siLMNA-2 treatment groups;(2)The changes of protein levels after LMNA knockdown were examined by Western Blot;(3)The changes of mRNA level after LMNA knockdown were examined by qRT-PCR;(4)CCK8 method was used to examine the effect of LMNA knockdown on cell proliferation;(5)Cell migration assays were used to examine the effect of LMNA knockdown on cell migration ability;(6)The gene expression profiles of 786-O with/without LMNA knockdown were analyzed by RNA-seq;(7)The effects of LMNA knockdown on Wnt/β-catenin and AKT signaling pathways were examined by Western Blot.(8)Western Blot was used to examine whether the tumor suppressor gene PBRM1 regulates the expression of lamin A/C in ccRCC cells;(9)The luciferase assay was used to further examine whether PBRM1 was a target gene of E2F1 in ccRCC cells.Results:(1)Knockdown of LMNA had no significant effect on the proliferation ability of 786-O and SN12C cells;(2)Knockdown of LMNA inhibited the migration of 786-O and SN12C cells;(3)LMNA knockdown decreased the protein levels of MMP2 and MMP9 in both cell lines;(4)Knockdown of LMNA had no significant effect on the protein level of β-catenin,but reduced the protein levels of AKT and p-AKT in ccRCC cells;(5)RNA-seq analysis identified 89 differentially regulated genes,some genes were downregulated(NUDT16,MSC-AS1,GGT2,SPIN1,ADORA2A)while some were upregulated(UTP14C,FKTN,MIEF1);and these lamin A/C regulated genes were involved in ribosome biogenesis,taurine and hypotaurine metabolism,ribosome,protein output,and mannose O-glycan biosynthesis in 786-O cells;(6)The protein level of lamin A/C was increased after PBRM1 knockdown;(7)PBRM1 is a target gene of E2Fl,and E2F1 can bind to the upstream response element of PBRM1 to inhibit its expression.Conclusions:(1)Knockdown of LMNA did not significantly affect the proliferation ability of 786-O/SN12C cells,but significantly inhibited its migration ability;(2)LMNA probably inhibited cell migration via Wnt/β-catenin and P13K/AKT signaling pathway;(3)PBRM1 inhibits the expression of lamin A/C at protein level;E2F1 can bind to the response element upstream of PBRM1 and inhibits the expression of PBRM1.