| Objectives: Erythrocyte membrane protein band 4.1-like 2(EPB41L2)belongs to the protein 4.1 family,which is involved in cell morphology maintenance,cell adhesion and other cellular biological behaviors.In recent years,protein 4.1 family members have been found to be abnormally expressed in breast cancer,non-small cell lung cancer and other malignant tumors.However,the role and mechanism of EPB41L2 in clear cell renal cell carcinoma(ccRCC)has not been studied.Therefore,this study intends to analyze the expression of EPB41L2 in ccRCC,explore the biological function and simple mechanism of EPB41L2 in ccRCC cells,and which might provide new therapeutic targets and biomarkers to predict prognosis for the treatment of ccRCC.Methods:(1)The relative expression level of m RNA of EPB41L2 in ccRCC and paracancerous tissues was analyzed by The Cancer Genome Atlas(TCGA)database,and the relationship between EPB41L2 and overall survival time(OS)of ccRCC patients was analyzed by Kaplan-Meier Plotter.Real-time fluorescence quantitative PCR(qRT-PCR)and Western blotting were used to verify the expression of m RNA and protein of EPB41L2 in 20 pairs of clinical ccRCC and paracancerous tissues.(2)The expression of EPB41L2 was detected by immunohistochemical method(IHC)in 110 pairs of ccRCC and paracancerous tissues.The correlation between the expression and the clinicopathological features of renal cell carcinoma was analyzed.(3)qRT-PCR and Western blotting were used to detect the m RNA and protein expression of EPB41L2 in different renal cell carcinoma cells(ACHN,769-p,786-O,A498,Caki-1).The lentivirus transfection method was used to construct the renal cell lines ACHN and 769-P that stably up-regulated EPB41L2 and the renal cell lines 786-O and A498 which stably knocked down EPB41L2.(4)The transfection efficiency was verified by qRT-PCR and Western blotting.The cell proliferation,survival,migration and invasion of the stable cell line were detected by Cell Counting Kit-8(CCK-8)method,Survival test,scratch test and Transwell invasion test.(5)ccRCC cells overexpressing EPB41L2 were sequenced by single cell transcriptome based on illumina sequencing platform,and differential signal pathways were enriched and analyzed by Kyoto encylopaedia of genes and genomes(KEGG).Western blotting assay was used to detect the effect of EPB41L2 up-regulation and knockdown on the expression of MAPK signaling pathway-related molecules in renal cell carcinoma cell lines.In addition,the response experiment was conducted to verify whether U0126-Et OH and Adezmapimod could reverse the promoting effect of EPB41L2 knockdown on the migration and invasion ability of ccRCC cells.Results:(1)Through database analysis,it was found that the expression level of EPB41L2 in renal cell carcinoma was lower than that in normal paracancerous tissues.The higher the expression of EPB41L2 in renal cell carcinoma,the higher the OS(P <0.001).The expression levels of m RNA(P < 0.001)and protein of EPB41L2 in 20 pairs of clinical renal cell carcinoma tissues were lower than those in adjacent tissues.(2)The results of IHC assay showed that the expression of EPB41L2 was lower in renal cell carcinoma with higher pathological grade,and the expression level of EPB41L2 was significantly correlated with clinical T stage and pathological Fuhrman grade of renal cell carcinoma.(3)qRT-PCR and Western blotting assays showed that the expression levels of m RNA and protein in ccRCC cell lines 786-O and A498 were higher than those in ccRCC cell lines ACHN,769-P and Caki-1(P < 0.01).Stable overexpression of EPB41L2 in ccRCC cell lines ACHN-EPB41L2 and 769-P-EPB41L2 in control cells and stable knockdown of EPB41L2 in ccRCC cell lines 786-O-sh EPB41L2 and A498-sh EPB41L2 and control cells 786-O-Ctrl and A498-Ctrl were successfully constructed.(4)The results of cell function test in vitro showed that up-regulation of EPB41L2 could significantly inhibit the proliferation,survival,migration and invasion of renal cancer cell lines ACHN and 769-p,whereas knockdown EPB41L2 could significantly promote the proliferation,survival,migration and invasion of renal cancer cell lines 786-O and A498.(5)KEGG signal pathway enrichment analysis suggested that MAPK pathway was significantly enriched after EPB41L2 overexpression.Western blotting results showed that after EPB41L2 overexpression,the phosphorylation levels of MAPK pathway related molecules ERK and p38 were significantly down-regulated in ccRCC cell line 769-P.On the contrary,after EPB41L2 was knocked down,the p-ERK and p-p38 were significantly up-regulated in ccRCC cell line 786-O.Response experiments showed that the ERK/p38 MAPK inhibitor U0126-Et OH and Adezmapimod could reverse the knockdown effect of EPB41L2 on promoting migration and invasion ability of ccRCC cells.Conclusion:(1)The expression of EPB41L2 is low in renal cell carcinoma,and its expression level is related to the clinical stage and malignant grade of ccRCC.Patients with high expression of EPB41L2 have higher survival rate.(2)In vitro experiments showed that EPB41L2 could inhibit the proliferation,survival,migration and invasion of renal cancer cells.(3)The inhibition of malignant characterization of renal cancer cells by EPB41L2 may be achieved by regulating the phosphorylation level of ERK and p38 proteins in MAPK pathway.The results of this study clarify the role and mechanism of EPB41L2 in the occurrence and development of renal cell carcinoma,which will provide a new idea for the diagnosis and treatment of renal cell carcinoma. |
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