Expression Of Capn4and Its Role In Invasion And Metastasis Of Clear Cell Renal Cell Carcinoma

Renal cell carcinoma (RCC) is the second most prevalent urological cancer，with70%of patients are clear cell renal cell carcinoma（ccRCC）.Metastasis is a major cause ofdeath among RCC patients, but the mechanism is still unclear. The μ-form of the calpainfamily are widely expressed in mammals, which is heterodimers with a distinct catalyticsubunit and a common regulatory subunit (Calpain small subunit-1, Capn4). Capn4havebeen implicated in a variety of biological functions, including signal transduction, cellproliferation and cell differentiation. Furthermore, Capn4has been shown high expressionin some carcinoma, such as hepatocellular carcinoma, intrahepatic cholangiocarcinoma andbreast carcinoma, and to correlate with the metastasis/invasion of them. The relationshipbetween Capn4and RCC，however，have not been investigated at home and abroad. Wetherefore examined the expression of Capn4in ccRCC tissues and cell lines, and analyzedthe role and mechanism of Capn4in ccRCC. Then, we evaluated its correlation withsurvival potential of ccRCC patients.PartⅠ The clinical significance of Capn4expression in clear cell renalcell carcinomaObjective To investigate the expression of Capn4in ccRCC and matched adjacentnon-tumor tissues. Analyze the relationship among Capn4and the clinicopathologicfeatures of human clear cell renal cell carcinoma. To explore whether Capn4play animportant role in the outcome of renal cell carcinoma.Methods Capn4expression was measured in75pairs of ccRCC and matched adjacentnon-tumor tissues by qRT–PCR, western blotting and immunohistochemistry. The prognostic role of Capn4in ccRCC was evaluated by Kaplan-Meier（K-M）and Coxregression analyses.Results Capn4expression in the ccRCC tissues was significantly higher than in theadjacent non-tumor tissues; the expressions of Capn4were closely related with theFuhrman grade, T stage, the size of tumor of clear cell renal cell carcinoma. Survivalanalysis (Kaplan-Meier test) showed that Capn4expression was also correlated withprognosis in ccRCC.Conclusion The expression of Capn4are over expressed in clear cell renal cellcarcinoma. Capn4likely plays an important role in clear cell renal cell carcinoma.PartⅡ The role and mechanism of Capn4in invasion andmetastasis of clear cell renal cell carcinomaObjective Detect the expression of Capn4in ccRCC, and assessed cell proliferation,motility, adhesion and migration ability, in order to explore the possible mechanism, andfind a new renal cell carcinoma prognostic marker and therapeutic target.Methods1. qRT-PCR analysis showed expression of Capn4in all the four ccRCC cell lines(786-0、ACHN、Caki-1、Caki-2).2. We transfected786-0cells with small RNA intefrence plasmids to disrupt Capn4expression, which was identified by qRT-PCR analysis.3. Using CCK-8to detect the influence of Capn4down-regulation on786-0cellsproliferation.4. Wound healing assay were performed to detect the role of Capn4in chemotaxisability and cell movement.5. Adhesion assay was used to examine the effect of Capn4expression on the abilitycell adhesion of ccRCC cells to the extracellular matrix.6. Tanswell invasion assay was used to examine the effect of Capn4downregulationon786-0cells invasion ability. 7. Western blotting analysis was used to detect the expression of ERK1/2、p-ERK1/2、MMP-2and MMP-9after knockdown Capn4, further identified the molecularmechanism of Capn4in invasion and metastasis.Results Capn4mRNA was detected in four ccRCC cell lines by qRT-PCR analysis.The human RCC cell lines,786-0, expressed higher level of Capn4than the others. Aftertransfected Capn4cells by small RNA technology, the expression of Capn4significantlydecreased. Capn4-reduced786-0cells showed a significant decreased chemotaxis, invasionand adhesion ability Compared with control cell. Western blotting assay demonstrated thatknockdown of Capn4reduced MMP2, MMP9, and phosphorylation of ERK which is animportant factor for cell signaling pathways.Conclusion Down-regulation of Capn4impaired ccRCC cells chemotaxis,proliferation, migration, invasion ability, demonstrate that Capn4is involved in theregulation of ccRCC cells chemotaxis and metastasis. Metal matrix protein can regulateccRCC cells migration and invasion, playing a very important role in ccRCC cells invasion.Capn4stimulation promotes786-0ccRCC cell invasion and that the effect is partly due toERK1/2-dependent up-regulation of MMP2and MMP9. Capn4may serves as a novel andpromising metastasis therapeutic target for ccRCC.