Human endogenous retrovirus subfamily H long terminal repeat associating protein 2 expression and its contribution to the regulation of biological function of human clear cell renal cell carcinoma cell line

Abstract

Objective To investigate the clinical significance and biological function of human endogenous retrovirus subfamily H long terminal repeat associating protein 2 (HHLA2) in human clear cell renal cell carcinoma (ccRCC). Methods Down-regulation of HHLA2 was performed by using RNA interference (RNAi) method to investigate the role of HHLA2 in regulation of biological behaviors in human clear cell renal cell carcinoma cell lines 786-O and ACHN. The cell counting kit-8 (CCK-8) assay, wound healing assay, and transwell invasion assay were used to examine the cellular function after HHLA2 knockdown of these cells. We also identified the differentially expressed genes upon HHLA2 knockdown in ccRCC cell lines by using gene microarray analysis. Results To investigate the functions of HHLA2 in human ccRCC, we successfully constructed HHLA2 knockdown expression in human ccRCC cell lines by using the RNAi method. CCK-8 assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell proliferation. The wound healing assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell migration (LV-HHLA2-sh1: 786-O: F=99.340, P<0.01, ACHN: F=113.500, P<0.01; LV-HHLA2-sh2: 786-O: F=35.320, P<0.01, ACHN: F=26.470, P<0.01). Transwell invasion assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell invasion. The cell cycle arrest at G1 phase was induced and the expressions of Cyclin D1, c-Myc and Cyclin E1 were decreased. In addition, according to the microarray data, the expressions of epithelia-to-mesenchymal transition markers, such as E-cadherin, N-cadherin and Vimentin, were significantly changed after knockdown of HHLA2 expression. After knockdown of HHLA2 in 786-O and ACHN, the expression of E-cadherin was significantly increased, meanwhile the expression of N-cadherin and Vimentin were significantly decreased. Conclusion Our findings indicated that HHLA2 was involved in the progression of human ccRCC and could be used as an important prognostic predictor for this malignancy. Key words: Human endogenous retrovirus subfamily H long terminal repeat associating protein 2; Human clear cell renal cell carcinoma; RNA interference