RNAi-mediated PIN1 silencing inhibits HCC tumorigenicity

Abstract

Background The phospho-Ser/Thr-Pro specific prolyl-isomerase PIN1 has been implicated in multiple aspects of cell cycle regulation. Our previous findings showed that PIN1 was over-expressed in more than 50% of hepatocellular carcinoma (HCC), and its expression correlated significantly with β-catenin and cyclin D1 expression, suggesting the involvement of PIN1 in hepatocarinogenesis. This study aims to investigate whether inhibition of PIN1 expression would block cell proliferation and inhibit the transformed phenotype of HCC cells both in vitro and in vivo. Materials and Methods To examine the oncogenic role of PIN1 over-expression in HCC, siRNA eukaryotic expression vector of PIN1 and PIN1 cDNA under the control of a human CMV promoter were constructed and stably transfected into a HCC cell line - PLC/PRF/5 and a non-tumourigenic human liver cell line - MIHA, respectively. Tumourigenicity of siRNA stable PIN1 and PIN1 transfectants were determined by soft agar assay, colony formation assay and nude mice injection. The effect of PIN1 on cell growth was examined by MTT assay and flow cytometry analysis. cDNA microarray (Affymetrix array) analysis was employed to detect the differential gene expressions between PIN1 gene-silenced and vector controlled PLC/PRF/5 cell line. Results Using RNAi technology, we found that the expression of double-stranded RNA led to efficient and specific inhibition of endogenous PIN1 expression in PLC/PRF/5 cells as determined by RT-PCR and Western blotting, resulting in deregulated cell cycle progression and decreased proliferation by 57.6 ± 4.7%. Both colony formation and growth in soft agar were inhibited by PIN1 gene silencing. Conversely, stable expression of PIN1 in MIHA promoted cell transformation. Subcutaneous injection of PIN1 transfected MIHA cells into nude mice also conferred tumour formation whereas PIN1 gene silenced PLC/PRF/5 cells did not. By cDNA microarray analysis, several oncogenes including cyclin D1 and hnRNP A2 were significantly down-regulated by PIN1 gene silencing, suggesting an oncogenic role of PIN1 over-expression in hepatocarcinogenesis. Conclusion Our study suggests that PIN1 plays a functional role in the transformed phenotype of HCC cells and contributes to HCC carcinogenesis. PIN1 may therefore represent a potential target for HCC cancer therapy.