Effect of PPM1H on malignant phenotype of human pancreatic cancer cells.

Abstract

The objective of this study was to investigate the effect of silencing gene protein phosphatase 1H (PPM1H) on malignant phenotype of human pancreatic cancer cell line BxPC-3. In order to explore the function of PPM1H in pancreatic cancer cells, real-time PCR and western blotting were used to detect the expression of PPM1H in different pancreatic cancer cell lines. Human pancreatic cancer cell line BxPC-3 was treated with 10ng/ml TGF-β1 and 200ng/ml BMP2 for 72h, respectively, and the mRNA and protein expression levels of PPM1H and EMT-related markers (E-cadherin, vimentin) were detected by real-time PCR and western blotting, respectively. Using exogenous RNA interference technology to silence the PPM1H gene, the expression of PPM1H and EMT-related markers at mRNA and protein levels were detected by real-time PCR and western blotting. The cell migration and invasion were measured using Transwell assays. Finally, cell counting kit-8 (CCK-8) and flow cytometry were used to determine the effect of PPM1H on cell proliferation and apoptosis of BxPC-3 cells. The expression levels of PPM1H in all of the examined pancreatic cancer cell lines (BxPC-3, MIA-PACA2, PANC-1, SW1990, PANC-03.27) were lower than that of normal pancreatic ductal epithelial cells (HPDE6-C7) at both mRNA and protein levels. Both TGF-β1 and BMP2 treatment induced EMT and downregulation of PPM1H in BxPC-3 cells. By using RNA interference to transiently knock down PPM1H expression in BxPC-3 cells, we found that the expression of E-cadherin was downregulated while vimentin was up-regulated. The data suggested that silencing PPM1H gene can induce EMT in BxPC-3 cells. In addition, Transwell migration assays showed that silencing PPM1H gene can promote the invasion and metastasis of BxPC-3 cells. Cell proliferation and apotosis detection demonstrated that silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. In conclusion, PPM1H is aberrantly expressed in human pancreatic cancer cell lines and can be downregulated when EMT is induced by cytokine stimulation. Silencing PPM1H gene can induce EMT in BxPC-3 cells, and promote the invasion and metastasis of BxPC-3 cells. Moreover, silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. PPM1H may be a new tumor-suppressor factor for pancreatic cancer and provides new insight into molecular targets for gene therapy of pancreatic cancer.