2015; 34:856C80. It was also accountable for RPN2-stimulated elevated expression of MMP-9 and for invading HCC cells. It can be concluded that over-expression of RPN2 in HCC aggravated SR 11302 the malignant progression into cancerous cells. This research provided new evidences that RPN2 could facilitate tumor invasion by increasing the expression of MMP-9 in HCC cells. < 0.05, ***< 0.001 vs control group. RPN2 mediates HCC cell proliferation To confirm that RPN2 regulates the proliferation rate of HCC cells, the overexpression of RPN2 in Huh-7 and HepG2 cells was exhibited with WB and qPCR (Physique 2AC2D). Further, MTT assay (Physique 2E and ?and2F)2F) revealed that multiplication of Huh-7 and HepG2 cells, 12C72 h post-transfection, was greatly increased when they were transfected with RPN2-expressing adenovirus (AD-RPN2). The RPN2 overexpression caused a noticeable increase in colony numbers, evaluated by the soft agar colony formation assay, while transfection with the control (AD-NC) did not affect colony numbers of HepG2 cells (Physique 2G and ?and2H).2H). To determine whether RPN2 overexpression promotes tumor phenotypes in normal hepatocytes, we performed RPN2 overexpression in normal hepatocytes (NHCs). However, there was no significant different in cell proliferation between RPN-overexpressing NHCs and control NHCs, indicating that RPN2 only exert its function in malignant cells (Physique 2I and ?and2J2J). Open in a separate windows Physique 2 RPN2 overexpression promotes proliferation of Huh-7 and HepG2 cells. The cell lines were transfected with AD-RPN2 and AD-NC (control). Western blotting (A, B) and qPCR (C, D) were conducted to confirm RPN2 overexpression in both the cell lines. (E, F) Multiplication of Huh-7 and HepG2 cells was measured at time points of 12, 24, 36, 48, 60, and 72 h after transfection by the MTT assay. (G, H) Soft agar colony formation assay of the Huh-7 and HepG2 cells expressing RPN2 and controls. (I) The NHC were transfected with AD-RPN2 and AD-NC (control). WB was conducted to confirm RPN2 overexpression in NHC. (J) Multiplication of NHC was measured at time points of 12, 24, 36, 48, 60, and 72 h after transfection by the MTT assay. The band SR 11302 of target protein was normalized to the density of action. The quantification was performed independently in a Slc4a1 single band. The experiments were performed three times. Data are recorded as mean SD. **<0.01 vs control group. Previous research had reported that invasion and migration of HCC cells is usually a major cause of mortality during HCC development and progression [9]. To determine whether RPN2 influences the invasion and migration of HCC cells, transwell migration and wound-healing assays were carried out after transfection of HepG2 and Huh-7 cells with the RPN2-expressing adenovirus (AD-RPN2) and control (AD-NC). In the wound healing assay, overexpression of RPN2 promoted migration of Huh-7 and HepG2 cells towards gap created by scratching of the cell monolayer (Physique 3A and ?and3B).3B). Overexpression of RPN2 clearly increased migration of HCC cells (Physique 3C and ?and3D),3D), especially in HepG2 cells, which is consistent with data from the wound healing assay. Moreover, we examined the effect of RPN2 overexpression on EMT; the ectopic expression RPN2 led to a decrease in E-cadherin and an increase in N-cadherin expression in both the cell lines, as determined by WB (Physique 3E and ?and3F).3F). These data suggested that RPN2 overexpression facilitates the metastatic and invasive attribute of HCC cells < 0.05, **< 0.01 vs control group. Next, the effect of RPN2 silencing SR 11302 in HepG2 and Huh-7 cells was determined by transfecting the cell lines with vector.