MiR-29c has been reported to be downregulated in various types of tumors [16–19], including gastric carcinoma [6, 20]. In nasopharyngeal carcinomas, miR-29c reportedly regulates the expressions of extracellular matrix proteins , implicating the involvement of miR-29c downregulation in tumor invasiveness. In esophageal squamous cell carcinoma, miR-29c has been shown to induce cell cycle arrest by regulating the expression of cyclin E . However, in gastric carcinoma, there have been no data to suggest the function and targets of miR-29c. In the present study, we showed that ectopic expression of miR-29c suppressed the proliferation of gastric carcinoma cells and their ability to form colonies on soft agar. Furthermore, the growth suppression was not accompanied by caspase activation, but occurred through a decrease in the rate of DNA synthesis, suggesting that miR-29c may suppress cell proliferation by regulating progression of the cell cycle. To our knowledge, this is the first report to describe the tumor-suppressive role of miR-29c in gastric carcinoma. The mechanism responsible for miR-29c downregulation in gastric carcinoma is still unknown. Since genomic loss at 1q32.2, where miR-29c is located, is rarely detected in gastric carcinoma, whereas 1q gain is frequently observed , it is unlikely that genomic copy number alteration is responsible for the downregulation of miR-29c. In addition, we showed that miR-29c downregulation is probably not associated with DNA methylation or histone deacetylation. Other factors, such as interaction of transcriptional suppressors with the miR-29c promoter and post-transcriptional regulation may play a role. Further studies will be needed to clarify these issues.
In this study, we showed that miR-29c reduced the expression of RCC2 at both the mRNA and protein levels. The luciferase assay with a reporter containing the miR-29c binding sequence at the 3’UTR of RCC2 mRNA suggested that miR-29c directly targets the 3’UTR of RCC2 mRNA. Although RCC2 (also known as TD-60) is reportedly a component of the chromosomal passenger complex, which is a crucial regulator of chromosomes, the cytoskeleton, and membrane dynamics throughout mitosis [22, 23], little has been known about the oncogenic relevance of RCC2. Mollinari and colleagues showed that RCC2 knockdown induced cell cycle arrest at prometaphase in HeLa cells due to failure of spindle assembly, suggesting that RCC2 regulates cell cycle progression by modulating chromosome segregation and cell cleavage . In this study, we showed for the first time that RCC2 is upregulated in gastric carcinoma tissues. Furthermore, knockdown of RCC2 induced growth suppression without caspase activation in gastric carcinoma cells. Together with the data reported previously by other groups, our results suggest that RCC2 contributes to the proliferation of gastric carcinoma cells by regulating progression of the cell cycle. Interestingly, growth suppression without caspase activation was also observed in miR-29c-transfected cells, suggesting that downregulation of RCC2 may be involved in the miR-29c-induced growth suppression. In addition, both miR-29c and RCC2 transfection decreased cell viability accompanied by a reduction of BrdU incorporation, suggesting that RCC2 may play a role in/around S phase of the cell cycle. Thus, our data suggest that miR-29c reduces the expression of RCC2 in gastric carcinoma cells, leading to suppression of their growth, not through induction of apoptosis but by a cell cycle regulation signal. Therefore, we can hypothesize that decreased expression of miR-29c results in enhanced expression of RCC2, and confers a growth advantage on gastric carcinoma cells. Because the contribution of RCC2 to cell cycle regulation signaling during S phase of the cell cycle is still poorly understood, further studies will be needed to clarify this issue.
On the other hand, PPIC siRNA-transfected cells did not exhibit growth suppression, although PPIC mRNA level was decreased. We demonstrated that PPIC is one of the target genes of miR-29c and was significantly upregulated in gastric cancer tissues. Therefore, PPIC may play some role as a downstream molecule of miR-29c, although it may not be involved in cell proliferation and apoptosis. It has been reported that PPIC, also known as cyclophilin C, is a cellular binding protein for the immunosuppressive drug cyclosporine A, which can suppress T-cell activation [25–27]. Moreover, it has been reported that the natural cellular ligand for PPIC, cyclophilin C-associated protein (CyCAP, also kinown as MAC2BP) , can act as a modulator of endotoxin signaling in vivo . These facts led us to speculate that aberrant expression of PPIC may affect some aspects of the immune response, such as inflammation, during gastric carcinogenesis. However, to assess the function of PPIC under miR-29c regulation, further studies will be needed.
In gastric carcinoma tissues, CDK6 upregulation was not observed, at least at the mRNA level, although CDK6 expression was suppressed at both the mRNA and protein levels by ectopic expression of miR-29c in MKN45 cells. These findings suggest that CDK6 expression in tissue is probably affected by other factors. Indeed, upregulation of CDK6 in gastric carcinoma has been reported by other research groups [30, 31], and a portion of cases showing CDK6 overexpression harbored chromosomal amplification of 7q21.2, where the CDK6 gene is located .