MiR 29c is the member of the miR 29 family composed of miR 29a, 29b and 29c. Deep sequencing has revealed that this miRNA family is that most highly expressed in gastric tissues, and that the read count of miR 29c is the highest among them, or suggesting that alteration of the miR 29 expression level has an impact on gastric cells. Therefore, Inhibitors,Modulators,Libraries we hypothesized that miR 29c could be a candidate tumor suppressor miRNA in gastric carcinoma, as well as miR 375. In the present study, to test this hypothesis, we attempted to clarify the function of miR 29c in the tumorigenesis and/ or progression of gastric cancer. Results Quantification of miR 29c in gastric carcinoma To confirm that miR 29c was downregulated in gastric carcinoma, as determined by miRNA microarray, we performed quantitative RT PCR using 12 paired samples of tumor tissue and the adjacent normal epithelium.
Indeed, miR 29c was significantly down regulated in the tumor tissues from all 12 cases, which comprised 7 intestinal type and 5 diffuse type car cinomas. The degree of miR 29c down regulation was not associated with clinicopathological features such as histological classification, patient age, Inhibitors,Modulators,Libraries sex, tumor location, depth and stage. It has been reported that epigenetic modifications such as DNA methylation might influence genome wide gene expression during tumorigenesis of gastric carcinoma. To determine whether epigenetic modification is associated with miR 29c downregulation, we treated MKN45 cells with 5 aza 2 deoxycytidine and trichosta tin A and assessed the subsequent miR 29c expression.
As shown in Additional file 2, the expression of miR 29c was Inhibitors,Modulators,Libraries not increased by treatment with these reagents, suggesting that epigenetic modifications may not be as sociated with the downregulation of miR 29c in gastric carcinoma Inhibitors,Modulators,Libraries cells. Exogenous expression of miR 29c suppresses the proliferation of gastric carcinoma cells To investigate the biological function of miR 29c in gas tric carcinoma, we first assessed the effects of Inhibitors,Modulators,Libraries its expres sion on cell viability using the MTS assay. We transfected precursor miR 29c or negative control into three gastric carcinoma cell lines MKN45, MKN74 and MKN7 which showed very low levels of miR 29c expression in comparison with normal epithelial tissues, and found that cell viabilities in pre 29c transfected cells were decreased in all of 3 cell lines tested.
The suppression of proliferation occurred in a time dependent manner in MKN45, and simi lar results were obtained in MKN74 and MKN7 cells. MiR 29c also reduced the ability of MKN45 and MKN74 to form colonies in soft agar. These results suggest that miR 29c may have tumor suppressive functions in gastric carcinoma cells. selleck chem inhibitor To determine whether the growth inhibition by miR 29c was associated with apoptosis, caspase activities in MKN45 cells were determined at 48 h after transfection with pre 29c.