| ObjectiveGastric cancer, with high morbidity and mortality, is one of the most common cancers inChina. Similar to other tumors, a variety of abnormal expressions of coding and non-coding genestake part in the occurrence and development of gastric cancer. Recent studies showed thatmicroRNAs, miRNAs, are associated with gastric cancer. However, their mechanisms are not clear.So, it is important to obtain more information about the relationships between miRNAs and gastriccancer. Through suppressing the expression of target genes, miRNAs play their biological roles.And the activation of oncogenes is one of the reasons of tumorogenesis. As a result, thedownregulated miRNAs whose targets are oncogenes play a crucial role in the tumorogenesis. Ourprevious studies showed that seven miRNAs were down-regulated in gastric cancer tissues. To thisend, the aims of this study are to reveal the down expression reasons of these miRNAs and themain molecular mechanisms of their roles in gastric cancer occurrence.Methods1. To screen the existence of CpG islands in the promoter region of miRNA genes,bioinformatics analysis was used to analyze the upstream sequences in promoter of sevendown-regulated miRNA genes.2. Real-time RT-PCR was employed to detect the expression of the screened miRNAsbetween gastric cancer cell lines (MGC-803, AGS and SGC-7901) and normal gastric epithelialcell line (GES-1), and gastric cancer tissues and paired-adjacent normal tissues from gastric cancerpatients.3. To release the expression of miRNA genes that are regulated by DNA methylation, gastriccancer cells (MGC-803and SGC-7901) were treated by5-aza-2’-deoxycytidine (5-aza-dC), aDNA methylase inhibitor, respectively.4. To increase or decrease the corresponding miRNA levels in cells, miRNA mimics orinhibitors were transfected to gastric cancer cells or normal gastric epithelial cell with liposome,respectively.5. Real-time RT-PCR was used to detect the expression of miRNAs in the cells treated with5-aza-dC or transfected with mimics or inhibitors. MTT assay and/or real-time cell analysis(RTCA) were used to detect the proliferation on cells. Flow cytometry was used to detect the cellcycle distributions. Western blot was used to analyze the expressions of cell cycle-related proteins miRNAs associated target genes, respectively.6. The dual luciferase assay was used to confirm miRNAs’ targets.Results1. Bioinformatics results showed that the upstream regions in the promoters of two miRNAs(miR-195and miR-378), among seven down-regulated miRNAs, contained CpG islands.2. The real-time RT-PCR results showed that compared with GES-1the expressions ofmiR-195and miR-378were significantly down-regulated in MGC-803, AGS, and SGC-7901(P<0.001). Also, the expressions of miR-195and miR-378were significantly down-regulated ingastric cancer tissues, compared to paired-adjacent normal tissues (P<0.05). The expressions ofmiR-195and miR-378in MGC-803were significantly restored by5-aza-dC (P<0.05). However,the expression of the other four miRNAs had no significantly change miR-768-3p has been notfurther studied because of its absence in the updated miRNA database (http://www.mirbase.org/cgi-bin/query.pl?terms=miR-768).3. The cell cycle of gastric cancer cells MGC-803and SGC-7901treated with5-aza-dC for24h or48h were arrested in G2/M or S phase, respectively. The expressions of cyclin-dependentprotein kinase (CDK)1, CDK2and cyclin A were increased after treating with5-aza-dC.4. The growths of gastric cancer cells and normal gastric epithelial cells were suppressed bymiR-195/miR-378mimics and miR-195/miR-378inhibitors, respectively. The growth of normalgastric epithelial cells was significantly promoted by miR-195or miR-378mimic. As thebase-lines of miR-195and miR-378in gastric cancer cells were very low, their growths were notaffected by miR-195/miR-378inhibitors.5. The cell cycle of gastric cancer cells were arrested in G0/G1or G2/M phase by miR-195ormiR-378mimic.6. The Western blot results showed that tumor-associated protein CDK6or vascularendothelial growth factor (VEGF) were one of the targets of miR-195or miR-378, respectively.The dual luciferase assay further verified the target relationship between them.ConclusionsFirst, by combining bioinformatics analysis with the results of the use of DNA methylaseinhibitor, we found that the main reason of the down-regulated miR-195and miR-378in gastriccancer were hypermethylation in their genes’ promoters. Second, by transfecting miRNA mimicsor inhibitors to increase or decrease the levels of miRNA, our experiments indicated that miR-195and miR-378had the properties of tumor suppressor. Finally, through analysis of their relatedtargets, we suspect that one of the molecular mechanisms of miR-195and miR-378acting as atumor suppressor was by regulating the expressions of CDK6and VEGF, respectively, and then by... |
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