The Role And The Molecular Mechanism Of MiR-93 In Gastric Cancer Progression

Part oneObjective:To investigate the expression of miR-93 in 20 normal gastric mucosas,60 paired gastric cancers with their normal counterparts, as well as 6 gastric cell lines. Then explore the correlation between miR-93 levels and the clinicopathologic features of gastric patients.Methods:Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was applied to examine miR-93 levels in 20 normal gastric mucosas,60 paired gastric cancers with their adjacent counterparts, as well as 6 gastric cell lines. Next, Mann-Whitney Test was used to explore the correlation between miR-93 levels and the clinicopathologic features of gastric patients.Results:Compared with the normal gastric mucosas and peritumoral tissues, miR-93 expression significantly overexpressed in gastric cancer samples. Among them,76.7%(46 of 60) gastric cancer (GC) samples had more than 2-fold upregulated levels of miR-93 with respect to their paired counterparts. Next, we found that miR-93 expression was associated with tumor size, lymph node-metastasis and pTNM stages. Patients with the early stages (stage I and II) had a much lower miR-93 levels than that with the advanced stages (stage III and IV). Similarily, we also detected upregulated levels of miR-93 in six GC cell lines (AGS, SGC-7901, MKN-28, MKN-45, HTB135, and MKN-74) than in normal gastric epithelium cell lineGES-1.Conclusion:MiR-93 expression was overexpressed in both human GC tissues as well as in GC cell lines, and its dysregulation was associated with clinicopathological features of GC patients, which indicates that miR-93 may act as a keyl role in GC development.Part twoObjective:To explore miR-93 effects on GC cells proliferation, cell cycle distribution, apoptosis, invasion, and migration through enforced or silenced miR-93 expression in vitro.Methods:To determine whether miR-93 expression affecting GC cells proliferation, cell cycle distribution, apoptosis, invasion, and migration, then miR-93 mimics was transiently transfected into AGS for its upregulation, and HTB135 cells was transfected with miR-93 inhibitor for its downregulation.24h,48h,72h, and 96 h after transfection, AGS and HTB135 cells proliferation was investigated with CCK-8 analysis.48 h after transfection, flow cytometry analysis was conducted to assess AGS and HTB135 cell cycle distribution and apoptosis ability.24 h after transfection, transwell analysis was conducted to evaluate miR-93 influences on AGS and HTB135 cells invasion and migration..Results:The proliferation ability of AGS cell was remarkable enhenced 48 h,72 h, and 96 h after transfected with miR-93 mimics. On the contrary, the proliferation capability was greatly impared when the miR-93 inhibitor was transfected for 48 h,72 h, and 96 h. However, no significant differences were observed 24 h after the miR-93 mimics and its inhibitor were transfected. In addition,flow cytometry analysis shewed that miR-93 mimics significantly decreased the percentages of AGS cells at the G0G1 stage, while obviously increased their proportions at the S phase. On the contrary, miR-93 inhibitor dramatically increased the percentages of HTB135 cells at the G0G1 phase, yet significantly reduced their proportions at the S stage. Yet no significant differences were observed the percentages of cells at the G2/M stage, transwell analysis revealed that enforced miR-93 significantly enhanced AGS cell invasion and migration ability, while miR-93 inhibitor greatly impared HTB135 cell above ability.Conclusion:Above gain- and loss-of-function analysis showed that enforced miR-93 expression significantly enhances GC cells proliferation, invasion, and migration, and these data suggest that miR-93 may function as an oncogene in gastric cancer development and progression.Part threemiR-93 regulates gastric cancer cell growth and metastasis through targeting P21 and PTENObjective:To explore the mechanism by which miR-93 regulating gastric cancer cell growth and metastasis.Methods:We used two online bioinformatic algorithms (miRBase and TargetScan) to predict putative target gene of miR-93 in gastric cancer cells and paid highly attention to these tumor suppressor genes which are closely related to cell growth and metastasis. Then dual-luciferase reporter assay was performed to confirm its potential target genes. Western blot analysis was applied to test whether the expression of this gene was affected by miR-93 upregulation or downregulation. Moreover, we silenced above target genes in gastric cancer cells to validate whether miR-93 mediated gastric cancer cell growth and metastasis through regulating these target genes.Results:P21 and PTEN are potential target genes of miR-93 by above online database. Dual-luciferase reporter assay revealed that enforced expression of miR-93 obviously reduced the luciferase activity of pGL3-P21-WT reporter and pGL3-PTEN-WT reporter when compared with their miR-NC groups. However, the luciferase activity of pGL3-P21-mut reporter and pGL3-PTEN-mut reporter were not significantly affected in the cells transfected with miR-93 or miR-NC. Western blot assay showed that the protein level of P21 and PTEN were also significantly influenced by miR-93 expression. Enforced expression of miR-93 markedly diminished, while its inhibitor obviously elevated protein levels of P21 and PTEN in AGS and HTB135 cells. Moreover, knockdown of P21 and PTEN expression markedly enhanced AGS cell proliferation, invasion and migration.Conclusion:These results strongly indicate that miR-93 may function as an oncomiRNA in gastric cancer cells, at least, through targeting P21 and PTEN. Therefore, miR-93 might act as a potential molecular target for controlling gastric cancer development.