MiR-320a Inhibits Gastric Carcinoma By Targeting Activity In The FoxM1-p27^KIP1 Axis

BackgroundGastric cancer is the fourth most commonly diagnosed cancer and the second leading cause of cancer death worldwide, especially in developing countries Although the incidence has gradually decreased, there are still many confirmed mortalities annually worldwide. Gastric cancer is generally diagnosed at an advanced stage, which is the primary cause of its poor prognosis. Infection with Helicobacter pylori is one of the most important factors contributing to the development of gastric carcinoma. In order to improve the outcome of gastric cancer, identification of genetic and epigenetic events regulating the proliferation of gastric cancer cells is required. System biological analysis of gastric cancer samples have shown the importance of microRNAs in this process.ObjectiveIn this study, we aimed to identify the role of miR-320a in gastric carcinoma and the down-stream FoxM1 and P27KIP1 regulatory mechanisms both in vitro and in vivo.Materials and Methods1 Patients and tissue specimensResected tissues from 22 gastric cancer patients and distal normal gastric tissues (>5 cm from the margin of the tumor) were harvested at surgery. The patients underwent the surgeries at Qilu Hospital, Shandong University during 2014 and 2015. None of the patients had received adjuvant chemotherapies before surgery.2In vitro studies2.1 The effect of miR-320a on FoxMl expression was evaluated using overexpression of miR-320a mimics and inhibitors. In AGS, BGC-823, and HGC cells, we examined the mRNA and protein level of FoxMl and P27KIP1 by the overexpression and inhibition of miR-320a.2.2 We detected the foci number after transfected with miR-320a mimics and inhibitors in AGS, BGC-823, and HGC cells.2.3 We validated whether FoxMl is a direct target of miR-320a by luciferase report assay. In AGS, BGC-823 and HGC cells, miR-320a mimics were co-transfected with FoxM1 wild-type or mutant-type 3’UTR plasmids to detect whether miR-320a can directly targeted the binding site located at FoxMl 3’UTR and FoxMl-P27KIP1 axis may be a direct target of miR-320a.2.4 In order to determine whether miR-320a affects the P27KIP1 expression by suppressing FoxMl, the recovery experiment was performed. We examined the expression of FoxMl and P27KIP1 and foci number after the co-transfection of miR-320a inhibitors and the special FoxMl siRNA.3In vivo studiesSixteen (8-10 weeks old) male nude mice were purchased from QING ZI LAN Animal Company and divided into two groups with one as control and the other as miR-320a inhibition. The mice were subcutaneously injected with 4×105 BGC-823 cells per mouse. One group was injected with miR-320a inhibitor stable transduction cells and the other group was injected with the matched control cells.Results1 Human gastric cancer tissues exhibit low miR-320a expression and the expression of miR-320a and FoxMl showed a negative correlation.1.1 Compared with normal human tissues, gastric cancer specimens showed significant inhibition of miR-320a expression (*P<0.05) and activation of FoxMl (*P<0.05) by qRT-PCR.1.2 There are a negative association of miR-320a and FoxMl expression in gastric cancer.1.3 The gene expression was not associated with age, gender, and specimen differentiation.1.4 The p27KIP1 protein expression was decreased (**P<0.01) with the increased FoxM1 protein expression (**P<0.01) in human gastric cancer specimens.2 MiR-320anegatively regulated the expression of FoxMl and positively regulated the expressionof p27KIP1.And FoxMl is a direct target of miR-320a.2.1 In AGS, BGC-823, and HGCcells, miR-320a mimics decreased the expression of FoxM 1 (*P<0.05)and restored the expressionof p27KIP1 (*P<0.05)bothat the mRNA and protein levels.while miR-320a inhibitor increased the expression of FoxM1 (*P<0.05) and decreased the expressionof p27KIP1 (*P<0.05) bothat the mRNA and protein levels.2.2 MiR-320a suppression can enhance the FoxMl protein expression (*P<0.05) and reduce the p27KIP1 protein expression (*P<0.05),and the effect of miR-320a inhibitor on FoxMl and p27KIp1 was greater than FoxM1 siRNA.2.3 Co-transfection of miR-320a and wild-type 3’UTR plasmid reduced the luciferase activity by approximately 62% relative to the control (*P<0.05), whereas mutant 3’UTR co-transfection almost restored the luciferase activity.3 MiR-320a regulates proliferation of gastric cancer cells.3.1 Transfection of miR-320a mimics reduced the number of colonies (**P<0.01), while the inhibition of miR-320a markedly increased the number of colonies (**P<0.01)3.2 MiR-320a suppression can reduce the number of colonies (**P<0.01) and the effect of miR-320a inhibitor on colonies was greater than FoxM1 siRNA.3.3 MiR-320a suppression increases gastric tumor growth in nude mice.3.4 Silencing of miR-320a increased expression of FoxMl (**P<0.01) and decreased expression of P27KIP1 (**P<0.01) in nude mice.Conclusion1 Human gastric cancer tissues exhibit low miR-320a expression and increased FoxMl expression and the expression of miR-320a and FoxMl showed a negative correlation.2 MiR-320a directly bounds to the 3’UTR of FoxMl and inhibits the expression of FoxMl.3 MiR-320a regulates proliferation of gastric cancer cells through FoxM1-P27KIPI axis.In this study, we demonstrate that miR-320a has an anti-tumor role in gastric cancer. miR-320a directly inhibits the expression of FoxMl through the binding of FoxMl 3’UTR, resulting in the increased expression of P27KIP1. Our in vitro and in vivo data indicate that the biological activity miR-320a is to inhibit the gastric cancer cell proliferation.