Up-regulation Of MicroRNA-375 Inhibits The Proliferation Of Gastric Cancer Cell Through Targeting AEG-1

Gastric cancer(GC)is the third leading cause of cancer death and a major public health care problem worldwide,including in Western populations.Gastric cancer was the one of the most common type of cancers,it could cause about 800,000 patients die every year.The clinical outcome of gastric cancer had gradually improved nowadays,but the prognosis is still unsatisfied.This is because the mechanism of the gastric cancer development was still poorly understood despite of a few oncogenes and tumor suppressor genes have been reported in gastric cancers.MicroRNAs(miRNAs)are small(18-25 nucleotides)RNAs with nonprotein-coding capacity that function in regulating the expression of genes.MiRNAs exert their regulatory roles at the level of post-transcription by stimulating target messenger RNA(mRNA)degradation and translational repression.Dysregulation of miRNAs has been detected in a great variety of disorders,particularly in malignancies.Increased evidence has shown that certain miRNAs function as tumour suppressors or oncogenes in different human cancers.Recently studies showed that miR deregulations could regulate the cell proliferation and apoptosis.Studies also reported that 43 miRs were up-regulated and 37 miRs were down-regulated in gastric cancer,which indicating the dysregulations of miRs were associated with the progression and prognosis of gastric cancer.A few studies indicated that miR-375 expression was down-regulated in many human cancers;however the biological role of miR-375 in gastric cancer is still not be understood.In the present study we try to indicate that the transfection of miR-375 mimic significantly decreased the expression of AEG-1 gene both in mRNA level and protein level,which suggest miR-375 regulate the gastric cancer cell through the regulation of AEG-1 gene.Our findings suggested that miR-375 targeted AEG-1 gene in gastric cancer cell and regulated the gastric cancer cell growth in vitro,which highlight the therapeutic potential of miR-375 in gastric cancer clinical treatment.PART I The biological effects of miR-375 on gastric cancer cellsObjective:In this study,we screened the difference between miR-375 in SGC-7901,BGC-823 and MKN-28 gastric cancer cells and normal tissue differences in the expression of the stomach,the expression levels of miR-375 in gastric carcinoma was further validated by quantitative real time PCR technique for clinical samples,and may establish the theoretical basis for subsequent experiments.Methods:The normal gastric epithelial cells and gastric cancer cell lines were cultured in a specific cell culture medium.The miR-375 was detected by TaqMan MicroRNA Reverse Transcription Kit in SGC-7901,BGC-823 and MKN-28 gastric cancer cells and normal gasltric mucosal epithelium The expression of miR-375 was up-regulated by siRNA transfection with siRNA-MiR-375 and control siRNA-mimic.The cells were transfected into SGC-7901 cells by Lipofectamine 3000 kit.The cells were incubated with MTT assay every 24 hours The cell viability was measured and the absorbance was measured at 570 nm as the expression level of cell viability.The cultured cells were transfected with Annexin V and Propidium iodide and the apoptosis was detected by flow cytometry equipped with CellQuest softwareResults:The expression of miR-375 in gastric cancer cells was significantly lower than that in normal gastric mucosal epithelial cells.The expression of SGC-7901 cells was the lowest in normal gastric mucosal cells(0.24),and then SGC-7901 gastric cancer cells were selected.The expression of miR-375 in SGC-7901 cells was significantly higher than that in the control group(P<0.05),and miR-375 overexpression measured with MTT assay significantly inhibited the proliferation of SGC-7901 cells,especially in the time-dependent 36h after transfection.Cell apoptosis of miR-375 overexpressing in SGC-7901 was detected by flow cytometry.The apoptosis rate of miR-375 cells was 6.8%,while no significant changes were observed in the control group.The difference was statistically significant,and overexpression of miR-375 promoted apoptosis.Conclusion:MiR-375 is a key factor in inhibiting the proliferation and inducing apoptosis of SGC 7901 gastric cancer cells,therefore miR-375 may play an important role in the apoptosis of gastric cancer cells.PARTⅡ Effects of miR-375 and AEG-1 on the growth regulation of gastric cancer cells in vitroObjectives:We evaluated the biological role of miR-375 in gastric cancer and investigated the expression and mechanisms of miR-375and AEG-1 in gastric cancer,Methods:To culture gastric cancer SGC-7901 normal differentiation growth in conventional way and SGC-7901 gastric cancer cells were transfected through Lipofectamine 3000 kit,making miR-375 expression up,siRNA-MiR-375 and control siRNA-mimic were transfected into SGC-7901 cells.Using Western Blot and RT-PCR assay to confirm the relation between miR-375 and the AEG-1 mRNA level。The AEG-1 mRNA relative expression was measured by RT-PCR assay.GAPDH was used as the internal control gene.Results:Our RT-PCR results demonstrated that when up-regulation of miR-375 in SGC-7901 cells,the AEG-1 mRNA level was significantly decreased and the similar AEG-1 expression change also observed in protein level with Western Blot assay。Conclusions:The expression levels of miR 375 is closely related to AEG-1 in mRNA levels and proteins of gastric cancer cells.Therefor miR-375 regulates the gastric cancer cell proliferation and apoptosis through the regulation of AEG-1 gene.