The Preliminary Study On The Function And Mechanism Of MiR-339-5p In Gastric Cancer

Backgroud:The morbidity of gastric cancer(GC)ranks 4th in men and 5th in women,its mortality rate after the lung cancer in the world.Approximately 70%deaths occured in the developmenting countries,and China accounts for 40%of them.Most of patients were diagnosed at advanced stages of tumor,led to a poorer prognosis for GC patients.Therefore,searching for effective biomarker for early diagnosis and prognosis and exploring the potential mechanism responsible for the development and meatstasis of GC,would provide novel therapeutic target and strategy for diagnosis and threament of GC.MicroRNAs(miRNAs)are a class of short(approximately 18-22 nucleotides in length),non-coding,endogenously expressed small RNAs.By binding to the 3'-UTR of target gene mRNA,miRNAs negatively modulate the expression of target via suppressing translation or degrading messenger RNAs(mRNA).MiRNAs have critical roles in various tumor-related biological processes including cell proliferation,apoptosis?migration and invasion.Theoretically,one miRNA directly target hundreds of mRNAs,which involveld in various kinds of activated or inactivated cancer pathways.Thus,deregulated miRNAs,mRNAs and downstream effectors constituted a complex network that modulating the initiation and progression of GC.Increasing evidence showed that miRNAs function as oncogene or tumor suppressor in tumors depending on the nature of their target gene.However,the biological function and mechanism of miRNAs in gastric carcinogenesis are still not very detailed.we aim to explore the function and mechanism of miRNAs in GC development by in vitro and in vivo analysis,providing novel insight into the diagnosis and therapy of GC.Objetives:To explore the function and mechanism of miR-339-5p in gastric carcinogenesis,as well as novel target and potential molecular mechanism involved in the progression of GC.Methods:1.The expression of miR-339-5p was detected by qRT-PCR in GC tissues and paired non-tumor tissues.We also analysed correlations between miR-339-5p and clincopathological characteristics including age,gender,differentiation,tumor size,TNM stage,lymph node metastasis,The completely followed-up data of patients were used to evaluate the effect of miR-339-5p on overall survival rate.2.MiR-339-5p mimics?inhibitor and corresponding negative control were transfected to SGC-7901 and BGC-823 cells.MTT and plate colony formation assays were performed to evaluate the effect of miR-339-5p on cell proliferation.Flow cytometry was conducted to determine the effects of miR-339-5p on cell cycle distrubution.Woung-healing and transwell invasion assays were used To determine whether miR-339-5p could regulate GC cell migration and invasion ability,respectively.Western blot assay was used to detected the protein expression of cell growth marker Cyclin D1 and migration marker MMP2 and MMP9.3.Three publically avaliable bioinformatic databases were used to predict the potential target of miR-339-5p.Constructs containing wild-type or mutant-type of NOVA1 mRNA 3'-UTR and miR-339-5p were cotransfected into GC cells and luciferase reporter assay were performed to evaluate the effect of miR-339-5p on NOVA1 luciferase activity.The regulating effect of miR-339-5p on NOVA1 was detected by qRT-PCR and Western blot.The mRNA level of NOVA1 in GC tissues and nontumorous tissues was detected by qRT-PCR.The stably expressing NOVA1 vectors was constructed and transfected into GC cells to reveal the biological effect of NOVA1 on tumor cell growth,migration and invasion.4.The xenograft model analysis was performed to verify the tumor suppressor role of miR-339-5p in vivo.Western blot assay was conducted to measure the protein levels of NOVA 1 in the harvested tumors.5.MSP and BSP assays were performed to reveal the methylation status of miR-339-5p-related CpG island in GC cells and tissues.GC cells were treated with different concentrations of demethylation agent 5-AZA to verify whether the miR-339-5p-related CpG island methylation correlated with its downregulation.Results:1.The expression of miR-339-5p was significantly decreased in GC tissues compared to that in adjacent nontumorous tissues(p<0.05).The downregulated miR-339-5p was significantly correlated with the larger tumor size(p=0.01 1),TNM stage(p=0.005)and lymph node metastasis(p=0.035).The patients with low expression of miR-339-5p had low overall survival rate comparison with patients with high expression of miR-339-5p(p=O.044).2.We transfectd GC cells with miR-339-5p mimic,inhibitor and corresponding negative control,respectively.We found that upregulated miR-339-5p promoted cell proliferation and colony formation,while downregulated miR-339-5p inhibited cell proliferation and colony formation(p<0.05).The results of flow cytometry assay showed that transfection with mimic induced G1 cell cycle arrest(p<0.05),whereas inhibitor decreased the cellular population of G0/G1 phase and a sharp increase in S phase.Woung-healing and Transwell invasion assays showed that overexpression of miR-339-5p obviously suppressed cell migration and invasion,while decreased miR-339-5p enhanced the migratory and invasive ability of GC cells(p<0.05).Furthermore,overexpression of miR-339-5p led to a reduction of protein levels of Cyclin D1,MMP2 and MMP9,while inhibition of miR-339-5p exhibited the opposite effect.3.NOVA1 was predicted as a potential target of miR-339-5p.The results of luciferase reporter assay showed that miR-339-5p mimic decreased the luciferase activity of wild-type of NOVA 1 mRNA 3'-UTR,but not the mutant-type(p<0.05).The data of qRT-PCR and western blot showed that miR-339-5p mimic obviously led to a lower mRNA levels of NOVA1,as well as protein levels.The mRNA expression of NOVA1 was significantly increased in GC tissues,but not correlated with any clinicopathological features of patients.In vitro analysis indicated that overexpression of NOVA1 impaired the inhibition effect of miR-339-5p on cell proliferation,migration and invasion.4.In vivo xenograft model experiment showed that stably expressing of miR-339-5p vectors significantly inhibited tumor growth compared to control vectors(p<0.05).The protein levels of NOVA1 were obvilously decreased in overexpression of miR-339-5p mice tumors.5.By using public avaiable database,a CpG island was observed around miR-339-5p.The result of MSP indicated that the methylation level of miR-339-5p was increased in GC cell compared to GES-1 cells.In comparison with adjacent nontumor tissues,the methylation level of miR-339-5p enhanced in GC tissues.BSP analysis confirmed that methylation of miR-339-5p-related CpG island was more frequent in GC cells.The expression of miR-339-5p could be reversed by 5-AZA treatment(p<0.05).Conclusions:MiR-339-5p inhibited GC cell proliferation,migration and invasion by targeting NOVA1,and the downregulated miR-339-5p indicated poor prognosis of GC patients.Moreover,CpG island methylation may be an explanation for the deregulated miR-339-5p in GC development.Thus,miR-339-5p/NOVA1 axis expanded the knowledge of molecular mechanism of gastric tumorigenesis,providing an novel therapeutic target for treatment.