MiR-1301-3p Promotes Cell Proliferation In Gastric Cancer Via Targeting SIRT1

Background:Gastric cancer(GC)represents one of the most common malignant diseases around the world.In China,the morbidity of this malignancy is increasing obviously and accounts for nearly fifty percent of the global incidence rate.Oncogenes and tumor suppressive genes which interact with each other play essential roles in the development of diverse tumors.SIRT1,a class ? histone deacetylase which depends on nicotinamide,could regulate the physiological and pathological process by creating proteins and DNA methylation,playing dual effects on the development of neoplasms.The established studies recognized it as a tumor suppressor in gastric carcinogenesis.A previous study found that SIRT1 leads to G1-phase arrest via NF-kB/Cyclin D1 signaling,retarding proliferation of gastric cancer cells.As a result,SIRT1 could affect tumorigenesis by regulating cell cycle progression.MicroRNAs(miRNAs)are short non-coding RNAs which regulate gene expression by binding to the 3'-UTR of target gene,resulting in its inhibition in transcriptional level or degradation.Accumulating studies have unveiled the critical biological functions of miRNA in tumorigenesis.We lucubrated the global miRNA expression profiles in GC derived from The Cancer Genome Atlas(TCGA)and identified miR-1301 was up-regulated significantly in GC.miR-1301-3p was recognized as a tumor suppressor gene inhibiting the migration and invasion of HepG2 cells in previous study.However,not long after the discovery,others proved that miR-1301-3p was highly expressed in liver cancer cells and promoted tumorigenesis.The paradoxical results veiled the mysterious functions of miR-1301-3p,making the researches focused on its role in cancers more fascinating.Besides that,the correlations between miR-1301-3p and prostate cancer progression as well as proliferation of glioma cells were revealed,and miR-1301-3p may act as oncogene that accelerate the process of prostate carcinogenesis through targeting PPP2R2C and anti-oncogene inhibiting cell proliferation in glioma.Notably,miR-1301 could suppress particular tumor cell migration and invasion in a p53-dependent manner.But the roles and potential mechanisms of miR-1301-3p in gastric cancer are still unclear.Objective:To evaluate the expression of miR-1301-3p in gastric cancer tissues and cells,to discuss the roles and related molecular mechanisms of miR-1301-3p in gastric cancer,to identify the target gene of miR-1301-3p and to clarify the function of miR-1301-3p/SIRT1 in the proliferation of gastric cancer cells,providing noveldirections for finding new biomarkers,improving the early diagnosis of gastric cancer and target therapy.Methods:1.The expression levels of miR-1301-3p and SIRT1 were detected in 60 pairs of GC tissues and adjacent normal tissues using the methodology of qRT-PCR.The relationships between the relative expression of miR-1301-3p and clinicopathological characteristics were assessed.2.SGC-7901 and MGC-803 were transfected with miR-1301-3p mimics and inhibitor lentivirus.qRT-PCR was utilized to verify the efficacy of transfection.The CCK-8 assay and clone formation assay were used to determine the effect of miR-1301-3p on proliferation of gastric cancer cells.Flow cytometry was used to evaluate the cell cycle guided by miR-1301-3p.Western blotting was employed to detect the level of SIRT1 protein and cell cycle related proteins.3.Luciferase reporter assay was used to prove whether miR-1301-3p could bind to the 3'UTR region of SIRT1.4.SIRT1 was downregulated using siRNA in SGC-7901 and MGC-803 cells transfected with miR-1301-3p-inhibitor and CCK-8 assay,clone formation assay and cell cycle detection in flow cytometry were performed.5.Tumorigenesis assay was used to testify the role of miR-1301-3p in tumor growth in vivo.Results:1.miR-1301-3p is up-regulated in both gastric cancer tissues and cells compared with normal gastric tissues and GES-1.2.miR-1301-3p promotes the proliferation and cell cycle progression of GC cells,accelerating the transformation of G1/S stage.3.SIRT1 was a direct target of miR-1301-3p in GC and miR-1301-3p promotes cell proliferation in gastric cancer via targeting SIRT1.SIRT1 is down-regulated in gastric cancer tissues and its expression level is negatively associated with miR-1301-3p.4.Knockdown of SIRT1 could eliminate the partial effects of miR-1301-3p-inhibitor on GC cell proliferation and cell cycle progression.5.miR-1301-3p facilitates tumor growth of GC cells in vivo.Conclusion:miR-1301-3p promotes cell proliferation in gastric cancer via targeting SIRT1 through cell cycle progression and it could become a novel serum biomarker for early diagnosis and a potential therapeutic target.