Molecular Mechanism And Function Of MicroRNA-1260b In The Proliferation And Invasion Of Colorectal Carcinoma

BackgroundColorectal cancer(CRC)as the third most common malignancy in the world,is an important public health problem that threatens human health.Revealing the complex pathogenesis of colorectal cancer is a frontier issue in the field of cancer research.MicroRNAs are a class of highly conserved small noncoding RNAs that play an important role in the development of tumors and can function as oncogenes or tumor suppressor genes,depending on regulating the downstream tumor-associated target genes and signaling pathways.Based on this,miRNAs become a new research hotspot in tumor diagnosis,treatment and prognosis.Previous studies have reported that miR-1260b is abnormally expressed in various solid tumors and participates in tumorigenesis and development.However,the biological function and related mechanisms of miR-1260b in colorectal cancer proliferation and invasion have not been fully elucidated.ObjectivesIn the present study,we aim to explore the effects of miR-1260b on biological functions such as proliferation and invasion of colorectal cancer cells,and to predict and verify the downstream target genes and related molecular mechanisms of miR-1260b.Methods1.Expression of miR-1260b in CRC tissues and cells was investigated by real-time quantitative PCR analysis.2.The miR-1260b inhibitor was transfected into RKO and SW620 CRC cell lines.We carried out CCK-8 method,colony formation assay and transwell matrigel invasion assay to detect the cell proliferation and invasion abilities in vitro after transfection of miR-1260b inhibitor.3.Five bioinformatics databases including MiRanda,TargetScan,DIANA-microT,Pictar,and MICROCOSM were used to predict the downstream targets of miR-1260b.TSC22D2 with high predictive values was selected as candidate target.Luciferase reporter system and real-time quantitative PCR were used to identify the interaction of TSC22D2 and miR-1260b.4.The plasmids carrying the miR-1260b sequences and TSC22D2 without 3'UTR region were co-transfected into SW480 and HCT116 cell lines.And then we detected the expression of TSC22D2 by Western blot.CCK-8 method,colony formation assay and Matrigel invasion assay were carried out to detect proliferation and invasion abilities in vitro after transfection of miR-1260b and miR-1260b/TSC22D2.Results1.We detected the expression of miR-1260b in 48 pairs of matched fresh CRC tissues by Real-time PCR.Results showed that the expression of miR-1260b in CRC tissue was significantly higher than that of corresponding normal mucosa(P<0.05).And miR-1260b expression was significantly higher in the cancer tissues with metastasis than those without metastasis(P<0.05).MiR-1260b expression was also detected in six CRC cell lines by Real-time PCR.Results showed that miR-1260b expression level in CRC cell lines were significantly higher than normal human colon mucosal epithelial cell line(NCM460)(P<0.05).The statistical comparison analysis indicated that miR-1260b expression was highest in SW620 cells,and was gradually decreased in RKO,Caco2,HT29,HCT116 and SW480 cells.2.After predicting by five common databases,TSC22D2 had the higher predictive values and was associated with proliferation and metastasis.So we choose TSC22D2 as the potential downstream target of miR-1260b.In SW620 and HCT116 cells,luciferase reporter system showed that the luciferase activity was decreased significantly respectively in miR-1260b/TSC22D2-3'UTR group(P<0.001,P<0.001).Western Blot results showed that compared to NC group,the expression of TSC22D2 was significantly up-regulated after transfecting miR-1260b inhibitor in RKO and SW620 cells.The expression of TSC22D2 was significantly down-regulated in SW480 and HCT116 cells after transfection of miR-1260b,while TSC22D2 expression was up-regulated again in miR-1260b/TSC22D2 group compared to miR-1260b group.3.CCK-8 assay showed that compared to NC group,the proliferation abilities of miR-1260b inhibitor group in RKO and SW620 cells were significantly decreased(all P<0.05).The cell proliferation abilities of miR-1260b group were significantly increased compared to mock group(all P<0.05),and proliferation abilities were significantly reduced in miR-1260b/TSC22D2 group than miR-1260b group(all P<0.05).4.Colony formation assay showed that transfection of miR-1260b inhibitor significantly suppressed the colony formation abilities of RKO and SW620 cell lines(P=0.001,P=0.003).And miR-1260b overexpression could significantly enhance colony forming abilities compared to mock group(P<0.001,P<0.001),while the colony forming abilities of miR-1260b/TSC22D2 group were decreased again compared to miR-1260b overexpression group(P<0.001,P<0.001).5.Boyden Chamber transwekk assay results showed that invasive abilities of RKO and SW620 cells were markedly decreased after transfection of miR-1260b inhibitor(P<0.001,P<0.001).In vitro invasion assay results showed that numbers of invading cell in miR-1260b group were significantly increased compared to mock group(P<0.001,P=0.002),while invading cell numbers of miR-1260b/TSC22D2 were significantly decreased compared to miR-1260b overexpressing group(P<0.001,P<0.001).ConclusionsMiR-1260b is up-regulated in colorectal cancer and promotes proliferation and invasion of colorectal cancer cells by inhibiting the target gene TSC22D2.