MiR-1246 Knockdown Via Lentivirus Inhibits Tumor Growth And Promotes Apoptosis Of Cervical Cancer Cells By Targeting Thrombospondin-2

MicroRNAs(miRNAs)are a family of small non-coding RNA molecules that down-regulate the expression of their protein-coding target genes.These RNAs are typically 19–25 nucleotides(nt)long and are cleaved from 70–100 nt hairpin pre-miRNA precursors.They are widely considered to be the master regulators of many important biological processes,including apoptosis,cell growth,viral infection,and cancer development.For example,studies have reported that miR-1246 has oncogenic effects and is elevated in serum and tumor tissues isolated from patients with CSCC with lymph node metastasis.Squamous cell carcinoma accounts for about 85% of all types of cervical cancer,adenocarcinoma accounted for about 10 to 15%,adenosquamous carcinoma 3%.the five year survival rate of patient without lymph node metastasis in early stage of cervical cancer,is up to 80 ~ 90%,while patients with lymph node metastasis in the same stage,the five-year survival rate is only 40 ~ 50%.Cervical cancer(CC)is a leading cause of cancer-related death in women worldwide and has significant societal,economic,and familial consequences.Therefore,enhancing early diagnosis and treatment options for patients with CSCC and other types of cervical cancer has been the focus of many gynecological oncology studies.At present,surgery and radiotherapy are standard treatments for cervical cancer.However,the clinical outcomes of these treatments vary dramatically and are difficult to predict.Therefore,it is necessary to identify new,effective treatment approaches,particularly for patients with radio-insensitivity and those susceptible to relapse.In our own laboratory,we previously screened the full mi RNA profile in patients with cervical squamous cell carcinoma(CSCC)and demonstrated that miR-1246 is expressed at similar levels in both the serum and cervical tumor tissues in patients with lymph node metastasis,indicating that this miRNA may also have applications as a serum marker for lymph node metastasis in CSCC.Unfortunately,even though significant efforts have been made to understand the function of miR-1246 in CSCC and other cancers,very little is known concerning its mechanism.Thrombospondin-2(THBS2,TSP2),a member of the thrombospondin family,was previously shown to be a target of miR-1246 in cervical cancer.THBS2 is known to regulate cell adhesion and migration via hydrolysis of the extracellular matrix(ECM).THBS2 may also play a role in inhibiting angiogenesis by regulating matrix metalloproteinases(MMPs)and ECM proteins.While it is possible that a miR-1246/THBS2/ECM signaling cascade is involved in cervical cancer metastasis,direct evidence of this pathway has never been reported.In this study,we utilized miRNA inhibitor via lentiviral infection to evaluate the role of mi R-1246 in CSCC tumorigenesis and progression.Human cervical cancer cell line,SiHa.Knockdown of this miRNA appeared to inhibit tumor growth and promote apoptosis of cervical cancer cells in vitro by interacting with the THBS2/MMPs/ECM pathway and by targeted regulation of THBS2 expression.Furthermore,inhibition of miR-1246 expression in tumor xenografts slowed tumor development and growth in vivo.The research is divided into three sections:Section one:To construct the lentivirus which regulates target genes,and to observe regulative effect of lentivirus in vitro and in vivo experiment would be conduct.The effects of stable infection of the miR-1246 inhibitor letivirus are observed by methods of CCK8,Flow cytometry technique and Xenografts in nude mice.Section two:To identify miR-1246 inhibits the THBS signaling pathway,expression of THBS2 and related genes are detected by qRT-PCR、Western blotting assay after the expression of miR1246 is receded.The purpose of this study is to further know the molecular mechanism miR-1246 affect the invasion and metastasis of tumor and cell apoptosis and biology effect of THBS2,in order to establish targeted therapy with the target of mi R-1246 and its target genes.Part I1.Down-regulation of miR-1246 promotes cell apoptosis and invasion resistance in vivo and in vitroObjective:To identify the ability of miR-1246 knockdown promotes cell apoptosis and invasion resistance for cervical carcinoma cells.Methods : miR-1246-inhibitor was synthesized and cloned into recombinant lentiviral vector(LV-miR-1246-Inh),which was then used to infect SiHa cervical cancer cells.The effects of LV-miR-1246-Inh infection on cell invasion,proliferation,and apoptosis were evaluated by transwell assays,cell count assays,and flow cytometry.Results : CCK8 assay showed that proliferation rate of group LV-miR-1246-Inh were slow down compare to control group,indicating that miR-1246 down regulation significantly restains cervical carcinoma cell growth.The Rate of apoptosis in group LV-miR-1246-Inh was about 16.1% higher than the others.Conclusion:miR-1246 inhibition induces cell apoptosis.2.miR-1246 knockdown inhibits SiHa tumor cell growth in vivo.Results: The cells were then injected subcutaneously into the left flanks of the mice..Tumors volume was measured every 3 days after tumor cell implantation.Mice were sacrificed 4 weeks later for evaluation.Tumor volume was markedly decreased in LV-miR-1246-Inh group compared to the Blank and LV-NC group.Conclusion: Down-regulate of mi R-1246 inhibited tumor growth of SiHa cells in vivoPart II1.Inhibition of mi R-1246 increased THBS2 protein expression and induced downstream changes in the THBS2/MMP signaling pathwayTo evaluate the role of THBS2,a predicted mi R-1246 target gene,its expression was examined by western blot.THBS2 protein expression in cells infected with LV-miR-1246-Inh was significantly higher than that observed in the control groups.These data suggested that Lentivirus-mediated miR-1246 knockdown upregulated the expression of THBS2 protein in SiHa cells in vitro.Part IIITo find the correlation between THBS2 and clini-pathological in cervicalcarcinoma tissues.Results : The expression of THBS2 were detected in 52 case cervical carcinoma tissues.level of THBS2 protein in cervical cancer was the lowest and has significance compared with those in others cervical tissues(P<0.05),concerned with depth of infiltration,metastasis of lymphoid node and clinical stage.The relationship between THBS2 expression and the prognosis was analyzed by follow-up.Low expression of THBS2 suggests poor prognosis.Conclusion:1.Down-regulation of miR-1246 promotes cell apoptosis and restrain invasion.2.Down-regulation of miR-1246 affects the expression of THBS2,ECM and MMPs.3.Decreased THBS2 expression is inversely associated with malignant potential or differentiation of cervical cancer as a prognosis biomarker and targeted therapy point.Low expression of THBS2 suggests poor prognosis.