Function And Mechanism Of MiR-130a In The Development Of Serous Ovarian Cancer

Ovarian cancer is a common gynecologic malignant tumor,the mortality rate has been ranked first in gynecologic malignant tumor.Serous ovarian carcinoma(SOC)is the most common and most malignant subtype,with a five year survival rate of only 30%.The fundamental reason is the lack of understanding of the molecular mechanism of serous ovarian cancer,and the lack of clinical practical early diagnostic methods and effective treatment strategies.Therefore,to elucidate the molecular mechanism of SOC,and to screen related molecular markers,is of great value for early diagnosis,prevention and treatment.The mechanistic target of rapamycin(mTOR)signaling pathway regulates many major cellular processes and is implicated in an increasing number of pathological conditions,including cancer,obesity,type Ⅱ diabetes mellitus and neurodegeneration.mTOR signaling is altered in many human cancers.mTOR activation is common in human epithelial ovarian cancers.According to The Cancer Genome Atlas(TCGA),mTOR mutations were identified in 1.9%of ovarian cancers.Activation of mTOR signaling pathway is associated with poor prognosisof epithelial ovarian cancer.The tuberous sclerosis 1(TSC1)/TSC2 tumor suppressor complex serves as a repressor of the mTOR pathway.TSC1/TSC2 mutations or low expression of TSC1/TSC2 results in dysfunction TSC1-TSC2 complex and activation of mTORC1.TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2.TSC1 has been reported to be low expressed in many cancers,such as breast cancer,colorectal cancer and bladder cancer and so on.What’s more it is reported that TSC1 was decreased in serous ovarian cancer,suggesting that the TSC1 may become an new targets for diagnosis and treatment of SOC.MicroRNA(miRNA)is a kind of non-coding small molecule RNA that participates in post transcriptional regulation.It can regulate target genes by binding to mRNA 3’ untranslated region(UTR).We found that the 3’UTR of TSC2 is only 117nt,however the 3’ UTR of TSC1 is 4887nt.So we speculate TSC1 is more likely to be regulated by microRNAs.Therefore,this study is committed to clarify the expression of TSC1 in HGSOC,and search for miRNA targeting TSC1,to achieve regulation of mTOR pathway,and provide a new target for diagnosis and treatment of ovarian cancer.Therefore,this study is divided into the following three parts:Part Ⅰ:miR-130a suppresses TSC1 expression in serous ovarian cancerObjective:Although studies have shown that TSC1 plays a role as tumor suppressor gene in many tumors.However it is not clear in HGSOC yet.This study aims to prove its role in HGSOC and find miRNAs that can target TSC1.Methods:Western blot and immunohistochemical staining were used to detect the expression difference of TSC1 in HGSOC tissues and normal fallopian tube.The relationship between TSC1 and ovarian cancer staging was analyzed.We use Targetscan,miRDB and PicTar software to analyze the 3 ’UTR area of TSC1 to predict microRNAs that may regulate TSC1.We find miR-130a,miR-27 and miR-204 are candidates for regulating TSC1.The transient transfection method was used to transfer the candidate miRNAs into the A2780 and SKOV3 cell lines to detect the expression of TSC1 and preliminarily determine that miR-130a may regulate the TSC1.The level of the key proteins in the mTOR signaling pathway was further examined by western blot after the up and down regulation of miR-130a in the A2780 and SKOV3 cell lines.To prove that TSC1 is the target gene of miR-130a we conducted the dual luciferase reporter assay.We also examined the miR-130a expression by QPCR and analyze the correlation of the miR-130a and TSC1.Results:TSC1 showed loss or decreased expression in HGSOC tissues(n=28)compared with FT(n=12).We then examined TSC1 expression in HGSOC(n=109)and FT(n=43)by immunohistochemistry and analyzed the clinicopathologic parameters.It was shown that TSC1 had significantly lower intensity of immunostaining in HGSOC than in fallopian tubes.Low TSC1 expression level was significantly correlated with advanced tumor stage.Overexpression of miR-130a,but not miR-27 or miR-204,significantly reduced TSC1 expression.In contrast,miR-130a inhibitor restored the expression levels of TSC1.Moreover,overexpression of miR-130a significantly increased levels of p-mTOR,p-p70S6K and p-4E-BP1 whereas miR-130a inhibitor suppressed mTOR and its downstream signaling effectors.To confirm that TSC1 is a direct target of miR-130a,we then introduced TSC1 3’UTR(containing three putative miR-130a-binding sites)and corresponding mutant counterparts into pmiRGLO vector.These reporter vectors were then transfected into cells with and without miR-130aexpression.We found that miR-130a overexpression reduced the luciferase activity in cells transfected with the wild-type3’UTR of TSC1 but not in cells with mutant 3’UTR in A2780cell lines.Furthermore,we measured miR-130a expression level in HGSOC and found that miR-130a is significantly higher in HGSOC tissues(n=31)than normal FT tissues(n=11).Importantly,a significant correlation between high miR-130a and low TSCllevels was detected in the same collection of HGSOC specimens.Conclusion:miR-130a can suppress TSC1 expression by targeting its 3’UTR,which inhibits its expression and then activates the mTOR signaling pathway.And the decreased expression of TSC1 in HGSOC could be partly due to the overexpression of miR-130a.Part II:miR-130a promotes proliferation/invasion and inhibits autophagy of serous ovarian cancerObjective:in the first part,the targeted regulation relationship between miR-130a and TSC1 has been identified,so the effect of miR-130a over or down expression in ovarian cancer cells and what role miR-130a plays in the development of SOC are still remain unclear.Therefore,this part mainly explores the functional role of miR-130a in serous ovarian cancer cell lines.In addition,As miR-130a could activate mTOR signaling pathway by targeting TSC1 based on the conclusion of part I,we speculated that miR-130a might affect mTOR-dependent autophagy.Methods:3 kinds of ovarian cancer cell lines were constructed with miR-130a stable overexpression by lentivirus infection for the following experiment.And the effect of miR-130a on the proliferation of ovarian cancer cell lines was detected by the clonogenic assay,soft agar assay and MTT test.The effects of miR-130a on cell invasion were investigated by Transwell test and Western blot was used to detect the EMT related proteins.To further prove TSC1 downregulation as a mediator of miR-130a in ovarian cancer cells,we performed rescue experiment by co-transfecting miR-130a and TSC1 overexpression construct without 3’UTR into A2780 and SKOV3 cells respectively.In order to further clarify the effect of miR-130a on tumor growth and metasis in vivo,cells with and without miR-130a overexpression were subcutaneously inoculated in nude mice or injected into the nude mice by tail vein.Rapamycin and cell starvation treatment were used to induce autophagy.Autophagy related molecular markers were examined after over expression or down expression of miR-130a,to prove the effect of miR-130a on autophagy.Moreover,we monitored autophagic flux by confocal microscopy in A2780 cells infected with GFP-RFP-LC3 adenovirus.Results:Overexpression of miR-130a significantly increased the colony-forming efficiency and anchorage-independent cell growth in serous ovarian cancer cell lines,whereas miR-130a inhibitor reduced the clonogenicity in A2780 cells.Overexpression of miR-130a could also significantly enhance the proliferation of ovarian cancer cells.Moreover,miR-130a dramatically enhanced the invasive capacity of ovarian cancer cell lines,and inhibition of miR-130a decreased invasion potential in HEY cells.Inaddition,we measured epithelial-mesenchymal transition(EMT)markers by western blot.Overexpression of miR-130a decreased epithelial markers and elevated the levels of mesenchymal markers.The experiments in vivo also indicate that miR-130a overexpression in ovarian cancer cell lines enhances tumor growth and metastasis.Ectopic TSC1 expression attenuated the increase in colony-forming efficiency and the invasiveness caused by miR-130a mimics in A2780 and SKOV3 cells.On the other hand,miR-130a over expression inhibited the autophagy induced by Rapamycin or starvation.The autophagy flow test showed that miR-130a over expression decresed both the autophagy bodies and autophagic lysosomes,while inhibition of miR-130a,on the other hand,increased both autophagy bodies and autophagic lysosomes in Rapamycin or starvation induced autophagy.Conclusion:miR-130a over expression enhances proliferation/invasion and induces EMT in serou ovarian cancer cell lines in vitro and vivo.On the other hand,miR-130a can block rapamycin or starvation induced autophagy through down regulation of TSC1 in serous ovarian cancer cells.Part Ⅲ:miR-130a is transactivates by NF-κB in serous ovarian cancer.Objective:miR-130a has been shown to be transcriptionally upregulated by NF-κB in human biliary epithelial cells and cervical cancercells.But they haven’t clarify the direct regulation between them.So we next determined whether overexpression of miR-130a in SOC was regulated by NF-κB.Methods:the NF-κB pathway was activated by LPS and TNF-a,and the changes of miR-130a-TSC1-mTOR pathway were detected by QPCR and Western blot.We next investigated whether the miR-130a pri-miRNA is directly regulated by NF-κB.We examined the role of the predicted transcriptional binding sites in induction of miR-130a expression by promoter constructs(PGL4.21)in a luciferase reporter assay.And then we performed chromatin immunoprecipitation(ChIP)analysis using anti-NF-κB p65 antibody in A2780 cells in the presence or absence of TNF-a treatment.Results:LPS stimulation significantly increased miR-130a expression in A2780 cells whereas inhibition of NF-κB activation by pyrrolidine dithiocarbamate(PDTC)significantly reduced miR-130a expression triggered by LPS.Importantly,TNF-a treatment increased miR-]30a expression,suppressed TSC1 expression and activated mTOR signaling pathway in A2780 cells.And NF-κB p65 siRNA significantly repressed miR-130a expression and attenuated TNF-a induced miR-130a expression.The dual luciferase assy showed tha fragment2,which is located close to the transcriptional start site,yielded a robust transcriptional activity induced by TNF-a.The ChIP assay showed that treatment with TNF-a significantly induced NF-κB binding to miR-130a promoter region(sitel,site3 and site 4).Site 4 showed significant NF-κB binding in the absence of TNF-a.Conclusion:NF-κB transcriptionally upregulates miR-130a expression and consequently activates mTOR signaling pathway in response toinflammatory stimuli.This study focuses on the molecular mechanism of miR-130a to promote the development of serous ovarian cancer and elucidates its upstream and downstream regulatory mechanisms:TSC1 is frequentlydownregulated in HGSOC and low TSC1 expression level isassociated with advanced tumor stage.miR-130a is overexpressed in HGSOC and suppresses TSC1 expression bytargeting its 3’UTR.Furthermore,in response to inflammatorystimuli,NF-κB transcriptionally upregulates miR-130a expression in ovarian cancer cells.The main innovation points:1.The molecular mechanism of TSC1 low expression in serous ovarian cancer is clarified for the first time from point of miRNA,which provides a new target for the diagnosis and treatment of serous ovarian cancer.2.The molecular mechanism of miR-130a to promote the development of serous ovarian cancer was identified,and miR-130a was found to be a new type of autophagy inhibitor.3.The relationship between the NF-κB signaling pathway and the mTOR signaling pathway was established,and establish a new link between inflammation and mTOR pathway in HGSOC.The defects and future directions:1.We haven’t establish transgenic mice with miR-130a over or down expression.If we can establish this animal model,it will greatly enrich our understanding of the work mechanism of miR-130a.2.The ovarian cancer cell lines used in this study are older,and some researchers believe that some of the cell lines can not reflect the characteristics of serous ovarian cancer.Therefore,constructing new cell lines which can represent the characters of high and low grade serous ovarian cancer,is important and meaningful.