The Effects And Mechanism Of MiR-320a Target Regulating RASSF8 On The Proliferation And Metastasis Of Epithelial Ovarian Cancer

BackgroundOvarian malignant tumor is the most common malignant tumor in female genital tract tumors.It includes a variety of pathological types.Epithelial ovarian cancer(EOC)is the most common ovarian malignant tumor.Located at deep pelvic cavity,early diagnosis of ovarian cancer is difficult,and the lack of effective screening means,so most ovarian cancer patients were diagnosed at advanced stage.In addition,ovarian cancer is prone to extensive pelvic and abdominal metastasis,lymph node metastasis,so epithelial ovarian cancer is the most lethal of all female reproductive system tumors.The tumorigenesis and development of this tumor are affected by many factors,including cell movement,misregulation of tumor suppressor or oncogene expression,and abnormal migration regulation.The current standard of treatment for ovarian cancer is primary tumor resection combined with adjuvant chemotherapy.Although most ovarian cancer patients initially respond to this treatment regimen,but most eventually die from relapse or chemotherapy resistance.Further exploration of the molecular mechanisms of ovarian cancer pathogenesis and progression is urgently needed.It is known that the occurrence and development of malignant tumors are affected by many factors,including oncogene activation,inactivation of tumor suppressor genes,resulting in abnormalities in the relevant signaling pathways in the body,promoting cells proliferation,formation of new blood vessels,distant metastasis of tumor cells,etc.At present,the specific mechanism of tumor metastasis has been widely investigated but remained unclear.It is concluded that tumor cells are involved in the blood circulation system during the movement and supported by the tumor microenvironment,so it is considered to be the initiator of most tumor metastasis,and when the tumor microenvironment promotes tumor cell movement,the main mechanism is Epithelial-Mesenchymal Transition(EMT),which is the morphological process of epithelial cells transforming into mesenchymal cells under specific conditions,playing a role in cancer invasion-metastasis.The process involves reversible,rapid changes in many genes.More and more studies have found that there is a close relationship between tumor cells and Epithelial-Mesenchymal Transition(EMT).EMT plays an important role in the process of cancer invasion and metastasis,and involves reversible and rapid changes of many genes.EMT-related transcription factors,such as Slug,Snail and Zeb1,can induce EMT in tumor cells and promote early invasion and metastasis of tumors.A number of studies have revealed the influence of EMT on tumor cells.In addition to enhancing the ability of cell invasion and migration,EMT induced by transcription factor transfection also showed stem cell characteristics,which enhanced their apoptosis resistance.The research on EMT-related transcription factors is still in the early stage,and the mechanism of EMT is extremely complicated.Not only the transcription factors are involved,microRNAs(miRNAs)also become a hotspot in EMT and tumor progression research.Increasing evidence showed that microRNAs(miRNAs)play an important role in tumor genesis,progression and metastasis,and may provide effective therapeutic strategies for cancer patients.miRNAs are small non-coding RNAs(19-24 nt in length)that regulate the expression of target genes by binding to the 3’-untranslated region(3’-UTRs),leading to translation inhibition or mRNA degradation.The expression of miRNAs and their regulated target genes constitute a regulatory network that dynamically regulates various biological behaviors.To date,miR-320a has been found either up-regulated or down-regulated in many types of cancer,such as hepatocellular carcinoma,colon cancer,gastric cancer and prostate cancer.However,miR-320a related studies in EOC have rarely been reported.A recent study analyzed data of ovarian cancer samples downloaded from GEO(Gene Expression Omnibus),and obtained 17 significantly differentially expressed miRNAs.The co-expressed network showed that the higher the expression of miR-320,the worse the prognosis of EOC,but the specific mechanism of 320a in EOC has not been reported yet.The purpose of this study was to investigate the expression of miR-320a and its biological effects in EOC.Bioinformatics software predicted that miR-320a has a potential binding site in the 3’-UTR region of RASSF8 mRNA,suggesting that miR-320a may have a targeted regulation of RASSF8 expression.RASSF8 was found essential for maintaining AJs(adhesion adhesion sites)of epithelial cells and plays a role in epithelial cell migration.RASSF8 was found widely expressed in normal tissues of mouse embryos and human adults.RASSF8 promotes apoptosis and regulates actin cytoskeleton to prevent the genesis and metastasis of lung tumors,possibly through Wnt and NF-κB signaling pathways,and was described as a potential tumor suppressor for lung cancer,cutaneous melanoma,and esophageal squamous cell carcinoma.However,the role of RASSF8 in EOC has not been reported.Here we conduct this current research,in order to reveal the expression and the role of RASSF8 plays in EOC,the possible interaction between RASSF8 and miR-320a,and the underlying regulatory mechanism,and therefore provide theoretical basis for developing novel treatment strategies for EOC.This study aims to investigate the expression and biological effects of miR-320a in EOC,that whether it has an effect on cell proliferation and metastasis,and further elucidate the underlying mechanisms of these effects,that if this effect was achieved by targeting regulation of RASSF8.Part I:Expression and functional study of miR-320a in epithelial ovarian cancer tissues and cell linesObjectives1.To study the expression of miR-320a in epithelial ovarian cancer and the relationship with clinicopathological features;2.To explore the biological role of miR-320a in epithelial ovarian cancer cells and to explore its possible mechanisms in the development of epithelial ovarian cancer;Methods1.Real-time quantitative PCR was used to detect the expression of miR-320a in normal ovarian tissues,benign ovarian tumors and epithelial ovarian cancers,and the relationship between miR-320a expression and clinicopathological features were analyzed.2.The expression of miR-320a in ovarian cancer cell lines(SKOV3,OVCAR3,A2780)and normal ovarian cell line IOSE80 were examined.3.The SKOV3 cell line stably overexpressing miR-320a was constructed by using lentivirus in SKOV3 cells with low miR-320a expression level,and miR-320a-inhibitor was transiently transfected into OVCAR3 cells with higher miR-320a expression level,and CCK-8 assay,Transwell chamber model,and cell flow technique were used to evaluate the metastasis,neogenesis and apoptosis of ovarian cancer cells.The tumorigenicity test of nude mice was used to evaluate the tumorigenic ability of ovarian cancer cells in nude mice after miR-320a overexpression,and the role of miR-320a in ovarian cancer cells was further verified from in vivo experiments.4.Western blot analysis was used to detect changes in EMT-related indicators in ovarian cancer cell lines after miR-320a overexpression or downregulation.HE staining was used to observe the morphology and infiltration of tumor cells in the tumor tissues of nude mice,and immunohistochemistry was used to evaluate the expression changes of E-cadherin and Vimentin in the tumors of nude mice.Results1.The expression level of miR-320a in EOC tissues were higher than those in normal ovarian tissues and benign ovarian tumors;the expression level of miR-320a in advanced ovarian cancer tissues(stage Ⅲ,Ⅳ)were higher than those in early ovarian cancers(stage Ⅰ,Ⅱ)(P<0.05),the expression level of miR-320a in the lymph node metastasis group was higher than that in the no lymph node metastasis group(P<0.05),but there were no difference in age,histological classification,and pathologic grade in the expression level of miR-320a(P>0.05).2.The expression of miR-320a in three cell lines(SKOV3,OVCAR3,A2780)was higher than that of normal ovarian cells(IOSE80).3.miR-320a can promote metastasis and neogenesis of ovarian cancer cells,but there are no obvious effect on the apoptosis of ovarian cancer cells.Tumor formation in nude mice also confirmed that miR-320a can promote the proliferation of ovarian cancer cells.4.miR-320a can promote the EMT behavior of ovarian cancer cells.Compared with the control cells,overexpression of miR-320a decreased the expression of E-cadherin in SKOV3 cells,while the expression level of Vimentin increased.Down-regulation of miR-320a increased the expression of E-cadherin in OVCAR3 cells,while the expression level of Vimentin is reduced.The expression levels of E-cadherin and Vimentin in nude mice were detected by immunohistochemistry,The results showed that,compared with SKOV3/miR-NC group,the expression levels of Vimentin were relatively higher in SKOV3/miR-320a tumor group,but E-cadherin expression levels were lower.These findings also support the hypothesis that miR-320a may promote EMT in EOC cells.Conclusion1.miR-320a’s high expression in epithelial ovarian cancer tissues and cell lines suggests that miR-320a may be a potential oncogenic gene in ovarian cancer,which plays an important role in the occurrence and development of epithelial ovarian cancer.2.miR-320a can promote metastasis and neogenesis of ovarian cancer cells,but has no obvious effect on the apoptosis of ovarian cancer cells.3.miR-320a can promote the EMT behavior of ovarian cancer cells.Part Ⅱ:Prediction and identification of miR-320a target gene RASSF8ObjectiveOur previous study found that miR-320a was up-regulated in epithelial ovarian cancer tissues and cell lines,suggesting that miR-320a may be involved in the development of ovarian cancer and it can be a Oncogene.So which target gene does miR-320a play its biological function in ovarian cancer?This part of the study predicts the target gene of miR-320a by bioinformatics methods,and further verifies how miR-320a targets the target gene in ovarian cancer cells.The clarification of this issue will further deepen our understanding of the pathogenesis of ovarian cancer.Methods1.Use the miRNA bioinformatics prediction software database(PicTar,TargetScan,miRanda)to find the target genes for preliminary prediction.2.The mRNA expression levels of RASSF8 in ovarian cancer cell lines(SKOV3,OVCAR3,A2780)and normal ovarian cell lines(IOSE80)were detected by RT-PCR.The role of RASSF8 in EOC was preliminarily investigated.3.We used dual luciferase reporter gene vector to Construct wild-type pGL3-RASSF8-WT and mutant pGL3-RASSF8-Mut,co-transfecting cells with miR-320a to observe the luciferase activity of miR-320a on pGL3-RASSF8.And further to determine whether miR-320a can directly regulate the 3’-UTR region of RASSF8.4.To investigate the relationship between the expression levels of miR-320a and RASSF8 in ovarian cancer cells,we transiently transfected miR-320a mimic in SKOV3 cells and detected the expression of RASSF8 in miR-320a transfected cells by qRT-PCR.Then,we examined the effects of RASSF8 mRNA and protein expression in SKOV3 cells after miR-320a overexpression by RT-PCR and Western Blot.Further verify the relationship between the two gene.Results1.Bioinformatics analysis revealed that RASSF8 is a target gene of miR-320a.2.The expression of RASSF8 in ovarian cancer cells(SKOV3,OVCAR3,A2780)were lower than those of normal ovarian cells(IOSE80).3.miR-320a can target the 3’-UTR region of RASSF8.4.Overexpression or downregulation of miR-320a can negatively regulate mRNA and protein levels of RASSF8 in ovarian cancer cells.Conclusion1.RASSF8 is the target gene of miR-320a.2.miR-320a can down-regulate the expression of RASSF8 in ovarian cancer.Part III The preliminary study on the influence of RASSF8 on the biology of epithelial ovarian cancer and its regulation mechanismObjectives1.To study the expression of RASSF8 in epithelial ovarian cancer tissues;2.To investigate the biological role of RASSF8 in epithelial ovarian cancer cells and to explore its possible regulatory mechanisms in epithelial ovarian cancer.Methods1.Immunohistochemistry was used to detect the expression of RASSF8 in normal ovarian tissue,benign ovarian tumor and epithelial ovarian cancer.2.Transfect siRNA targeting RASSF8 and Negative Control into SKOV3 cells,and CCK-8 assay and Transwell chamber model were used to evaluate the proliferation,invasion and migration of ovarian cancer cells after RASSF8 down-regulation.3.Western blot analysis showed that the expression of NF-κB signaling pathway P65,p-P65 and IκBα was down-regulated after RASSF8 down-regulation.Results1.Immunohistochemistry results showed that the expression of RASSF8 in ovarian cancer tissues were significantly lower than those in normal ovarian epithelium tissues and benign ovarian tumors,and the difference was statistically significant(P<0.05).The expression of RASSF8 mRNA in 20 ovarian cancer tissues and 20 adjacent normal tissues were studied by RT-PCR.The results showed that the mRNA level of RASSF8 was significantly decreased in ovarian cancer(P<0.01),suggesting that RASSF8 may be a tumor suppressor gene in EOC.2.RASSF8 can inhibit the proliferation,invasion and migration of ovarian cancer cells.Compared with the negative control group,down-regulation of RASSF8 promoted the proliferation of SKOV3 cells,and the number of invasion and migration cells was higher.3.RASSF8 exerts a tumor suppressor effect by regulating NF-κB signaling pathway.The results showed that the expression of P65 and p-P65 increased and the expression of IκBα decreased after RASSF8 down-regulation,compared with the control group,the difference was statistically significant.ConclusionRASSF8 plays a tumor suppressor role in epithelial ovarian cancer and may play a role in regulating NF-κB signaling pathway.