The Mechanism Of MiR-652 In The Development And Progression Of Endometrial Cancer

BackgroundEndometrial cancer(EC)is the most common gynecologic malignant tumor,is the fourth most common among all women malignancy.The morbidity of EC is on the rise in recent years.The EC is classified into two type according to the clinicopathologic feature and pathogenesis:estrogen-dependent type(type ?)and estrogen-independent type(type ?).Recent studies found that there are molecular and morphological heterogeneity among the same type of EC,therefore,researchers divided EC into subtypes based on the immonohistochemical and molecular characteristics,in order to further stratify risk categories and get better survival.Though the 5-year survival rate of EC is up to be 82%,contrasting with the declining mortality rate in other type of cancers,the death rate of EC rose about 2%per year from 2010 to 2015.There are many factors that can influence the prognosis of EC,such as the stage,grade and metastasis of lymph node.The development and progression mechanism of EC is unclear,and the effective treatment for advanced and recurrent EC is still lacking.Further mechanism studies for EC is urgent needed to get better prognosis of EC.MicroRNA(miRNA)is a type of non-coding RNA,with the length of 22 nucleotides.The miRNA has been demonstrated to play important roles in the development of malignant tumors through binding to un-translational region(UTR)of the target gene,which can lead to the degradation of the mRNA or repression the translational process.MiRNA is involved in many biological processes,such as proliferation,metastasis and chemoresistance.Approximately one third of the human genes can be regulated by miRNA at the post-transcriptional level.Though recent studies have found the aberrant expressions of miRNA between EC tissue and normal endometrium tissue,the mechanism the miRNA in EC is still unclear.Studies have found that miR-652 can play important roles in different malignant tumors,such as act as an oncomir in human breast cancer and osteosarcoma,and a tumor suppressor gene in malignant pleural mesothelioma.But the mechanism of miR-652 in EC was still unclear.Hence,in this research,we focus on the aberrant expression of miR-652 in EC and study the mechanism of miR-652 in EC carcinogenesis.The discovery will improve the treatment for EC patients,especially those at advanced stage.The research include three parts as follows:1.The aberrant expression of miR-652 in EC and the clinical significance;2.The role of miR-652 in proliferation and metastasis in EC;3.The downstream target of miR-652 and its function in EC.Part ? The expression of miR-652 and its clinical significance in ECObjective:Recent studies found that miR-652 expressed abnormally in various malignant tumors,the expression of miR-652 is higher in lung cancer,breast cancer,osteosarcoma than normal tissues and the expression of miR-652 in malignant pleural mesothelioma is lower than normal tissues.Through analyzed the expression of miRNA in the TCGA database,we found that the expression of miR-652 is higher in EC tissues than normal endometrium tissues.But the functional role of miR-652 in EC is largely unknown.In this study,we verified the aberrant expression of miR-652 in EC and analyze the correlation between miR-652 expression and the EC clinical characteristic,to estimate the value of application in EC treatment.Methods:Through analyze the TCGA database,find out the aberrant expression miRNAs in EC.The tissue samples was collected from April 2015 to December 2017 at Qilu Hospital,74 tissues was collected,including 22 normal endometrium tissues and 52 EC tissues.RNA was extracted and the expression of the miRNA was verified through qRT-PCR.Finally,considering both the qRT-PCR results and the published articles,we selected miR-652 for further research.Cox regression test was used to analyze the role of miR-652 in EC prognosis.We also analyzed the correlation of miR-652 with the tumor differentiation,stage,lymph node metastasis.Results:Through the analysis of TCGA database,we found the expression of miR-652 was higher in EC tissues than normal endometrium tissues.The results of qRT-PCR also get the same results.Also,the expression of miR-652 was higher in endometrial serous carcinoma(ESC)tissues than endometrial endometrioid carcinoma(EEC)tissues.The expression of miR-652 in EC had no relationship with the level of estrogen,which means the up-regulation of miR-652 was tumor-specific.Based on the analysis of the TCGA database,Cox Regression Test was analyzed,we found that the pateints with high expression level of miR-652 exhibited shorter overall survival(OS)than those with low miR-652 expression(p=0.03).We further analyzed the correlation between miR-652 expression and other clinicopathological parameters,besides histological grade,miR-652 also had correlation with tumor recurrence.Conclusion:The expression of miR-652 is higher in EC tissues than normal endometrium tissues.The higher expression of miR-652 is correlated with poor prognosis of EC.miR-652 can be a novel marker to evaluate the recurrence of EC.Part ? The role of miR-652 in proliferation and metastasis in ECObjective:Recent studies found that miRNA can be involved in various processes of cancers,such as proliferation,metastasis,apoptosis,autophagy etc.miR-652 expressed aberrantly in different kind of malignant tumors and involved in the process of carcinogenesis.miR-652 can act as an oncogene in tumors like lung cancer,breast cancer and osteosarcoma.But in malignant mesothelioma,the expression of miR-652 is downregulated and can act as a diagnostic maker.In EC,we found the expression of miR-652 was higher in EC tissues than that in normal endometrium tissues,but we still have no idea about the effect of miR-652 in EC.In this study,we mainly investigate the effect of miR-652 in EC through up and down regulate the expression of miR-652 in EC cells.Through,we hope to find out the value of miR-652 in the diagnosis and therapy of EC.Methods:Firstly,EC cells was transient transfected with miR-652 mimics and inhibitors to up and down to regulate the expression of miR-652.Then,in vitro,MTT assay and plate clone formation assay was performed to measure the proliferation effect of miR-652 on EC cells,7AAD-PE apoptosis experiment was used to test the effect of miR-652 on apoptosis.Through transwell assay,we investigate the effect of miR-652 on migration and invasion in EC cells.In vivo,we constructed EC cells stably expressed miR-652 with the vector of PGIPZ.We injected the mice in subcutaneous and intraperitoneal with miR-652 overexpressed and the control Ishikawa cells,respectively.In the subcutaneous injection assay,the size of the tumor mass was measured to evaluate the effect of miR-652 on tumor growth;and in the intraperitoneal injection assay,the number and volume of the tumor mass was analyzed to know the effect of miR-652 on tumor metasis.Results:To investigate the possible role of miR-652 in EC,we established three EC cell lines with transient miR-652 up-regulation and down-regulation,and another three cell lines with stable miR-652 overexpression.we observed the morphological changes in miR-652 overexpressed cells.AN3 CA and Ishikawa cells overexpressing miR-652 exhibit mesenchymal morphology.In EC cell lines,the cell proliferation assays and colony formation assays revealed that overexpression of miR-652 can significantly promote EC cell proliferation,whereas RNAi-mediated silencing of miR-652 decreased cell growth ratio.the migration and invasion effect of miR-652 was analyzed using transwell assay and wound healing assay,miR-652 can promote migration and invasion in EC cells,and as predicted,transfection of inhibitory miR-652 can markedly impair the migrating and invading capacity.Western blot showed that up-regulation of miR-652 can promote the expression of proteins that can promote EMT.Up-regulation of miR-652 can accelerate the cell cycle period,but no influence was showed on apoptosis.In vivo,in the subcutaneous injection assay,the size and weight of the tumor mass in the miR-652 overexpressed group was significantly larger than that in the control group;In the intraperitoneal injection assay,the number and volume of the tumor mass in the miR-652 overexpressed group was larger than that in the control group.All the results showed that up-regulation of miR-652 can promote tumor growth and metastasis both in vivo and in vitro.Conclusions:miR-652 can promote proliferation and metastasis in EC both in vitro and in vivo,which act as an oncomir in EC and promote the progression of EC.Part III The downstream target and regulation mechanism of miR-652 in ECObjectives:It was known that miRNA can bind with the 3'UTR region of the target gene to decay the mRNA or repress the translation to regulate the expression of the target.On miRNA can bind with the 3'TUR region of various genes,also,one target gene can be regulated by hundreds of miRNAs.Researchers have found that miR-652 can exert different effects through binding with different targets in various tumors.The effect of miR-652 on proliferation and metastasis in EC has been demonstrated,but the mechanism of how miR-652 effects was still unclear.In this study,we aim to find out the downstream target of miR-652,and investigate the mechanism of miR-652 in EC.Methods:Through screening the predictive database such as Targetscan,miRNAMap,miRDB and combined with the results of RNA-sequence in EC cells,we found that RORA may be one of the downstream target of miR-652.The 3'UTR region of RORA was analyzed to find the combination site of miR-652.qRT-PCR and western blot assay was performed to investigate the RNA and protein level of RORA in EC up and down regulated EC cells,respectively.The RNA and protein level of RORA in EC tissues and normal endometrium tissues was studied.IHC was used to test the expression of RORA in mice subcutaneous tumor mass and EC tissues.To investigate whether RORA was directly targeted by miR-652,we insert the 3'UTR region of RORA into pmirglo vector and performed luciferase assay.Rescue assay was also performed to verify that miR-652 exert oncogenic effect in EC through target RORA.Results:Through screening the database of Targetscan,miRNAMap and miRDB,we found that RORA may be one of the target gene,and in the results of RNA-sequence,RORA was downregulated in the miR-652 overexpression group.The RNA and protein level of RORA was lower in EC tissues than normal endometrium tissues.In the IHC assay,the expression of RORA was also higher in normal endomterim tissues than the EC tissues.In EC cells,the expression of RORA was downregulated in miR-652 overexpression EC cells both at the RNA and protein level.In the suncutenous tumor mass,IHC assay showed the expression of RORA was weaker in miR-652 overexpression group than the control group.Luciferase assay showed that the fluorescence intensity of the wild-type pmirGLO-RORA vector was weaker when miR-652 was overexpressed,but not in the mutant vector.Rescue assay showed the overexpression of RORA can inhibit the proliferation of Ishikawa cell and the promotion effect of miR-652 can also be reversed.Interfering of RORA can promote the proliferation and metastasis effect of EC cells.The Western blot assay showed that interfering RORA can promote the expression of p-catenin in EC.Conclusions:RORA was the downstream target of miR-652 in EC.miR-652 exerts its oncomir effect through targeting RORA.