Role Of MARCH7 Regulating Malignant Progress Of Endometrial Cancer And Its Mechanism

Endometrial cancer(EC)is a tumor originating from endometrium,one of the most common gynecological tumors,and ranks the third leading cause of death of female genital cancers.Most women with endometrial cancer are diagnosed early,usually after vaginal bleeding.Although the overall prognosis of endometrial cancer is favorable,some women still have invasive neoplasms,such as deeply aggressive tumors,which increase the risk of progression and death.Even if detected early,metastasis and recurrence may be the main causes of high mortality.About 30%of cases with endometrial cancer have been diagnosed at stage ? or ?,thereby having a poor prognosis.Of those factors that lead to terrible clinical outcome,metastasis plays the determining role.Therefore,there is still an urgent need to investigate the mechanism of metastasis and progression of endometrial cancer,which will help to improve the overall survival rate of cancer patients.Exploring metastasis-related genes and the underlying mechanisms may develop anti-metastasis strategies.The formation of metastasis of malignant tumors requires a series of steps,including invasion,infiltration,extra osmosis and metastasis.The cellular adhesion of tumor cells is decreased.Tumor cells detach from the primary tumor site,acquire migration ability and leave the primary tumor site,infiltrate vascular endothelium,invade blood vessels and lymphatic circulation,and then infiltrate into distant areas to form metastasis.Epithelial-mesenchymal transition(EMT)is closely related to oncogenesis.Cancer cells acquire the ability of migration and invasion through EMT.Epithelial cancer cells obtain some changes through EMT,concluding loss of cell polarity,loosing of cellular junction,and cytoskeletal reorganization.The adhesion of cells is decreased,migration and difusion of cells is increased,which promotes the invasion and metastasis of tumors.Therefore,EMT plays an important role in cancer metastasis.Transcription factors(TF)are essential for EMT.Many transcription factors,including Snail,Zeb and Twist,induce EMT.And which are associated with invasion and metastasis of tumors.Many signaling pathways are involved in EMT during oncogenesis.Tyrosine kinase receptors,TGF-?,Wnt,Hedgehog,?-catenin,Stat3,Notch and Nanog signaling pathways have been proved to be involved in regulating EMT.EMT is an instantaneous and dynamic process.At present,there is no definite evidence on the efficacy of anti-cancer therapy for EMT in vivo.To explore the mechanism of EMT and tumor evolution in depth,which will provide a new strategy for treating advanced or metastatic tumors. microRNAs are a group of small RNAs,and consist of 19-25 nucleotides.They are highly conserved in the evolutionary process.They regulate gene expression through posttrans-lational modulations.In the process of oncogenesis,microRNAs play the role of oncogene or anti-oncogene through their own up-regulation or down-regulation.Many kinds of microRNAs have been proved to regulate EMT.microRNAs can directly target EMT transcription factors and regulate tumor metastasis.Therapeutic strategies for EMT may be an important direction of anti-cancer therapy.Understanding the physiological functions of microRNAs may promote the development of prognostic assessment and therapeutic strategies for cancer.E3 ubiquitin ligase MARCH7 is a member of the membrane-associated RING finger protein family.MARCH7 has numerous cellular functions,including immune regulation,quality control of protein,membrane trafficking,and spermatogenesis.Previous studies have showed that MARCH7 may promote cell proliferation,migration,invasion and autophagy.Some studies have found that autophagy promotes EMT.EMT plays an important role in cancer progression.EMT may be a potential mechanism for MARCH7 regulating cancer progression.Further exploration the microRNAs of the upstream regulation factors will be helpful to understand the mechanism of cancer progression.At present,there is no study on MARCH7 in endometrial cancer.In this study,the role of MARCH7 in invasion and metastasis of endometrial cancer and its underling mechanisms are investigated,which provides experimental and theoretical basis for exploring new molecular markers of prognosis evaluation and follow-up,and new targets for treatment of metastatic tumors in endometrial cancer.Part one Expression and significance of MARCH7 in endometrial cancerObjectiveTo detect the expression of MARCH7 protein in endometrial cancer tissues,and explore the relationship between the expression of MARCH7 and clinicopathological characteristics in endometrial cancer.MethodsImmunohistochemistry was used to detect the expression of MARCH7 in non-neoplastic endometrial tissues(n=20)and endometrial cancer tissues(n=46).To analyze the correlation between the expression of MARCH7 and clinicopathological characteristics.To analyze the correlation between the expression of MARCH7 mRNA and clinicopathological characteristics of endometrial cancer tissues(n=54)from TCGA database.Results(1)MARCH7 protein was predominantly localized on the plasma membrane and cytoplasm.The expression of MARCH7 was remarkably increased in endometrial tumor tissues compared with normal tissues(p<0.05).To determine the correlation of MARCH7 expression with tumor stage and tumor grads,all cancer samples were grouped according to tumor stage(stage ?/?,and stage ?/?)and pathological grade(grades 1,and grades 2-3).The MARCH7 level of advanced tumor(stage ?/?)was higher than that of early stage lesions(stage ?/?)(P<0.05).Furthermore,the staining intensity was significantly correlated with the pathological grade(grade 2-3 versus grade 1;P<0.05).However,the association between the expression of MARCH7 and age was not significant(P>0.05)(2)The relation between the expression of MARCH7 mRNA and clinicopathological characteristics of endometrial cancer tissues form TCGA database was analyzed.In consistence with our IHC analysis,the expression level of MARCH7 of advanced endometrial cancer(stage ?/?)was higher than that of early endometrial cancer(stage ?/?)(p<0.05);the higher the grade of tumors(grade 2 or grade 3 versus grade 1),the higher the expression level of MARCH7(p<0.05)Conclusion The expression level of MARCH7 was higher in endometrial cancer tissues,and the expression of MARCH7 was associated with the FIGO stage and pathological grade of endometrial cancer.Part two Effects of MARCH7 on malignant behavior of the endometrial cancer HEC-1-A and Ishikawa cell linesObjectiveTo investigate the malignant biological behavior including the proliferation,invasion,metastasis of endometrial cancer cell lines,when silencing of MARCH7 in Ishikawa cells or over-expression of MARCH7 in HEC-1-A cells was done.Methods(1)The expression of MARCH7 mRNA in human endometrial cancer cell lines(Ishikawa,HEC-1-A,AN3CA,RL95e-2)was detected by qPCR.(2)CCK-8 assay was used to detect the cell proliferation after silencing of MARCH7 in Ishikawa cells or over-expression of MARCH7 in HEC-1-A cells.(3)Transwell assay was used to detect the cell migration and invasion after silencing of MARCH7 in Ishikawa cells or over-expression MARCH7 in HEC-1-A cells.(4)A metastasis nude mice model was employed to validate the role of MARCH7 in the invasion and metastasis of endometrial cancer cells in vivo,after silencing of MARCH7 in Ishikawa cells.Results(1)The basal level of MARCH7 was investigated in four cell lines(Ishikawa,HEC-1-A,AN3CA,and RL95-2)at the mRNA level by real-time quantitative PCR(qPCR).Of these,a higher level was noted in Ishikawa and RL95-2 cells,and a lower lever was detected in HEC-1-A and AN3CA cells.(2)CCK-8 assay showed that the cell proliferation of HEC-1-A cells was enhanced after over-expression of MARCH7(P<0.05).However,the cell proliferation of Ishikawa cells was inhibited after silencing of MARCH7(P<0.05).(3)Transwell assay showed that down-regulation of MARCH7 with sh-MARCH7-1 or sh-MARCH7-2 could reduce the migration and invasion of endometrial cancer Ishikawa cells(P<0.01).On the other hand,over-expression of MARCH7 resulted in an increase of cell migration and invasion compare to infection with control vector only in HEC-1-A cells(P<0.05).(4)The sh-MARCH7 group of Ishikawa cells showed less abdominal tumor burden and number of metastatic lesions in the liver and lung,compared with the sh-NC group after injection.ConclusionThe cell proliferation,invasion,and metastasis were decreased,after silencing of MARCH7 in Ishikawa cells.The cell proliferation,invasion,and metastasis were increased,after over-expression of MARCH7 in HEC-1-A cells.Part three Mechanisms of MARCH7 in malignant behavior ofendometrial cancerObjectiveTo explore the potential mechanism of MARCH7 regulating invasion and metastasis of endometrial cancer.Methods(1)Immunohistochemistry was used to detect the expression of EMT-related proteins(Snail,Vimentin and E-cadherin)in non-neoplastic endometrial tissues(n=20)and endometrial cancer tissues(n=46).And analyze the correlation between the expression of MARCH7 and that of EMT-related proteins.(2)Western blot analysis was used to detect the expression of EMT-related proteins(Snail,Vimentin and E-cadherin)after silencing of MARCH7 in Ishikawa cells or over-expression of MARCH7 in HEC-1-A cells.(2)Infection of Ishikawa cells by Snail siRNA,the cell invasion,metastasis and EMT-related proteins were detected.(3)Infection of Ishikawa cells by MARCH7 lentiviral vector,and infected with Snail siRNA.the cell invasion,metastasis and EMT-related proteins were detected.(4)The expression of MARCH7,Snail and E-cadherin in the tumor of intraperitoneal transplantation was detected by immunohistochemical staining.Results(1)To determine the correlation between the expression of MARCH7 and that of EMT-related proteins;E-cadherin,Snail,and Vimentin in endometrial cancer tissues and normal tissues were detected by imunohistochemical staining.The expression level of Snail,and Vimentin was increased in endometrial cancer tissues,compared with that in normal tissues;however,the level of E-cadherin was decreased in endometrial cancer tissues(P<0.05).The expression level of Snail and Vimentin was positively associated with FIGO stage and tumor grade(P<0.05),but the level of E-cadherin was negatively associated with FIGO stage and pathologic grade(P<0.01).Meanwhile,the level of MARCH7 was positively correlated with that of Snail or Vimentin,and negatively correlated with that of E-cadherin(P<0.05).(2)Western blot analysis showed that the level of E-cadherin was decreased in HEC-1-A cells,accompanied with the increase of Vimentin and Snail,due to over-expression of MARCH7;while these effects were reversed in Ishikawa cells after silencing of MARCH7(P<0.05).(2)Silencing of Snail inhibited invasion of Ishikawa cells,accompanied with the decrease of Snail and Vimentin,and the increase of E-cadherin(P<0.05).(3)Over-expression of MARCH7 promoted cell invasion,accompanied with the decrease of E-cadherin and the increase of Vimentin and Snail in Ishikawa cells;and these effects were partially restored after knockdown of Snail(P<0.05).(4)Immunohistochemical staining results indicated that the level of E-cadherin was increased,and the level of Snail and MARCH7 was decreased in xenograft tumor tissues formed from Ishikawa cells infected with sh-MARCH7-1.(P<0.05).ConclusionThe expression of EMT-related proteins(Snail,Vimentin and E-cadherin)was associated with the FIGO stage and grade of endometrial cancer.The expression of MARCH7 was associated with that of EMT-related proteins in endometrial cancer tissues.MARCH7 may regulate cell invasion,metastasis and the EMT-related proteins of Ishikawa cells.MARCH7 could partially regulate cell invasion and EMT-related proteins through Snail-dependent pathwayPart four Effects of miR-27b-3p targeting MARCH7 on malignant behavior of endometrial cancer Ishikawa cell linesObjectiveTo study the effects of miR-27b-3p regulating MARCH7 and its effect on malignant behavior of endometrial cancer Ishikawa cells.Methods(1)The binding site between miR-27b-3p and MARCH7 was predicted by bioinformatics analyses.(2)Infected with miR-27b-3p mimics,the expression level of MARCH7 protein was detected in Ishikawa cells.(3)For MARCH7 3' UTR luciferase reporter assay,wild type or mutant reporter was constructed(termed MARCH7-wt or MARCH7-mut).Infection of Ishikawa cells by miR-27b-3p mimics,then the luciferase activity was detected by dual luciferase reporter gene detection system.(4)Infection of Ishikawa cells by miR-27b-3p mimics,and infected by MARCH7 lentiviral vecton the invasion,metastasis the EMT-related proteins(Snail and E-cadherin)of Ishikawa cells were detected.Results(1)Bioinformatics analyses showed that MARCH7 was a predicted target of miR-27b-3p(http://www.targetscan.org/);(2)The expression of MARCH7 was significantly decreased after infection of endometrial cancer Ishikawa cells by miR-27b-3p mimics(P<0.05).(3)It was found that the luciferase activity was inhibited in miR-27b-3p mimics group when Ishikawa cells were transfected with MARCH7-Wt,compared with mimics-NC group,while there is no suppress effect in Ishikawa cells transfected with MARCH7-mut.(4)It was found that miR-27b-3p mimics could down-regulate the protein level of Snail,up-regulate the protein level of E-cadherin,and inhibit the migration and invasion of Ishikawa cells(P<0.05).The phenotype can be partially restored by over-expression of MARCH7(P<0.05).ConclusionmiR-27b-3p could regulate the metastasis and invasion of endometrial cancer Ishikawa cells.MARCH7 is a direct target gene of miR-27b-3p.