Studies On The Mechanism Of MiR-206 Inhibiting Proliferation And Invasion Of Endometrial Cancer Cells By Targeting HDAC6

Endometrial cancer(EC)is one of the three most common malignancy of the female reproductive tract,and the sixth most common cancer in women worldwide.With the increase of the average life expectancy and the change of life habits,the incidence of EC has continued to rise and rejuvenate in the past two decades.In China,EC is the second common gynecological malignant tumor after cervical cancer,accounting for about 20-30%of gynecological malignant tumors.In some developed cities,the incidence of EC has surpassed cervical cancer,ranking the first among female reproductive tract malignant tumors.The etiology of EC is still not yet unclear,the main risk factors including genetics,obesity,diabetes,hypertension,estrogen medication history,polycystic ovary syndrome,etc.EC has been classically classified into two broad subtypes,the so-called types I(estrogen dependent)and II(not estrogen dependent),of which type I tumors account for the majority(85-90%),characterized by low grade and good prognosis.Therefore,for EC,if it can be diagnosed early,it will benefit the prognosis of most patients.At present,the latest studies found that the traditional classification could not accurately reflect the biological behavior of endometrial carcinoma.According to TCGA genome data,EC could be divided into POLE mutation,microsatellite instability(MSI),low copy type(CN-low)and the high copy type(CN-high),the prognosis of first three is good,and the prognosis is worst for the last one.Therefore,there are still many topics worth exploring for the mechanism of EC development.In recent years,microRNAs(miRNAs)have become more and more important in research.Many studies have shown that miRNAs have important effects on cell proliferation,mitosis,apoptosis,and differentiation.A variety of miRNAs expression abnormalities have been confirmed in EC,such as up-regulation of miR-182 and down-regulation of miR-152.As an important member of the miRNA family,mir-206 is abnormally expressed in a variety of human malignant tumors,including lung cancer,gastric cancer,colorectal cancer,kidney cancer,breast cancer,glioma,neuroblastoma,rhabdomyosoma and osteosarcoma,etc.,its role involves various aspects of tumor development and metastasis.However,whether miR-206 is involved in the development of EC is still unclear.Through bioinformatics analysis,we predicted that histone deacetylase 6(HDAC6)is one of the potential target genes of miR-206.HDAC6 belongs to class ? histone deacetylase,unlike other subtypes of this family,HDAC6 possesses a variety of protein substrates other than histones,and its role is mainly in the cytoplasm.HDAC6 can regulate the transport and degradation of proteins,thus affect the shape and migration of cells.It has been documented that HDAC6 is involved in the regulation of AKT,WNT and other signaling pathways,affecting the formation and metastasis of several human cancers.including ovarian cancer and breast cancer.Therefore,whether HDAC6 is involved in the regulation of the occurrence and development of EC is worth exploring.Part I Evaluation of expression of miR-206 and HDAC6 in EC and their clinical correlation0 bjectives:1.To screen the differentially expressed miRNAs in EC tissue and to identify its targetgenes;2.To detect the expression of miR-206 and HDAC6 in EC tissues and cells;3.To explore the interaction between the expression of HDAC6 and clinical characters;4.To analyze the relationship between HDAC6 expression and EC prognosis.Methods:1.The miRNA chip of the online database(BCGSC)was used to screen differentially expressed miRNAs in EC tissue and adjacent normal control;2.Bioinformatics prediction were performed to predict the potential target genes of miR-206;3.The relationship between HDAC6 expression and prognosis of EC patients were explored by data mining through expression profiles from The Cancer Genome Atlas(TCGA);4.Real-time quantitative PCR(qRT-PCR)and Western blot were used to verify the expression of miR-206 and HDAC6 in EC tissues and cells;and the localization and expression of HDAC6 in EC tissues and its relationship with tumor grade were detected by immunohistochemistry.Results:1.We found 159 differentially expressed miRNAs between EC tissue and adjacent normal control through miRNA chip analyzing,among which,72 were down-regulated and 87 were up-regulated.And miR-206 was included in these differentially expressed miRNAs;2.We predicted that HDAC6 might be a potential target gene for miR-206 through bioinformatics prediction;3.The results of qRT-PCR showed that the expression of miR-206 in EC was lower than that in the adjacent control group(n=46,P<0.001),and the expression of miR-206 in four kinds of EC cells(HEC-1-A,Ishikawa,AN3C,RL95)was significantly down-regulated compared with endometrial stromal cells(P<0.001);4.The results of qRT-PCR and Western blot showed that HDAC6 was more highly expressed in EC than in the adjacent control group(n=46,P<0.001,P<0.01);and also highly expressed in four kinds of EC cells(HEC-1-A,Ishikawa,AN3C,RL95)than endometrial stromal cells(P<0.001,P<0.01);5.Spearman correlation test showed that there is a strong negative correlation linear relationship between miR-206 and HDAC6 mRNA expression in EC tissues(P=0.0032);6.Using the tool websites Human Protein Atlas(https://www.proteinatlas.org)and UALCAN(http://ualcan.path.uab.edu/index.html),we analyzed the related data in the TCGA database,and found that the expression of HDAC6 was associated with stage,Stage ? patients have higher HDAC6 expression in cancer tissue when compared with normal endometrial tissue(P=0.04);and the HDAC6 high-expression group(n=423)had a significantly worse prognosis(P=0.0024)compared with the low-expression group(n=118);7.Immunohistochemical test showed that HDAC6 was mainly expressed in the cytoplasm of tumor cells;and its expression was correlated with pathological grade of EC,the HDAC6 staining in the Grade3 patients is deeper than that of the Grade 1 patients.C onclusions:1.HDAC6 is one of potential target genes of miR-206,and its expression might be inhibited by miR-206;2.MiR-206 expression was decreased in EC tissues and cells:high HDAC6 expression might associate with poor prognosis in EC.Part ? Effects of miR-206 and HDAC6 on proliferation and invasion of EC cells and their related mechanismsObjectives:1.To confirm the interaction between miR-206 and HDAC6;2.To investigate the effects of miR-206-HDAC6 axis on the proliferation,migration,invasion and other biological behaviors of EC cells;3.To explore the mechanisms of miR-206-HDAC6 axis influencing the biological behaviors of EC cells.Methods:1.Dual luciferase reporter assay was used to identify the interaction between miR-206 and HDAC6;2.Lentivirus was transfected into the Ishikawa cell line which is a kind of highly differentiated EC cell to establish stable cell lines with high/low expression of miR-206,high/low expression of HDAC6,simultaneous low expression of miR-206 and HDAC6,and simultaneous high expression of miR-206 and HDAC6:IshikawaminR206_NC,IshikawamiR206-,IshikawamiR206+,IshikawasiHDAC6,IshikawasiHDAC6_NC,IshikawaHDAC6+,Ishikawa HDA6+_NC,Ishikawa mR206-/siHDAC6,IshikawamiR206-/siHDAC6,AN3C cell line which is a kind of low differentiated EC cell were transfected by the same way.The transfection efficiency was confirmed by qRT-PCR and Western blot.3.The cell proliferation ability was detected by CCK-8 and plate clone formation assay;the horizontal migration ability was detected by cell scratch;the cell migration and the infiltration ability were detected by Transwell;the expression of HDAC6 and other signaling pathway proteins were detected by Western blot.4.Next-generation sequencing analysis was used to search for the differentially expressed genes(DEGs)after the abnormal expression of miR-206,and KEGG pathway enrichment analysis was then performed.Results:1.MiR-206 could affect the biological behaviors of EC cells:(1)The results of qRT-PCR confirmed that,compared with IshikawamiR206_NC and AN3CmiR206_NC cell line,the expression of miR-206 was significantly decreased in IshikawamiR206-and AN3CmiR206-cell line,while the expression of miR-206 was significantly increased in IshikawamiR206+ and AN3CmiR206+ cell line;(2)The results of CCK8 and plate cloning assay showed that compared with Ishikawa/AN3CmiR206_NC cell,Ishikawa/AN3CmiR206-cell exhibited an enhanced proliferation capacity,while Ishikawa/AN3CmiR206+exhibited an impaired proliferation capacity;(3)The wound healing and Transwell assay results suggested that the motility of IshikawamiR206-and AN3CmiR206-cell was enhanced when compared with IshikawamiR206_NC and AN3CmiR206_NC cell,and the motility of lshikawamiR206+and AN3CmiR206+cell was inhibited when compared with IshikawamiR206_NC and AN3CmiR206_NC cell:(4)A global gene expression analysis was conducted to s identify aberrantly expressed genes between AN3CmiR206-,AN3CmiR206+ and AN3CmiR206_NC cells,the top 25 differentially expressed genes(including HDAC6)were displayed in a heat map.Then KEGG pathway analysis were performed to explore the possible molecular mechanism of its action;2.Next-generation sequencing analysis showed that there are 25 differentially expressed genes between AN3CmiR206-,AN3CmiR206+and AN3CmiR206_NC cells,among them,12 were oncogene and 13 were antioncogene,most of them are taget genes of miR-206.The KEGG pathway analysis shown that these differentially expressed genes were distributed mainly in IL-17,AKT,et al pathway;3.Dual luciferase reporter assay showed that compared with the negative control RNA,miR-206 significantly reduced the luciferase activity of the wild-type HDAC6 3'-UTR(P=0.006),which means that miR-206 could directly bind the 3'-UTR of HDAC6 mRNA and regulate its expression;4.HDAC6 could affect the biological behaviors of EC cells:(1)The results of qRT=PCR confirmed that compared with the control group,the expression of HDAC6 in IshikawasiHDAC6 and AN3CsiHDAC6 cell lines was significantly decreased,while that in IshikawaHDAC6+and AN3CHDAC6+cell lines the expression was significantly increased.The same results were obtained by Western blot at the protein level;(2)The results of CCK8 showed that compared with IshikawasiHDAC6_NC and AN3CsiHDAC6_NC cell,IshikawasiHDAC6 and AN3CsiHDAC6 cell exhibited an impaired proliferation capacity,while IshikawaHDAC6+ and AN3CHDAC6+ cell exhibited an enhanced proliferation capacity when compared with IshikawaHDAC6+_NC and AN3CHDAC6+_NC cell.The same results were obtained in colony formation assay;(3)The wound healing assay showed that the motility of IshikawasiHDAC6 and AN3CsiHDAC6 cell was inhibited when compared with IshikawasiHDAC6_NC and AN3CsiHDAC6_NC cell;while the motility of lshikawaHDAC6+ and AN3CHDAC6+ cell was enhanced when compared with IshikawaHDAC6+_NC and AN3CHDAC6+_NC cell.Transwell assay showed similar results;5.The effects of miR-206 on cell proliferation,invasion and migration depends on activation of HDAC6:(1)Western blot analysis showed that when compared with IshikawamiR206+and AN3CmiR206+ cell,HDAC6 expression was significantly increased in IshikawamiR206+/HDAC6+ and AN3CmiR206+/HDAC6+cell(P<0.01);CCK-8 showed that the suppressive effect of miR-206 on cell proliferation rate could be abolished after co-transfected with miR-206 mimics and HDAC6 plasmid(P<0.01);wound healing assay and Transwell assay showed that overexpression of HDAC6 could reverse migration and invasion ability of cancer cells,which was inhibited by miR-206;(2)Western blot analysis showed that when compared with IshikawamiR206-and AN3CmiR206-cell lines,the expression of HDAC6 was significantly decreased in IshikawamiR206-/siHDAC6 and AN3CmiR206-/siHDAC6 cell lines(P<0.01);CCK-8 showed that low expression of HDAC6 could partially reverse the enhancement of cell proliferation ability caused by low expression of miR-206(P<0.01);wound healing assay and Transwell assay showed that silencing HDAC6 resulted in reversed inhibition of cell migration and invasion ability strengthened by miR-206 inhibitor;6.Western blot analysis showed that after the overexpression of HDAC6,the expression of PTEN was decreased,while the expression of p-AKT and p-mTOR was increased.After knocking down the expression of HDAC6,the expression of PTEN was increased,and the expression of p-AKT and p-mTOR was decreased,which means miR-206 might directly bind to the HDAC6 3'-UTR and that HDAC6 could exert its inhibitory effects on EC cell proliferation and metastasis via the HDAC6/PTEN/AKT/mTOR pathway.Conclusions:1.MiR-206 might inhibit the proliferation,migration and invasion of EC cells by targeting HDAC6 to regulate the PTEN/AKT/mTOR pathway;2.MiR-206 and HDAC6 might play roles in the occurrence and development of EC.