Sohlh2 Inhibits The Hypoxia-induced Epithelial-mesenchymal Transition By Downregulation Of HIF1? In Epithelial Ovarian Cancer Cells

Background Ovarian cancer is the most lethal gynecologic malignancy.Since the ovary is in the deep site of the pelvic and lack of effective early diagnosis methods,it can not be diagnosed until it has been at advanced stage and the five-year survival rate is less than 30%.Epithelial ovarian cancers are the most common type of all ovarian cancers,usually the distant metastasis has occurred when it is diagnosed.That makes the traditional treatment ineffective,like surgery,radiotherapy and chemotherapy.Therefore,to clarify the molecular mechanisms,especially the migration and invasion ability is important for early diagnosis and treatment of epithelial ovarian cancer.Hypoxia,the typical acidosis of tumor microenvironment,is resulted from uncontrolled growth of cancer cells.Hypoxia parts through all the tissues in solid cancer.Hypoxia can induce the expression of hypoxia-inducible factor 1(HIF1),promote the epithelial-mesenchymal transition,migration and invasion of cancer cells.Carbonic anhydrase(CA9)is controlled via the hypoxia inducible factor 1 a which regulates the intracellular and extracellular pH by catalyzing the hydration of carbon dioxide into bicarbonate and protons.Adaptation of tumor cells to hypoxia and acidosis microenvironment is a critical driving force in tumor progression and metastasis.Epithelial-mesenchymal transition,plays a critical role in the occurrence and progress of tumor metastasis including epithelial ovarian cancers.EMT is a process characterized by loss of cell-cell adhesion,increase of cell motility and invasiveness.Hypoxia can promote EMT.To inhibit the hypoxia-induced epithelial-mesenchymal transition may be the key to inhibit the invasiveness and metastasis of epithelial ovarian cancers.Sohlh2(Spermatogenesis and oogenesis basic helix-loop-helix(bHLH)transcription factor 2)is an important transcriptional factor of bHLH family.According to the previous report of our lab,Sohlh2 expressed highly at the surface of ovary and cattle epithelium cells,but was expressed at very low levels in epithelial ovarian cancer samples.And also overexpressed Sohlh2 in epithelial ovarian cancer cells can inhibit the growth,migration and invasion by regulation of different ways.All these studies can show that Sohlh2 might act as a novel tumor inhibitor gene.Sohlh2 can inhibit epithelial-mesenchymal transition of human breast cancer cells via downregulation of IL-8.Otherwise,the effect and mechanism of Sohlh2 on epithelial cancer cells under hypoxia has been unkown.Our study aims to explore the role of Sohlh2 on epithelial ovarian cancer cells under hypoxia,by cultured epithelial ovarian cancer cells in vitro.Using Chemical lack oxygen agent CoCl2 hypoxia treatment,Sohlh2 gene transfection and RNA interference,wound healing assay,Transwell,Matrigel,immunofluorescence and Western Blot were performed to explore the effect of Sohlh2 on the invasion and migration of cancer cells and underlying molecular mechanism.Our study may provide a new target for clinical treatment of ovarian cancer.Methods1.To exam the effects of Sohlh2 on invasiveness and migration of epithelial ovarian cancer cells under hypoxia.Plasmid transfection and RNA interference were used to build stable over-expression and knock down Sohlh2 of epithelial ovarian cancer cell lines(HO8910 and SKOV3)in vitro.Cells were divided into four groups:?HO8910-pEGFP group;?HO8910-pSohlh2 group;?SKOV3-Con group;?SKOV3-pShSohlh2 group.??were screened with G418 and ?? were screened with puromycin.Four groups hypoxia by CoCl2 for 48 hours.The effect of sohlh2 on the cancer cells migration and invasion were assayed by Wound healing,Transwell migration and Matrigel invasion methods.2.To detect the effects of Sohlh2 on hypoxia induced epithelial-mesenchymal transition.Western Blot was performed to detect the changes of expression of epithelial-mesenchymal transition markers E-cadherin,Vimentin,N-cadherin and Sohlh2 on the protein level in HO8910-pEGFP,HO8910-pSohlh2,SKOV3-Con,SKOV3-pShSohlh2 groups after hypoxia treatment;Immunofluorescence assay was used to detect the changes of E-cadherin and Vimentin under hypoxia of these two groups:H08910-pEGFP group;HO8910-pSohlh2 group.3.To analyze the molecular mechanisms of Sohlh2 in the process of hypoxia induced epithelial-mesenchymal transition.Western Blot was used to detect the expression of HIFla and CA9 on protein level under normal(25%O2)and hypoxia condition of these groups:HO8910-pEGFP group;HO8910-pSohlh2 group;SKOV3-Con group;SKOV3-pShSohlh2 group.Results1.After treatment with CoCl2,compared with the H08910-pEGFP group,wound healing assay showed that the migration distance of HO8910-pSohlh2 group became shorter(P<0.05);The numbers of migrative and invasive cells became significantly less(P<0.05).After treatment with CoCl2,compared with the SKOV3-Con group,wound healing assay showed that the migration distance of SKOV3-pShSohlh2 group became longer(P<0.05);The numbers of migrative and invasive cells became more(P<0.05).2.In HO8910 cells,after 48h hypoxia,compared with HO8910-pEGFP group,immunofluorescence assay showed that the expression of epithelial phenotype marker E-cadherin was significantly increased,while the expression of mesenchymal phenotype marker Vimentin was significantly decreased.3.Compared with H08910-pEGFP group,the expression of Sohlh2 and epithelial phenotype marker E-cadherin were significantly increased in HO8910-pSohlh2 group after 48h hypoxia,while the expression of mesenchymal phenotype marker N-cadherin and Vimentin were significantly decreased.Compared with SKOV3-Con group,the expression of Sohlh2 and epithelial phenotype marker E-cadherin significantly decreased in SKOV3-pShSohlh2 group after hypoxia,while the expression of mesenchymal phenotype marker N-cadherin and Vimentin were significantly increased.4.Cultured H08910-pEGFP and HO8910-pSohlh2 groups in normal condition(25%O2)and hypoxia,the protein level of HIF1? and CA9 in hypoxia condition were higher than in normal condition;Compared with the H08910-pEGFP group,the protein level of HIF1? and CA9 of HO8910-pSohlh2 group was decreased.Cultured SKOV3-Con and SKOV3-pShSohlh2 groups in normal condition(25%02)and hypoxia,the protein level of HIFla and CA9 in hypoxia condition were higher than normal condition.While compared with the SKOV3-Con group,the protein level of HIFla and CA9 of SKOV3-pShSohlh2 group was increased.Conclusions1.Sohlh2 can inhibit the ability of metastasis and invasiveness of epithelial ovarian cancer cells induced by hypoxia;2.Sohlh2 can inhibit the hypoxia-induced epithelial-mesenchymal transition;3.Sohlh2 may inhibit the hypoxia-induced epithelial-mesenchymal transition through deregulation of HIF1?/CA9 pathways.