Effect And Mechanism Of REC8 In Hepatocellular Carcinoma

Liver cancer,one of the most common malignant tumors clinically,is much more common in men than in women.In men,it is the second leading cause of cancer death worldwide and in developing countries.In more developed countries,it is the sixth leading cause of cancer death among men.An estimated 782,500 new liver cancer cases and 745,500 deaths occurred worldwide during 2012,with China alone accounting for about 50% of the total number of cases and deaths.Most(70% to 90%)primary liver cancers occurring worldwide are hepatocellular carcinoma(HCC).However,the pathogenesis of HCC is still not clear,and there is currently lack of effective treatments.The primary curative treatment for HCC is surgery.After surgery,the 5-year recurrent rate and metastasis rate are high,which become problems of clinical treatment of HCC.Therefore,it is important and difficult to study the mechanism of HCC at molecular level in the research of HCC.REC8 is one member of cohesin,which is one kind of structural maintenance of chromosomes(SMC)proteins.Cohesin adjusts chromosome separation and homologous recombination in cell mitosis and meiosis and promotes faithful DNA repair,which functions as a protein complex that is essential for cell proliferation in all eukaryotic cells.Correct chromosome separation is important for cell proliferation,while abnormal segregation of chromosome is associated with many diseases,especially with tumor development.Cohesin can regulate the interaction of enhancer and promoter by binding to DNA in the process of transcription.Cohesin can participate in a variety of gene expression regulation,such as the expression regulation of proto-oncogenes and oncogenes.What’s more,it is colocalized with CTCF along the genomes,regulating gene transcription together with CTCF.Therefore cohesin plays an important role in tumorigenesis.REC8 is one member of newly discovered cohesin,which belongs to meiosis-specific subunits of cohesin complex.Upon entering meiosis I,a meiosis-specific subunit REC8 replaces RAD21.REC8 is one of Cancer/Testis genes,which are overexpressed in reproductive cell lines,and activated abnormally in cancers.Their role in the physiology of cancer cell needs to be further elucidated.Recent studies found the aberrant methylation of REC8 in the gastrointestinal stromal tumor,abnormal REC8 expression in polyploid tumor cells,and REC8 as a tumor suppressor in the gastric cancer and thyroid cancer.It is rarely reported that REC8 is expressed in HCC cells and the mechanism is unclear neither.We found the REC8 was expressed in HCC tissues and cells,and it functions in multiple biological characteristics of HCC cells,such as proliferation,apoptosis,migration,invasion and angiogenesis.We further explored the mechanism of REC8 in HCC cells,which provides a new therapeutic target for HCC.Part one Expression of REC8 in hepatocellular carcinoma tissues and cellsObjectives: To compare REC8 expression in HCC tissues with adjacent-carcinoma normal tissues,and compare REC8 expression in HCC cells with hepatocytes.Material and methods: 40 cases of HCC patients were chosen between the years from 2015 to 2016 from hepatobiliary surgery department,Second Hospital of Hebei Medical University were collected.80 cases of experimental specimens included 40 cases of fresh HCC tissues and 40 cases of adjacent-carcinoma normal tissues.REC8 expressions in tissues were detected by immunohistochemistry(IHC),Real-time PCR and western blot.REC8 expression in HCC cells was detected by cell immunofluorescence staining analysis and western blot.Results:1 REC8 mainly is located in the nucleus in HCC tissues,and is located in the cytoplasm in adjacent-carcinoma normal tissues,and the protein expression of REC8 in the nucleus in HCC tissues is increasedThe result of IHC showed REC8 was expressed in HCC tissues and in adjacent-carcinoma normal tissues,but it was mainly located in the nucleus in HCC tissues,and was located in the cytoplasm in adjacent-carcinoma normal tissues.The protein expression of REC8 in the nucleus in HCC tissues is higher than in adjacent-carcinoma normal tissues(P<0.01).REC8 m RNA expression had no significant difference between HCC tissues and adjacent-carcinoma normal tissues by Real-time PCR(P>0.05).The protein expression of REC8 in the nucleus in HCC tissues was higher than in adjacent-carcinoma normal tissues by western blot(P<0.01),which is suggested that REC8 may play a role in the development of hepatocellular carcinoma.2 REC8 mainly is expressed in the nucleus in HCC cells,and is expressed in the cytoplasm in hepatocytes,and the protein expression of REC8 in the nucleus in HCC cells is increasedREC8 was mainly expressed in the nucleus in Hep G2,Hu H-7 and BEL-7402 cells,and was mainly expressed in the cytoplasm in QSG-7701 cells by cell immunofluorescence staining analysis.The protein expression of REC8 in the nucleus in HCC cells was higher than in hepatocytes by western blot when the total protein was 150 μg(P<0.05),which is suggested that REC8 may play a role in the development of hepatocellular carcinoma.Part two Effects of REC8 on biological characteristics of HCC cellsObjectives: To examine the effects of REC8 on proliferation,migration,apoptosis and angiogenesis of HCC cells through establishing REC8 overexpression HCC cell model by pc DNA3.1-Flag-REC8 plasmid.Methods: Hep G2,Hu H-7 and BEL-7402 cells were transfected by pc DNA3.1-Fl-ag-REC8 plasmid and pc DNA3.1-Flag control plasmid.The expression of PCNA was used for detecting cell proliferation,as well as CCK8 assay.Cell apoptosis was detected by expression of cleaved caspase-3 and Annexin V and propidium iodide(PI)double staining.Cell migration was accessed by expression of MMP-9,wound healing assay and matrigel invasion assay.Cell angiogenesis was observed by expression of VEGF-C and tube formation assay.Results:1 REC8 was successfully overexpressed by pc DNA3.1-Flag-REC8 in HCC cellsHep G2,Hu H-7 and BEL-7402 cells were transfected by pc DNA3.1-Flag-REC8 plasmid.REC8 was detected successfully overexpressed by Real-time PCR and western blot(P<0.01).2 REC8 suppresses HCC cell proliferationWestern blot results showed PCNA expression was decreased after transfection by pc DNA3.1-Flag-REC8 plasmid in Hep G2,Hu H-7 and BEL-7402 cells relative to control cells(P<0.01),indicating that REC8 suppresses PCNA expression in HCC cells.Colony formation assay results showed colony formation was reduced after transfection by pc DNA3.1-FlagREC8 plasmid in Hep G2,Hu H-7 and BEL-7402 cells relative to control cells(P<0.01),indicating that REC8 suppresses colony formation in HCC cells.Results of CCK8 assay showed that cell viability reduced after transfection by pc DNA3.1-Flag-REC8 plasmid in Hep G2,Hu H-7 and BEL-7402 cells relative to control cells(P<0.01),suggesting that REC8 suppresses HCC cell viability.3 REC8 promotes HCC cells apoptosisWestern blot results showed cleaved caspase-3 expression increased after transfection by pc DNA3.1-Flag-REC8 plasmid in Hep G2,Hu H-7 and BEL-7402 cells relative to control cells(P<0.01),indicating that REC8 promotes HCC cells apoptosis.REC8 overexpressed cells and control cells were stained with Annexin V/PI and analyzed by flow cytometry.Ectopic expression of REC8 in Hep G2,Hu H-7 and BEL-7402 cells resulted in a significant increase of apoptotic cell population as compared with control cells(P<0.01),suggesting that REC8 promotes HCC cell apoptosis.4 REC8 promotes HCC cells migration and invasionWestern blot results showed MMP-9 expression increased after transfection by pc DNA3.1-Flag-REC8 plasmid in Hep G2,Hu H-7 and BEL-7402 cells relative to control cells(P<0.01),indicating that REC8 promotes migration and invasion in HCC cells.Matrigel invasion assay displayed REC8 was increased the invasiveness of HCC cells after transfection by pc DNA3.1-Flag-REC8 plasmid in Hep G2,Hu H-7 and BEL-7402 cells relative to control cells(P<0.01),indicating that REC8 promotes invasion in HCC cells.Wound healing assay displayed HCC cells treated with pc DNA3.1-Flag-REC8 plasmid healed much more quickly than control cells(P<0.01),indicating that REC8 promotes migration in HCC cells.5 REC8 promotes HCC cells angiogenesisWestern blot results showed VEGF-C expression increased after transfection by pc DNA3.1-Flag-REC8 plasmid in Hep G2,Hu H-7 and BEL-7402 cells relative to control cells(P<0.01),indicating that REC8 promotes angiogenesis in HCC cells.Tube formation assay showed tube formation was increased after transfection by pc DNA3.1-Flag-REC8 plasmid in Hep G2,Hu H-7 and BEL-7402 cells relative to control cells(P<0.01),indicating that REC8 promotes angiogenesis in HCC cells.Part three REC8 promotes migration,invasion and angiogenesis of HCC via PKA pathwayObjectives: To explore the mechanism of REC8 promoting migration,invasion and angiogenesis of HCC by filtrating the target proteins of REC8 interaction via Co-immunoprecipitation and mass spectrometry.Methods: The interaction of REC8 and the target protein was confirmed by Co-immunoprecipitation(Co-IP)and cell immunofluorescence staining analysis.Hu H-7 cells were intervened with inhibitors of the target protein after overexpression of REC8,and the functions were confirmed.Results:1 The interaction of REC8 and PKA RII-α is confirmed in HCC cells58 target proteins of REC8 interaction was filtrated via Co-immunoprecipitation and mass spectrometry.Among them,PKA RII-α is known to be related to the cell migration,invasion,and angiogenesis.The results of mass spectrometry were validated by Co-IP.PKA RII-α was detected as a positive result by Co-IP,showing the interaction of REC8 and PKA RII-α was present in HCC cells.Co-IP was conducted by immunoprecipitation with PKA RII-α and immunoblotted for REC8 to test the interaction of REC8 and PKA RII-α.The result showed REC8 interacted with PKA RII-α in HCC cells,which was supported by cell immunofluorescence staining analysis in HCC cells,as they were both located in the nucleus.2 The interaction between REC8 and PKA RII-α promotes the PKA activity in HCC cellsWestern blot results showed PKA RII-α expression was not affected after transfection by pc DNA3.1-Flag-REC8 plasmid in Hu H-7 cells compared to control cells(P>0.05).PKA activity was increased after transfection by pc DNA3.1-Flag-REC8 plasmid in Hu H-7 cells relative to control cells(P<0.01),indicating REC8 promotes PKA activity in HCC cells.The interaction between REC8 and PKA RII-α promotes the PKA activity in HCC cells,but does not affect its expression.3 REC8 promotes migration and invasion in HCC cells via PKA pathwayWestern blot results showed MMP-9 expression was decreased after H89 intervention in REC8 overexpressed HCC cells(P<0.01),indicating that REC8 promotes MMP-9 expression,migration and invasion in HCC cells via PKA pathway.After the application of PKA inhibitors H89 in REC8 overexpressed HCC cells,the invasion ability of the HCC cells was decreaed by transwell invasion assay(P<0.01),and the migration ability of the HCC cells decreaed by scratch healing assay(P<0.01).4 REC8 promotes angiogenesis in HCC cells via PKA pathwayWestern blot results showed VEGF-C expression decreased after H89 intervention in REC8 overexpressed HCC cells(P<0.01),indicating that REC8 promotes VEGF-C expression and angiogenesis in HCC cells via PKA pathway.After the application of PKA inhibitors H89 in REC8 overexpressed HCC cells,the tube-forming ability of the HCC cells decreaed by tube formation assay(P<0.01).Conclusions:1 REC8 mainly is located in the nucleus in HCC tissues and HCC cells,and is located in the cytoplasm in adjacent-carcinoma normal tissues and in hepatocytes,suggesting REC8 may play a role in the development of hepatocellular carcinoma.2 REC8 suppresses proliferation,promotes apoptosis,migration,invasion and angiogenesis in HCC cells.3 REC8 promotes migration,invasion and angiogenesis in HCC cells via PKA pathway.