BCL2L10 Inhibits Growth And Metastasis Of Hepatocellular Carcinoma Both In Vitro And In Vivo

HCC(hepatocellular carcinoma,HCC)is one of the five common malignant tumor in the world,It now ranks sixth in the world among all malignancies,contributing to the third leading cause of mortality attributed to cancer.It is often diagnosed in the late stage,due to its long asymptomatic period.The biological mechanisms of HCC are still unclear.Up to now,surgical resection is the only effective method for treatment of HCC.However,the high transfer rate and high recurrence rate after resection seriously affect its prognosis.So research on the pathogenesis of HCC has been a global hot topic,which is a pathological process involved by multi-factors,multi-stage and multiple gene alterations.Studies show that apoptosis and proliferation affect throughout the course of HCC,involving high expression and abnormal activation of anti-apoptotic genes and inactivation of pro-apoptotic genes.It is generally accepted that defective apoptosis has play an important role in the development of HCC.BCL2L10(Bcl2-like-10),a polypeptide containing an open reading frame of 204 amino acids,was discovered when isolating novel BCL-2 related genes.Its protein molecular weight is 23 KD.BCL2L10 belongs to BCL-2 family and widely expressed in human tissues.The evolutionarily conservative BCL-2 family is a key regulator of cell apoptosis,play an irreplaceable role during normal embryonic development,tissue homeostasis,and a variety of pathological conditions by promoting apoptosis or anti-apoptosis.BCL-2 family members are divided into two categories according to the impact on apoptosis: anti-apoptotic members and pro-apoptotic members.The anti-apoptotic effect of the former is antagonised by the latter.Being the latest identified member of BCL-2 family,BCL2L10 mRNA of human is typically found has a relatively high level in marrow,liver,brain and lung.Previous studies classified it into the anti-apoptotic members playing a similar function as BCL-2.For example,studies show a tumor-specific up-regulation of BCL2L10 in breast,prostate,gastric,colorectal adenocarcinomas and non-small cell lung cancers.However,recent reports demonstrated that loss of BCL2L10 was prevalent in gastric carcinoma tissue in contrast to non-carcinoma gastric tissues.Over-expression of BCL2L10 caused gastric cancer cell apoptosis and inhibited cell growth,indicating that it may be a tumor suppressor in gastric cancer.Basic research points out that the capacity of BCL2L10 can exert different functions under different tissue circumstances and cellular context,maybe an available explanation for the contradictory role of BCL2L10 in cancer.Although BCL2L10 have a relatively higher expression level in liver,analysis of BCL2L10 gene expression in HCC has not been clearly described.To elaborately study the role of BCL2L10 in hepatocarcinogenesis,firstly,we compared the expression difference of BCL2L10 in human HCC tissues with their adjacent normal tissues,as well as HCC cell lines with normal liver cell lines.Secondly,both in vitro and in vivo functional studies related to tumor proliferation,apoptosis and metastasis were performed to reveal the functional significance of BCL2L10 in HCC.Finally,human cancer pathway array and the following validated tests were completed to find out the molecular mechanism of BCL2L10 in HCC.Part 1 Expression of BCL2L10 in hepatocellular carcinoma tissues and cellsObjectives: To compare BCL2L10 expression in HCC tissues with para-carcinoma tissues,and HCC cell lines with human normal liver cells.Methods: 42 pairs of HCC and corresponding para-carcinoma tissues were collected by surgery from hepatobiliary surgery department,the Second Hospital of Hebei Medical University between 2014-5 to 2015-6.Expression of BCL2L10 mRNA and protein levels in HCC and corresponding para-carcinoma tissues were detected by real time PCR and Western blot analysis respectively.Expression of BCL2L10 in HCC and corresponding para-carcinoma tissues were also observed by HE staining and immunohistochemistry.Expression of BCL2L10 mRNA in five HCC cell lines(HepG2,Hep3 B,Huh6,Huh7 and SMMC7721)and one human normal liver cell(LO2)as well as one pair of HCC tissues and corresponding para-carcinoma tissues were detected by RT-PCR and Real time PCR.Expression of BCL2L10 protein in three HCC cell lines(HepG2,Huh7,SMMC7721)and human normal liver cells(LO2)as well as one pair of HCC tissues and corresponding para-carcinoma tissues were detected by Western blot analysis.Results:1 Expression of BCL2L10 mRNA in 30 HCC tissues were significantly lower than that of corresponding para-carcinoma tissues among 42 pairs of patients(30/42),and so as to BCL2L10 protein.2 In comparison of BCL2L10 expression between HCC and paracarcinoma tissues,we find significantly more BCL2L10 expression in para-carcinoma tissues than that in HCC tissues,which were mainly expressed in cell cytoplasm.3 Expression of BCL2L10 mRNA in HepG2,Hep3 B,Huh6,Huh7,SMMC7721 cells were lower than that of corresponding para-carcinoma tissue and LO2 cell.Differences were more obvious in HepG2,Hep3 B,Huh6,Huh7 cells.Expression of BCL2L10 protein in HepG2,Huh7,SMMC7721 cells were lower than that of corresponding para-carcinoma tissue and LO2 cell.Differences were more obvious in HepG2,Huh7 cells.Conclusions: Impaired BCL2L10 expression was observed in human HCC tissues and cell lines.Part 2 Effects of BCL2L10 on proliferation and apoptosis of HCC in vivo and in vitroObjectives: To examine the effects of BCL2L10 on proliferation and apoptosis of HCC cells through establishing BCL2L10 overexpression or knockdown HCC cell model,and then validate its effect on HCC growth by in vivo tumorigenicity test.Methods: BCL2L10 were overexpressed in human HepG2 and Huh7 cells and knockdown in SMMC7721 cell.CCK8 assay was used for detecting cell viability,colony formation assay for proliferation,flow cytometry for cell cycle,as well as Western blot for the expression of PCNA(Proliferating Cell Nuclear Antigen)and CDC25C(cell cycle protein);Cell apoptosis was detected by Annexin V and propidium iodide(PI)double staining and expression of cleaved caspase-3,caspase-8,caspase-9.Effect of BCL2L10 overexpression in HepG2 cell on transplant subcutaneous sarcoma growth was evaluated by in vivo tumorigenicity test.Results:1 BCL2L10 was successfully overexpressed in HepG2 and Huh7 cells and knockdown in SMMC7721 cellBCL2L10 was overexpressed by BCL2L10 recombinant plasmid with Flag-tag as well as knocked down by siRNA targeting BCL2L10 as shown by RT-PCR and western blot.2 BCL2L10 inhibited HCC cell proliferationBCL2L10 inhibited HCC cell viability/.Result of CCK8 assay shows that HCC cells treated with BCL2L10 recombinant plasmid revealed a weaker cell viability while these treated with BCL2L10 siRNA revealed an increased cell viability,suggesting that BCL2L10 inhibited HCC cell viability.BCL2L10 inhibited HCC cell colony formation.The colony formation number of HepG2/BCL2L10 group was 230.7±35.96,which was significantly lower than HepG2/Control group with the number of 588.0±51.05.The colony formation number of Huh7/BCL2L10 group was 153.0±29.57,which was significantly lower than Huh7/Control group with the number of 386.3±24.52.All above suggest that BCL2L10 inhibited HCC cell colony formation.BCL2L10 reduced PCNA expression of HCC cell.Western blot results showed upregulated BCL2L10 lead to a reduced expression of PCNA,indicating that expression of BCL2L10 correspond to inhibition of HCC cell proliferation.BCL2L10 inhibited cell cycle of HCC cell.Cell cycle analysis by flow cytometry revealed BCL2L10 overexpression induced increased cells in G2/M phase for both HepG2 and Huh7 cells with a corresponding decrease in phase S and phase G1,suggesting that BCL2L10 inhibited mitosis in HCC cell.BCL2L10 decreased the expression of CDC25 C in HCC cell.Western blot results showed that BCL2L10 plasmid exposure led to decreased expression of CDC25 C in both HepG2 and Huh7 cells,indicating BCL2L10 inhibited mitosis in HCC cell by reducing expression of CDC25 C.3 BCL2L10 promote HCC cell apoptosisAnnexin V/PI double staining showed that compared with control group more apoptosis were found in BCL2L10 group both in HepG2 and Huh7 cells,and less apoptosis were found in Si-BCL2L10 group of SMMC7721 cell when BCL2L10 was knockdown by siRNA.Western blot results showed that expression of cleaved caspase-3,caspase-8,caspase-9 was remarkably higher in BCL2L10 overexpression group compared with control group.4 BCL2L10 inhibited transplant subcutaneous sarcoma growth of HepG2 cells.In vivo tumorigenicity test showed that the volume of transplant subcutaneous sarcoma in BCL2L10 overexpression group was much smaller than that of control group.Conclusions: Ectopic expression of BCL2L10 inhibited cell proliferation,while promoted apoptosis in HCC both in vitro and in vivo.Part 3 Effects of BCL2L10 on migration,invasion and angiogenesis of HCC in vivo and in vitroObjectives: To examine the effects of BCL2L10 on migration,invasion and angiogenesis of HCC cells in vivo and in vitro,and to explore the role of BCL2L10 in HCC metastasis.Methods: BCL2L10 successfully overexpressed in human Hep G2 and Huh7 cells and knockdown in SMMC7721 cell.Wound healing assay was used by accessing cell migration,matrigel invasion assay for invasion,tube formation assay for angiogenesis as well as HE staining and immunohistochemistry expression of CD34 in transplant subcutaneous sarcoma for angiogenesis in vivo.Effect of BCL2L10 on HCC metastasis was evaluated by in vivo lung metastasis assay.Results:1 BCL2L10 inhibited migration of HCC cellWound healing test displayed that HepG2 and Huh7 cells treated with BCL2L10 recombinant plasmid healed much more slowly than control cells while BCL2L10 knockdown by siRNA speed up healing procession indicating that BCL2L10 inhibited HCC cell migration.2 BCL2L10 remarkably inhibited invasion of HepG2 cell.3 BCL2L10 inhibited angiogenesis of HCCBCL2L10 reduces angiogenesis in vitro.As compared with empty control vector-transfected cells,conditioned culture medium from BCL2L10-transfected Hep G2 cells significantly reduced the tube-forming capacity of HUVEC on Matrigel,indicating BCL2L10 inhibited angiogenesis in tumor.BCL2L10 reduces angiogenesis in vivo.For H&E staining,result showed that tube density of tumors formed by empty vector-transfected HepG2 cells was clearly higher than that of tumors formed by HepG2 cells with ectopic expression of BCL2L10.This observation was confirmed by CD34 IHC analysis,which showed CD34 positive area in tumors formed by control HepG2 cells was significantly higher compared with that in tumors formed by BCL2L10 over-expressed HepG2 cells,further indicating BCL2L10 inhibited tumor angiogenesis in vivo.4 BCL2L10 inhibits lung metastasis in nude miceHistological examination of the lungs by H&E staining showed that 83.3%(5/6)of the mice bearing empty vectortransfected Hep G2 cells exhibited lung metastases.In contrast,only 33.3%(2/6)of the animals bearing BCL2L10 over-expressed HepG2 cells developed lung metastases.Therefore,these results provide strong evidence that BCL2L10 inhibits lung metastasis of HCC cells in vivo.Conclusions: Ectopic expression of BCL2L10 inhibited cell migration,invation and angiogenesis of HCC in vitro and inhibits lung metastasis of HCC in vivo.Part 4 Molecular mechanism of BCL2L10 inhibit the growth and metastasis of HCCObjectives: To explore the molecular mechanism of BCL2L10 inhibit the growth and metastasis of HCCMethods: To evaluate the molecular mechanisms underlying the tumor inhibitory effect of BCL2L10 on HCC cell,gene expression profiles in GV141-BCL2L10 transduced HepG2 cells were analyzed using a human cancer pathway PCR array.Then,the variation gene was validated by Western blot.Finally,the gene expression profiles regulated by BCL2L10 in HCC was clearly described.Results:1 Compared with GV141 vector transduced cells,BCL2L10 overexpressing cells showed profound alterations in genes involved in cell growth,cell apoptosis,cell to cell adhesion,cell metastasis,and angiogenesis,all of which are critical to the neoplasia of cell.Of note,enhanced expression of BCL2L10 significantly increased the expression of cell death genes including tumor necrosis factors(TNF;2.5-fold),tumor necrosis factor receptor superfamily member 25(TNFRSF25;1.8-fold),tumor necrosis factor receptor superfamily member 10B(TNFRSF10B;1.5-fold),cell adhesion genes including Cadherin-11(CDH11;4.8-fold),E-cadherin(CDH1;1.5-fold),and two metastasis suppressors,Tissue Inhibitors of Metalloproteinase 2(TIMP2;20.9-fold),Tissue Inhibitors of Metalloproteinase 1(TIMP1;1.7-fold);whereas,the expression of cell migration gene such as Matrix metallopeptidase 9(MMP9;8.0-fold)and Matrix metallopeptidase 13(MMP13;13.1-fold)as well as key pro-angiogenic factors such as vascular endothelial growth factor C(VEGFC;3.3-fold)were suppressed.Among all the alteration genes we tested in PCR array,the change of JAK1 was the most with more than 23-fold decrease due to BCL2L10 over-expression.2 To validate this,Western blot was performed on HepG2 cells with or without over-expression of BCL2L10.Consistent with the result of the PCR array,ectopic expression of BCL2L10 up-regulated the protein expressions of CDH1 and CDH11,whereas down-regulated the expressions of MMP9 and VEGFC,as well as the phosphorylation of JAK1 and STAT3.3 Our results indicated that BCL2L10 suppressed HCC progression through inhibiting cell growth and metastasis.Thus,BCL2L10 functions as a tumor-suppressor in HCC.Conclusions:1 BCL2L10 suppressed HCC progression through up-regulating the expressions of TNF,DR3,DR5,caspase-3,caspase-8,caspase-9,CDH1,CDH11,TIMP1 and TIMP2,whereas down-regulating the expressions of PCNA,CDC25 C,MMP9,MMP13 and VEGFC.2 Re-expression of BCL2L10 reduced the activation of JAK-STAT3 signaling pathway,and BCL2L10 may function as a tumor-suppressor in HCC.