| Primary liver cancer is one of the most common neoplasm with high incidence and high death rate in the world. Metastasis is the leading cause of HCC-related deaths. Therefore, molecular mechanisms underlying Hepatoma cells metastasis need to be illustrated and conquered, by which the grave threat of HCC to human being can be alleviated.Cancer metastasis contains a series of successive incidents, primarily including epithelial-mesenchymal transition (EMT), malignant cell migration, anoikis resistance, angiogenesis and lymphangiogenesis. Anokis-resistance is referred to the ability of cancer cells which helps to survive after detachment from the extracellular matrix. Anokis-resistance ensures detached cancer cells to circulate in the peripheral blood and reattach to colonize at the distant organs successfully. Therefore, anoikis resistance plays a vital role in the metastasis of cancer cells. However, the complicated cellular and molecular mechanisms underlying anoikis resistance have not been well elucidated.In our previous research, we have constructed the metastastic hepatoma cells model using poly HEMA (2-hydroxyethyl methacrylate) coated plates. We found that expression of BNIP3was significantly upregulated in detached hepatoma cells by analyzing the microarray expression profile. BNIP3is a BH-3only protein, a subunit belonging to the bcl-2family. BNIP3expression is regulated by HIF-1α, which is induced to express by hypoxia. BNIP is overexpressed in manignant glioblastoma and cervix cancer cells, et.al, while downregulated in duct carcinoma cells. In general, BNIP3can induce apoptosis or apotosis-like cell death in tumor cells. But the functions and molecular mechanisms of BNIP3in metastatic hepatoma cells has not been clarified.In order to study the role of BNIP3in the anoikis-resistance of hepatoma cells, both of the pcDNA3.1-BNIP3and pSilencer-BNIP3plasmids were successfully constructed and the influence of BNIP3on autophagy of hepatoma cells were further explored.Meanwhile, by using the metastatic hepatoma cells model, the change of BNIP3in detached hepatoma cells were verified and the molecular mechanism of BNIP3regulation was further explored. Moreover, through detection of the signaling pathways, including PI3K/AKT pathway, MAPK pathway, and mTOR pathway, the influence of BNIP3on autophagy of metastatic hepatoma cells were confirmed and the subsequent effect and the molecular mechanism of anoikis resistance was elucidated. These results indicated that targeting hepatoma cell survival by triggering BNIP3mediated anoikis provides a unique molecular basis for innovative therapeutic targeting of tumors before initiation of HCC metastasis.object1To construct BNIP3expression recombinant and shRNA recombinant, and confirm their successful construction.2Combined with the detection of BNIP3expression, the simultaneous detection of autophagy in metastatic hepatoma cells was conducted.3To study the modulation of molecular mechanisms of both BNIP3expression and the influence of BNIP3on autophagy.4To study the cellular and molecular mechanisms of BNIP3involved in anoikis resistance in metastatic hepatoma cells.Method:1Construction of human BNIP3eukaryotic expression vector and shRNA expression vector and detection the influence on autophagy of HCC cell1.1Construction and confirmation of Human BNIP3eukaryotic expression vector and shRNA expression vectorBNIP3Genetic coding areas was amplified through RT-PCR from HCC cell line, inserted into pcDNA3.1-His-C vector after enzyme cutting, and then pcDNA3.1-BNIP3eukaryotic expression vector was constructed. Meanwhile shRNA expression vector for human BNIP3gene was constructed. The recombinant vector was confirmed via sequencing, enzyme cutting, RT-PCR and western blot.1.2Influence of Human BNIP3eukaryotic expression vector and shRNA expression vector on autophagy of HCC cells.HCC cell line(BEL7402) was seeded in12-well dishes at1.5×105/ml, and transfection was performed at75%fusion. BNIP3eukaryotic expression vector and shRNA expression vector were transfected into HCC cell according to Lipofectamine2000TM Transfection protocols.24hours later, western blot was used for the detection of LC3cleveage and influence of BNIP3on autophagy of HCC cells. 2Dynamic simulation of HCC cell metastasis process2.1Establishment of HCC cell metastasis modelBEL7402cells were cultured to exponential phase in1640medium supplemented with10%FBS at37℃in humidified air with5%CO2. The cells were digested with0.25%trypsin-EDTA solution, and then stopted with RPMI1640medium supplemented with10%FBS. The single-cell suspension was cultured for24h in wells covered with poly-HEMA(final concentration3.6mg/cm-) and exposed to ultraviolet ray overnight.2.2Dynamic simulation of HCC cell metastasis processBEL7402cells at exponential phase were digested to single cell suspension using pancreatic enzyme solution supplemented with0.02%EDTA. The suspension was cultured in cell culture plate with or without polyHEMA at3X105/ml for24h at37℃in humidified air with5%CO2. The adhesion cells in cell plate without poly HEMA as adhesion group was simulated as primary HCC, whereas the suspension cells in cell cuture with poly HEMA as suspension group was simulate as metastasis cell in blood vessel.The suspension HCC cell reseeded in general cell culture plate and cultured for24h as suspension to ashesion group was simulated as secondary HCC.3Molecular regulation mechanism of BNIP3expression in Anoikis-resistant HCC3.1Detect BNIP3expression in anoikis-resistant HCC cellThe suspendingly cultured HCC cell line(BEL7402) were divided into two groups. One group was cultured in common cell culture plate for24h and the other was suspended for24h. Then mRNA and protein was extracted. The mRNA and protein level of BNIP3were detected via RT-PCR and Western blot with the HCC cells, adherent cultured for24h as control. HCC cells were cultured under suspension and adhesion states for48h.BNIP3expression was detected via Western blot.3.2Molecular regulation mechanism of BNIP3expression in Anoikis-resistant HCCmRNA and protein level of HIF-1a, which is upstream regulatory factor of BNIP3in suspension cultured HCC cell line(BEL7402) for24h and adhesion to suspension BEL7402for24h via RT-PCR and Western blot. ERK was deteced in the same condition.Protein level of HIF-la was deteced in suspension cultured for lh,3h,8h,24h via Western blot. HCC cell line(BEL7402) was cultured in suspension supplemented with ERK inhibitor UO126(10uM) and PD98059(10uM) for18h. Expression of HIF-1a and BNIP3were detected via Western blot. 4Effect and molecular reulation mechanism of autophagy in aoikis-resistant HCC cells4.1Occurrence and effect of Autophagy in anoikis-resistance HCC cellsThere are threee groups in this experiment, including attached cells, detached cells, detached transferring to attached cells. Autophagy was detected via Western blot. HCC cell line(BEL7402) was cultured in suspension for18h and cultured for another6h supplemented with3-MA(final concentration5mM) with DMSO as control. Effect of3-MA on activity and cell death of HCCcells was assayed by CCK8kit and trypan blue assay.4.2Molecular regulatory mechanism of autophgy in anoikis-resistant HCC cellsmTOR pathway was deteced in HCC cells at three states and different time ponits as previously described. Si-BNIP3was cotransfeted with or without pcDNA-LC3-GFP into HCC cells for24h,and then HCC cells were cultured in suspension for another24h. LC3expression was detected via Western blot. HCC metastasis model was either stimulated with mTOR activator insulin(100nM) or its inhibitor rapamycin(lOOnM) for24h, western blot was used to detect p-mTOR and LC3II.5Molecular regulatory mechanism of BNIP3in anoikis-resistant HCC cells5.1Confirmation of interference effect of siRNA towards BNIP3HCC cells were seeded in12well plate at1.5×105/ml, and was transfected at75%fusion. Transfection of HCC cells with si-BNIP3and si-NC was performed using Lipofectamine2000TM according to the manufacturer’s protocols(siRNA40pmol/well, liposome2ul/well). After24h, total protein was extracted and interference effect of si-BNIP3was confirmed by Western blot.5.2Effect of siRNA targeting BNIP3on autophagy of anoikis-resistanc HCC cellsHCC cells were transfected with siBNIP3and/or pcDNA-LC3-GFP for24h,and further cultured for24h. Cleavage of LC3was detacted by Western blot.5.3Effect and mechanism of si-BNIP3in anoikis-resistant HCCHCC cells were transfected with si-RNA for24h,and then cultured in suspension for24h. CCK8and trypan blue assay were used to detect activity and cell death of HCC cells respectively. Caspase-3/cpp32colorimetric method and Western blot were used to detect caspase3activation.6Statistics analysisResults were shown in mean±SD, T test and one-way ANOVA were used to analyze the statistical significance of each group, p<0.05was considered statistical significant. Result1Construction of human BNIP3eukaryotic expression vector and shRNA expression vector, detecting the influence of autophagy in HCC cells1.1Construction of pcDNA3.1-BNIP3expression vector and pSliencer-BNIP3interference vector successfullypcDNA3.1-BNIP3was confirmed via sequencing and enzyme cleavage, RT-PCR and Western blot. pSliencer-BNIP3was confirmed via sequencing, Real-time PCR and Western blot.1.2BNIP3was overexpressed and inhibited by pcDNA3.1-BNIP3and pSliencer-BNIP3.Western blot was used to detect autophagy marker LC3. The results showed that pcDNA3.1-BNIP3increased BNIP3expression and consequently enhanced autophagy, whereas pSliencer-BNIP3decreased BNIP3expression and consequently inhibited autophagy.2Dynamic simulation of HCC cells metastasis process2.1Establishment of HCC cells metastasis process successfullyBEL7402cells extended and grew to monolayer cells in the regular culture plate; whereas, HCC cell became rounded up and aggregated after being cultured in suspension for18h, and aggregated more intensively after24h. HCC cells cultured in suspension for18-24h was used to perform the following experiments.2.2Dynamic simulation of HCC cells metastasis processBEL7402cells were cultured in regulatory plate for24h after culture in suspension for24h, HCC cells adhere to the plate and extend in accordance with HCC cells at normal adherent growing condition. We simulate HCC metastasis process in vitro:adhesion group simulate the primary tumor, suspension group simulate blood vessel matestasis or lymphatic metastasis HCC cells, suspention to adhesion group simulate metastatic secondary tumor.3Overexpression of BNIP3was regulated by ERK/HIF-1α signal pathway in anoikis-resistant HCC cells3.1BNIP3and HIF-1α was overexpressed in anoikis-resistant HCCmRNA and protein level of BNIP3and HIF-1α in suspending HCC cells were upregulated in adhesion group and suspention to adhesion group (p<0.0001). BNIP3expression was detected in suspension and adhesion group. BNIP3of suspension group was overexpressed in a time dependent manner. 3.2ERK signal was activated in anoikis-resistant-HCC and BNIP3expression was regulated via ERK/HIF-1a signalling pathwayWestern blot results showed Phosphor-ERK in suspension-cultured HCC was higher than that in adhesion cultured group and suspension to adhesion group(p<0.0001). HCC metastasis model was cultured in suspension with ERK inhibitor UO126(10uM) or PD98059(10uM) for18h, Western blot showed that phosphor-ERK, HIF-1a and BNIP3were downreugulted significantly.4Autophagy maintained the survival in anoikis-resistant HCC and was regulated by mTOR pathway4.1Autophagy maintained the survival of anoikis-resistant HCCWetern blot showed that LC3active cleavage LC3II was increased in suspension group,and higher than adhesion group at all the detected time points (1,3,8,24h). It indicated that autophagy was enhanced in anoikis-resistant HCC cells. Autophagy inhibitor3MA inhibited autophagy in suspension group, and CCK8and trypan blue were used to detect cell activity. The results showed that apoptosis was increased in anoikis-resistant HCC cells after autophagy was blocked.4.2Autophagy in anoikis-resistant HCC cells was regulated by mTOR pathwayWestern blot showed that upstream and downstream signal molecules, including p-mTOR, p-S6K1, p-S6and p-4EBP1were decreased in detached cells. Furthermore, mTOR activator insulin decreased autophagy in anoikis-resistant HCC cells; conversely, rapamicin increased autophagy in anoikis resistant HCC cells. Thus, increased autophagy in the detached HCC cells was mediated by inhibition of mTOR in anoikis-resistant HCC cells.5BNIP3mediated autophagy afforded HCC cell anoikis-resistant capability5.1Si-BNIP3blocked BNIP3expression in HCC cellssi-BNIP3completely blocked BNIP3expression as determined by Western blot at24h.5.2Si-BNIP3decreased autophagy in anoikis-resistant HCC cellssi-BNIP3decresed endogenous and exogenous LC3II expression in suspending HCC cells, wich indicated that autophagy was inhibited by si-BNIP3.5.3Si-BNIP3reverse anoikis-resistance in suspending HCC cells via caspase-dependent pathwayCell death was increased in suspending HCC cells transfected with si-BNIP3as determined by CCK8and trypan blue test. Active cleavage of caspase3and activity of caspase were increased as detected by Western blot and caspase-3/cpp32colorimetry kit detection, respectively. Thus, aopoptosis was increased by siBNIP3via caspase-dependent pathway.ConclusionIn conclusion, we successfully construced human BNIP3eukaryotic expression vector pcDNA3.1-BNIP3and shRNA expression vector pSliencer-BNIP3, and found that autopagy was enhanced by overexpression of BNIP3and inhibited by silence of BNIP3. We dynamically simulated the meastasis of HCC via HCC metastasis model.BNIP3expression was upregulated in suspending cells and was regulated by ERK-HIF-1α signal pathway. Autophagy was increased in anchorage-deprived cells via BNIP3mediated mTOR pathway inhibition, which further confer anoikis-resistant capability of hepatoma cells. Study of function and molecular mechanism of BNIP3in anoikis-resistant cells would contribute to prevension and treatment of HCC metastasis by targeting BNIP3.Inovations and significances1We Constructed Human BNIP3eukaryotic expression vector and shRNA expression vector successfully and confirmed BNIP3mediated autopahgy in HCC cells.2We simulated invasion, migration and metastasis of hepatocellular carcinoma from primary lesion to secondary lesion via HCC metastasis model.3We confirmed overexpression of BNIP3in anoikis-resistant HCC cells and was regulated by ERK/HIF-1α signal pathway4We confirm autophagy was enhanced in anoikis-resistant HCC and BNIP3regulate autophagy through inhibition of mTOR by means of RNAi technology.5We investigated the molecular mechanisms involved in anoikis-resistant HCC. |
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