| Background:Hepatocellular carcinoma（HCC）is one of the malignant tumors,with a recurrence and metastasis rate of more than 70%5 years after operation.Studies have shown that the high invasion characteristic of HCC is an important reason for its recurrence and metastasis.Therefore,in-depth study on the regulatory mechanism of HCC cell invasion and metastasis has an important theoretical significance and potential clinical application value for the prevention and treatment of HCC invasion and metastasis and the improvement of prognosis.Hypoxia is a common phenomenon in solid tumors,which can induce invasion and migration of tumor cells.Studies showed that hypoxia inducible factor-1α（HIF-1α）activated the relative signaling pathways and enhanced the expression of transcription factors to induce invasion and migration of HCC cells.But based on the relatived signal pathway and transcription factors related intervention measures did not effectively curb the invasion and metastasis of HCC cells,suggesting that there may be an unknown regulation pathway and mechanism plays an important role in it.Cytoskeleton is an intricate network of protein filaments throughout the cytoplasm,which plays an important role in maintaining cell morphology and movement.Actin filament（F-actin）is an important component of cytoskeleton,and its dynamic remodeling is the structural basis and power source for the morphological changes and invasion and migration of tumor cells.It has been reported in the literature that HIF-1α,as an important transcription factor mediating the cell hypoxia response,can activate the RhoA/ROCK signaling pathway to mediate cytoskeleton damage and remodeling,but its specific molecular mechanism remains to be further elucidated.Phosphatidylinositol-4,5-bisphosphate（PIP2）is a kind of phospholipid.PIP2 is a phospholipid.Although it accounts for less than 1%of the membrane phospholipids,its function is very complex and it is an important membrane molecule that transmits extracellular stimulation signals to the cell.It was found that,as an important component of lipid raft,PIP2 can participate in biological processes such as the regulation of the remodeling of actin cytoskeleton,which necessary for the cell migration.Recent findings reported that the RhoA/ROCK path-specific inhibitor Y-27632 inhibits PIP2-induced cardiac hypertrophy in mice,which suggest that PIP2 may be a key regulator downstream of the RhoA/ROCK signaling pathway.Therefore,we believe that PIP2 is an important functional molecule transmitting extracellular hypoxia signal to the cytoskeleton.Capping actin protein of muscle Z-line（Cap Z）is an actin binding protein,which can bind to the barbed end of F-actin and participate in the regulation of the remodeling of the actin cytoskeleton by preventing the binding of the globular actin（G-actin）to the barbed end of F-actin.CAPZA1 is the 1 subunit of CapZ.The author studied the effect of CAPZA1 on invasion and metastasis of hepatocellular carcinoma and its possible mechanism during his master’s degree and found that the down-regulation of CAPZA1 could promote the remodeling of actin filament cytoskeleton to drive EMT,thus enhancing the invasion and migration ability of HCC cells.However,as an important molecular marker for evaluating invasion,metastasis and prognosis of HCC,the upstream regulatory mechanism of CAPZA1is still unclear.Hartman,T J and Li J et al.found that in the model of ventricular myocyte hypertrophy,CAPZ is involved in the remodeling of actin cytoskeleton,which can be regulated by PIP2.Roman Pleskot et al.found that PIP2 and CAPZ have very strong affinity in animal and plant cells.However,the interaction between PIP2 and CapZ and its mechanism have not been reported in tumor cells,especially HCC cells.In order to further study the molecular mechanism of hypoxia-induced invasion and migration of HCC cells,we proposed the following research assumptions based on the previous research work that hypoxia can promote the synthesis of PIP2 on the cell membrane by activating the HIF-1α/RhoA/ROCK signaling pathway,while PIP2 on the plasma membrane interacts with CAPZA1 and causes CAPZA1 to be released from the barbed end of F-actin,thereby opening the barbed end and promoting the G-actin polymerize on it to drive F-actin prolongation on the barbed end,thus driving the development of EMT and ultimately enhancing the invasion and migration ability of HCC cells.Objectives:1.Through clinical case analysis,the expressions of HIF-1α,PIP2 and CAPZA1 in the HCC tissue samples were detected to explore the correlation among them,and to clarify the influence of their expression changes on the clinicopathological factors and prognosis of HCC patients,so as to provide a basis for subsequent mechanism studies.2.An in vitro hypoxia culture model was established to study the effect of hypoxia on PIP2 synthesis at the level of hypoxia cultured hepatocellular carcinoma cells,and to verify whether hypoxia can promote PIP2 synthesis by activating HIF-1α/RhoA/ROCK signaling pathway.3.To elucidate the potential mechanism of CAPZA1,which plays a key negative regulatory role in the invasion and metastasis of HCC cells by affecting cytoskeleton remodeling.The interaction between PIP2 and CAPZA1 and the regulation mechanism of PIP2 on CAPZA1 were studied.4.A nude mouse liver orthotopic transplantation model was established to investigate the effect of blocking the HIF-1α/PIP2/CAPZA1 signaling pathway on the invasion and metastasis of HCC at the animal level,and to explore the feasibility of the prevention and treatment strategy for the invasion and metastasis of HCC targeted by actin cytoskeleton remodeling.Methods:1.Immunohistochemistry was used to detect the level of HIF-1α,PIP2 CAPZA1 in HCC tissue samples,and Image J software was used to evaluate the positive rate of them in HCC slices（including the positive rate and staining depth of HCC cells）.According to their positive rates,HCC patients were divided into HIF-1αover-expression group（HIF-1α-OE）and HIF-1αunder-expression group（HIF-1α-UE）,PIP2 low-level group（PIP2-Low）and PIP2 high-level group（PIP2-High）,CAPZA1 under-expression group（CAPZA1-UE）and CAPZA1 over-expression group（CAPZA1-OE）,respectively.And the differences in HCC tumor size,TNM stage,pathological differentiation,vascular invasion and prognosis were statistically analyzed.IH was used to detect the expression level of HIF-1α,PIP2,CAPZA1,e-cadherin,and Vimentin in HCC tissue microarrays,and the linear relationship between CAPZA1 expression and EMT as well as the linear relationship among HIF-1α,PIP2 and CAPZA1 was statistically analyzed.2.HCC cells were cultured in vitro in a hypoxic environment with 400 cm cobalt dichloride（CoCl2）.Dot blot,PIP2 ELISA and immunofluorescence were used to detect the level of PIP2 in HCC cells.RhoA-GTP pull down assay was used to detect the level of activated RhoA.The expression of HIF-1α,RhoA,ROCK1 and CAPZA1 was detected by western blot and the status and distribution of actin cytoskeleton in HCC cells was observed by immunofluorescence.The existence of HIF-1α/RhoA/ROCK1 signaling pathway and its effect on the synthesis of PIP2 and the remodeling of actin cytoskeleton were verified by adding chemical inhibitors YC-1 to inhibit HIF-1α,C3 transferase protein to inhibit RhoA and Y-27632 to inhibit ROCK.3.The expression of CAPZA1 was manipulated by infected the CAPZA1 interference lentivirus,with LV10-CAPZA1 and the CAPZA1 overexpression lentivirus,LV8-CAPZA1 in HCC cells.Transwell,wound healing,Western blot,qPCR,co-immunoprecipitation and immunofluorescence were used to observe the effects of CAPZA1 on invasion,migration,EMT and F-actin levels of HCC cells.Affinity precipitation experiments were conducted with the amide beads coated with PIP2 to verify the interaction between PIP2 and CAPZA1.PIP2was closed with direct addition of Neomycin to block the binding of PIP2 to CAPZA1 and direct addition of PIP2 to enhance the binding of PIP2 to CAPZA1.Immunoprecipitation,cellular substructure localization and immunofluorescence were used to confirm that the combination of PIP2 and CAPZA1 can change the distribution of CAPZA1 in cytoskeleton and cytoplasm and its effect on the remodeling of actin cytoskeleton.4.HCC transplanted nude mice model was constructed by inoculation of SMMC-7721cells with stable low expression of CAPZA1 and MHCCLM3 cells with stable high expression of CAPZA1.After 6 weeks,the number of HCC lesions in nude mice was observed using small animal MRI.A nude mouse model of HCC in situ transplantation was constructed by inoculation of MHCCLM3 cells,and Neomycin was intraperitoneally injected into nude mice for 1 month.The effect of Neomycin on HCC invasion and metastasis was determined by analyzing the number of HCC lesions on the liver surface.Results:1.Compared with HIF-1α-UE group,tumor diameter of HIF-1α-OE group is significantly larger（7.96±3.56 cm vs.6.67±3.57 cm,P=0.046）,the cases number in the TNM stagingⅢperiod andⅣperiod（75.0%vs.50.0%,P=0.036）and the number of patients with pathological differentiation in poor differentiation（23.6%vs.5.6%,P=0.016）of HIF-1α-OE group were significantly more.The 5-year tumor-free survival rate and 5-year survival rate in HIF-1α-OE group were both lower than those in HIF-1α-UE group.The HCC tumor size of the PIP2-High group was significantly larger than that of the PIP2-Low group（8.52±4.00 cm vs.6.66±3.59 cm,P=0.018）,and the vascular invasion rate of the PIP2-High group was significantly higher than that of the PIP2-Low group（44%vs.23.9%,P=0.038）.In the TNM staging,PIP2-High group in phaseⅢandⅣsignificantly more than the number of cases in the PIP2-Low group（82%vs.52.1%,P=0.010）.Compared with the prognosis of the two groups,the 5-year postoperative survival rate in the PIP2-High group was significantly lower than that in the PIP2-Low group（P=0.0022）.In 60 pairs of HCC tissues and paracancer tissues,CAPZA1 expression in paracancer tissues was higher than that in HCC tissues in 44cases（P=0.001）.The vascular invasion rate of CAPZA1-UE group was significantly higher than that of CAPZA1-OE group（45.1%VS 28.9%,P=0.038）.In TNM staging,the number of patients withⅢandⅣstage in CAPZA1-UE group was more significantly than that in CAPZA1-OE group（77.5%VS 57.8%,P=0.002）.In terms of pathological differentiation of HCC,the CAPZA1-UE group had significantly more poorly differentiated HCC patients than CAPZA1-OE group（29.6%VS 7.2%,P=0.001）.The 5-year tumor-free survival rate and5-year survival rate in CAPZA1-UE group were lower than those in CAPZA1-OE group.In HCC tissues,the expression of CAPZA1 was positively correlated with the expression of E-cadherin（r=0.3129,P<0.001）,and was negatively correlated with Vimentin expression（r=-0.5923,P<0.001）.In HCC tissue microarray,the expression of CAPZA1 was positively correlated with E-cadherin expression（r=0.3129,P<0.001）,and was negatively correlated with Vimentin expression（r=-0.5923,P<0.001）.In HCC microarray,the expression of HIF-1αwas positively correlated with the level of PIP2（r=0.6371,P<0.001）,and there was no linear correlation between the level of PIP2 and the expression of CAPZA1（r=0.1111,P=0.2232）,and between the expression of HIF-1αand CAPZA1（r=0.1444,P=0.1126）.2.Compared with the 20%O2 culture group,the expression levels of HIF-1α,RhoA and ROCK1 in HCC cells in the 1%O2 culture group and the CoCl2-treated group were all increased,but the expression levels of CAPZA1 in the three groups were unchanged.With the prolonging of CoCl2 treatment time,the expression of HIF-1α,RhoA,ROCK1 and PIP2 in HCC cells increased in a gradient.The treatment of YC-1 can inhibit the storage of HIF-1αcaused by hypoxia,and also inhibit RhoA and ROCK1 expression in HCC cells.The activation of RhoA and the expression of ROCK1 could be inhibited by adding C3 transferase protein under hypoxia condition.Under hypoxia conditions,the addition of Y-27632 inhibited the synthesis of PIP2 and the assembly of F-actin in HCC cells.3.The level of CAPZA1expression was highest in HCCLM3 cells and lowest in SMMC-7721 cells.The interference of CAPZA1 expression can promote the invasion and migration of SMMC-7721 cells,while up-regulated CAPZA1 expression can inhibit the invasion and migration of SMMC-7721 and MHCCLM3 cells.By interfering with CAPZA1expression in HCC cells,E-cadherin expression could be down-regulated while Vimentin expression could be up-regulated.The up-regulation of CAPZA1 expression in HCC cells can up-regulate E-cadherin expression,while down-regulate Vimentin expression.CAPZA1 can interact with F-actin,and the down-regulation of CAPZA1 can increase the level of F-actin in HCC cells.PIP2 coated amide beads can pull CAPZA1 down from the cell lysate.Immunofluorescence showed that after Neomycin treatment,F-actin content in HCC cells was decreased and the cell morphology became round.After PIP2 treatment,the content of F-actin in HCC cells was increased,and the cell morphology became fusiform.The immunoprecipitation showed that the treatment of Neomycin and PIP2 did not affect the expression of CAPZA1.But after Neomycin treatment,the unmber of CAPZA1 combined with F-actin was increased.After PIP2 treatment,the number of CAPZA1 combined with F-actin was reduced.Cellular substructural localization,Transwell,and wound healing assays showed that after the addition of Neomycin to block the binding of PIP2 to CAPZA1,CAPZA1 was more bound to the cytoskeleton and less distributed in the cytoplasm,and inhibited the invasion and migration of HCC cells.After adding the synthetic PIP2 to enhance the binding of PIP2 and CAPZA1,CAPZA1 was less bound to the cytoskeleton and more distributed in the cytoplasm,and enhanced the invasion and migration of HCC cells.4.In the nude mice treated with Neomycin,tumor lesions on the liver surface were less and mainly confined to the primary site,while the liver surface of the nude mice treated with sterile water was covered with tumor lesions（P=0.0001）.Compared with the control group,nude mice inoculated with LV10-CAPZA1 SMMC-7721 cells had significantly more HCC lesions（P=0.0435）,while nude mice inoculated with LV8-CAPZA1 MHCCLM3 cells had significantly fewer HCC lesions（P=0.0399）.Conclusions:1.The expression of HIF-1α,PIP2 and CAPZA1 in HCC tissues is closely related to the invasion and metastasis of HCC and the prognosis of patients.High expression of HIF-1αand PIP2 can promote the malignant progression of HCC and lead to poor prognosis.Low CAPZA1 expression can promote the invasion and metastasis of HCC by positively regulating EMT.Correlation analysis showed that the expression of HIF-1αwas positively correlated with the level of PIP2,and there was no linear correlation between HIF-1αexpression and CAPZA1 expression and between PIP2 level and CAPZA1 expression,suggesting that hypoxia might induce the synthesis of PIP2 through hypoxic effector,HIF-1α,and thus promote the invasion and metastasis of HCC.However,the expression CAPZA1 is not regulated by HIF-1αand PIP2,suggesting that there may be other regulatory mechanisms between them.2.Hypoxia can promote the synthesis of PIP2 on the cell membrane of HCC cells by activating the HIF-1α/RhoA/ROCK1 signaling pathway,thus inducing actin filaments and cytoskeleton remodeling.Blocking the synthesis of PIP2 can inhibit the remodeling of actin cytoskeleton.3.The combination of PIP2 and CAPZA1 can make CAPZA1 to be released from the barbed end of F-actin,open the barbed end of F-actin,and promote its continuous polymerization and prolongation,thus driving EMT to enhance the invasion and migration ability of HCC cells.However,the interaction mechanism between PIP2 and CAPZA1 is not well explained.We speculate that PIP2 may alter the protein conformation of CAPZA1 by binding to CAPZA1,thus causing CAPZA1 to lose the ability to bind to the barbed end of F-actin.Relevant research work needs to be further advanced.4.The invasion and metastasis of HCC in nude mice could be inhibited by blocking the HIF-1α/PIP2/CAPZA1 signaling pathway.Through in-depth explore,this study investigated the influence of HIF-1α/RhoA/ROCK signaling pathway on the level of PIP2 and the regulatory mechanism of PIP2 on CAPZA1.It is the first time to propose and confirm the research hypothesis that hypoxia can regulate the actin cytoskeleton remodeling by HIF-1α/RhoA/ROCK/PIP2/CAPZA1 pathway to promote the invasion and metastasis of HCC cells,which further enriched the theoretical treasure house of the study on the mechanism of HCC invasion and metastasis,and provided a new potential target for the prevention and treatment of HCC invasion and metastasis in clinical practice. |
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