A Preliminary Study On Effect And Mechanism Of CAPZA1 In Hepatocellular Carcinoma Metastasis

Background:Hepatocellular carcinoma(HCC)is one of malignant tumors with the sixth most commonly diagnosed cancer and the second cancer-related mortality around the world.China is a high incidence of liver disease,according to statistics,the incidence and mortality of HCC in China accounted for 50% of the world,seriously affecting people’s lives and health.Surgical resection is the main treatment for HCC.In recent years,surgery based comprehensive treatment has made a good discovery,but the recurrence rate and mortality rate of HCC patients after 5 years is still high to 70%.High invasion and metastasis characteristic of HCC is an important factor for postoperative recurrence and metastasis.Epithelial-mesenchymal transition(EMT)is an important mechanism of tumor invasion and metastasis.EMT is a biological process in which epithelial cells lose their epithelial properties through a specific transformation process and are transformed into mesenchymal cells that have the properties of movement.In this process,the adhesion between tumor cells and the basement membrane of epithelial characteristics disappeared,and the polarity of epithelial cells was lost.It has been confirmed that EMT plays an important role in the invasion and metastasis of HCC.HCC cells acquire the movement ability through the EMT,and they can reach out invasive pseudopod to invade the surrounding tissue and blood vessels from the primary lesion,and then settle down in the distance through the blood transport and form the metastatic lesions.Cytoskeleton is an important component of the cell,which plays an important role in the maintenance of cell morphology,motility and function.Actin is one of the most important components of the cytoskeleton,and the actin cytoskeleton morphological changes are closely related to the occurrence of EMT.In the process of EMT,all kinds of factors stimulate the expression of actin related proteins through specific signaling pathways.The expression changes of actin related proteins can break down the dynamic balance of actin cytoskeleton,and make the actin cytoskeleton depolymerization and reorganization seriously imbalance,leading to the change of the cell morphology and the formation of the tumor invasive pseudopod.It has been releaved that the dynamic changes of actin cytoskeleton play an important role in the formation of the invasi ve psudopod.Capping protein,a protein complex referred to as Cap Z,is an actin-binding complex that can bind to the barbed-end of existing actin filaments.The binding can impede the addition of G-actin to the barbed-end of existing actin filaments and regulate actin cytoskeleton remodelling.Therefore,Cap Z is thought to play an important role in the regulation of actin cytoskeleton remodeling.CAPZA1 is the α1 subunit of this complex.Therefore,in theory,we can infer that CAPZA1 can inhibit tumor cell EMT by inhibiting actin cytoskeleton remodeling,thereby inhibiting the invasion and me tastasis of tumor cells.It is reported that CAPZA1 can promote the malignant progression of neuroblastoma and gastric cancer,but its mechanism is not clear.Whether CAPZA1 has the same effect in HCC has not been reported.Objective:1.Through the collecting HCC samples and detecting the expression of CAPZA1 in paraffin specimens of cancer tissues,we want to analyze the relationship between the expression of CAPZA1 and the clinicopathological factors of HCC and the prognosis in patients with HCC,and to clarify the inhibitory effect of CAPZA1 on invasion and metastasis of HCC.2.At the cellular level,we want to verify the inhibitory effect of CAPZA1 on the invasion and metastasis of HCC cells,and identif y the negative regulatory relationship between CAPZA1 and HCC EMT,and preliminarily study the mechanism of CAPZA1 inhibition in HCC EMT.3.We will establish the nude mice orthotopic liver transplantation model to observe the effect of CAPZA1 on the invasion and metastasis of HCC cells in vivo.Methods:1.Approved by the Southwest Hospital of Third Military Medical University,129 Patients with HCC who accepted the surgery resection in Southwest hospital from January 2011 to December 2011 were collected.Among them,there were 115 male cases and 14 female cases.The patient’s medical records were reviewed to summarize the clinical pathological information such as the size of HCC,pathological differentiation,TNM stage,vascular invasion,lymph node metastasis and extrahepatic metastasis.The patients were followed up until December 31,2015,and the recurrence and mortality of HCC patients were recorded.The expression of CAPZA1 in paraffin sections of 129 patients with HCC was detected by immunohistochemistry,and the relationship between the expression of CAPZA1 and the prognosis of patients and the clinicalpathological characteristics of HCC were analyzed.2.Hep G2 and MHCC97 H cells were cultured in vitro.The expression of CAPZA1 in Hep G2 cells which have weak invasion ability was interfered,and the CAPZA1 expression in MHCC97 H cells which have strong invasion ability was induced to over expression.CCK-8 was used to detect the proliferation ability of Hep G2 and MHCC97 H cells.Transwell migration and invasion assay were used to detect the migration and invasion ability of Hep G2 and MHCC97 H cells,respectively.Western blot and q PCR were used to detect the expression of CAPZA1 and EMT related markers E-cadherin,N-cadherin,vimentin in HCC cells.The interaction between CAPZA1 and actin was detected by immunoprecipitation.Western blot was used to detect the expression of EMT related transcription factor Snail family,ZEB family and Twist family.3.We estabilished CAPZA1 stably expressing low expression of HepG2 cells and high expression of MHCC97 H cells in vitro.The above cell lines were injected into liver subcapsule of nude mice to establish nude mouse orthotopic liver transplantation model.6 weeks later,the metastasis of tumor cells in nude mice was observed by small animal MRI,and the death rate of nude mice was observed.Results:1.In the TNM staging of HCC,the ratio of CAPZA1 low expression group in the Ⅲ-Ⅳ stage was significantly higher than that of CAPZA1 high expression group(P<0.001).In the pathological differentiation of HCC,the HCC differentiation in the CAPZA1 low expression group was lower than that in CAPZA1 high expression group(P<0.001).Compared with the CAPZA1 high expression group,CAPZA1 low expression group not only had a higher rate of lymphatic invasion(P=0.033)and vascular invasion(P=0.002),but also had a higher rate of extrahepatic metastasis(P=0.006).In addition,the recurrence rate and mortality rate of CAPZA1 low expression group were significantly higher than those of CAPZA1 high expression group 5 years after operation(P<0.001).2.Interference of CAPZA1 expression can promote the migration and i nvasion of Hep G2,up regulation of CAPZA1 expression can inhibit the migration and invasion of MHCC97 H.The expression of CAPZA1 had no effect on the proliferation of Hep G2 and MHCC97 H cells.After the down-regulation of CAPZA1 expression,the expression o f epithelial marker E-cadherin was decreased,and the expression of mesenchymal markers N-cadherin and vimentin were up-regulated.After the up-regulation of CAPZA1 expression,the expression of epithelial marker E-cadherin was up-regulation,and the expression of mesenchymal markers N-cadherin and vimentin were decreased.CAPZA1 interacts with actin in HCC cells.When the expression of CAPZA1 was down regulated,the expression of transcription factors Snail1 and ZEB1 was increased.The expression of transcription factors Snail1 and ZEB1 decreased when the expression of CAPZA1 was up-regulated.3.Compared with the control group,CAPZA1 stabl y low expression Hep G2 cells had a wide range of invasion and metastasis in nude mice liver,and the survival time was shorter in nude mice.The invasion and metastasis of CAPZA1 stably high expression MHCC97 H cells was significantly inhibited in nude mice,and the survival time of nude mice was longer compared with the control group.Conclusions:1.CAPZA1 expression levels werenegatively correlated with the biological characteristics of primary HCC and patient prognosis.CAPZA1 could be a useful biomarkerfor clinical determination of the prognosis of HCC patients.2.CAPZA1 can inhibit the invasion and metastasis of HCC cells by regulating EMT.CAPZA1 can regulate the remodeling of actin cytoskeleton,which is the mechanism by which CAPZA1 inhibits HCC EMT.Transcription factors Snail1 and ZEB1 are involved in CAPZA1 inhibition of HCC EMT.3.In vivo,CAPZA1 could inhibit the invasion and metastasis of HCC cells in nude mice,and the low expression of CAPZA1 could promote the death of nude mice.Through the study of clinical cases,cell experiments and animal experim ents,we defined the role of CAPZA1 in the invasion and metastasis of HCC.We can draw the following conclusions through the above three aspects: CAPZA1 inhibits EMT in HCC cells by regulating actin cytoskeletonremodelling,thereby reducing the metastatic ability of the cells.Together,our data suggest that CAPZA1 could be a useful biomarker for clinical determination of the prognosis of HCC patients.However,the study about detail mechanism of how CAPZA1 inhibits EMT in HCC cells by regulating actin cytoskeletonremodelling is lack.This will be the main task in our following study.