ANXA5 Regulating Hepatocarcinoma And Renal Cancer Progression And Metastasis Via ERK/RAC1

Background:Annexin A5（ANXA5）is a Ca²⁺-dependent phospholipid-binding protein which is the smallest member of the annexin family and consists of 319 amino acids with a molecular weight of 35 kDa.ANXA5 plays an important role in maintaining cell membrane stability,exocytosis,regulating cell proliferation,differentiation,apoptosis,anti-inflammatory and antithrombotic processes.More and more studies have reported that the deregulation of ANXA5 is associated with tumorigenesis,metastasis,drug resistance and so on.Hepatocarcinoma cancer（HCC）and renal cell cancer（RCC）are two common malignant tumors,with the characteristics of high incidence,high metastasis rate and poor prognosis.Tumor metastasis is the most important factor that affects the prognosis and survival of cancer patients.It is of great significance to explore the molecular mechanism of tumor development and find a new target for tumor diagnosis and treatment.Research work from our group established the positive correlation of Anxa5 expression level with hepatocarcinoma progression and lymphatic metastasis.Previous work shown that higher ANXA5 expression level enhanced the in vitro proliferation,migration and invasion behaviours and the in vivo tumor malignant progression as well as lymph node metastasis（LNM）potential of Hca-P,a murine hepatocarcinoma cell line.However,the underlying molecular regulation mechanism and clinical significance of Anxa5 in cancer progression and metastasis,especially in cancer lymphatic progression and metastasis,are poorly understood.This thesis mainly studies the molecular mechanism of ANXA5 regulating the malignant behaviors of hepatocarcinoma and renal cell carcinoma.Objective:1.To investigate the expression of ANXA5 in clinical samples of liver cancer and renal cell carcinoma and its role in the progression of tumors;2.To definite the influence of ANXA5 on proliferation,colony formation,migration and invasion of hepatocarcinoma cell line Hca-P,renal cell carcinama cell line 786-O and ACHN malignant behaviors in vitro and in vivo.3.Reveal the direct interaction between ANXA5 and MYH9;4.To explore the molecular mechanism of ANXA5 regulate the HCC and RCC progression and metastasis.Methods:1.Immunohistochemistry was used to detect the expression of ANXA5,CRKI/II and RAC1 in 46 cases of human hepatocellular carcinoma and their matched paracancer tissue,and the correlation with clinical parameters was analyzed.Western blot was used to detect the expression of ANXA5,MYH9 and RAC1 in 40 cases of renal cell carcinoma and their matched paracancer tissue,analyzed the correlation with clinical parameters,and the correlation between them;2.The plasmid pGPU6/GFP/Neo-shControl and pGPU6/GFP/Neo-shANXA5 were transfected into hepatocellular carcinoma cell line Hca-P and renal carcinoma cell line786-O and ACHN using LipofectamineTM 2000,then obtained their monoclonal cell lines against G418 screening combined with limited dilution method.Using Western blot and qRT-PCR to detected the expression level of ANXA5 monoclonal cell;3.Construction of pcDNA3.1-V5/HisB-Anxa5 recombinant vector,transfected into renal cell carcinoma cell line 786-O and ACHN,Western blot used to detect the expression of ANXA5;4.The effects of ANXA5 down-regulation on colony formation and proliferation capacity of Hca-P;the effects of ANXA5 expression changing on 786-O and ACHN in vitro were detected by plate clony formation assay,trypan blue counting method and MTT assay;5.Transwell chamber assay and FITC-phalloidin cytoskeleton staining were used to detect the effect of ANXA5 down-regulation on the migration and invasion ability of Hca-P;the effects of ANXA5 expression changing on 786-O and ACHN;6.The effect of ANXA5 down-regulation on the adhesion of Hca-P was detected by in situ lymph node adhesion assay.The hanging drop aggregation assay was used to analyze the cell-cell adhesion ability of Hca-P cell;7.WesternblotwasusedtodetecttheinfluenceofANXA5on CRKI/II/DOCK180/RAC1 and MEK/ERK/C-MYC signaling pathways,qRT-PCR assay was used to detect VIMENTIN and MMP9 expression in Hca-P cell;Western blot was used to detect the influence of ANXA5 on PI3K/AKT/MEK/ERK/MMP pathway and CRKL/RAC1 expression in renal cell carcinoma cell line;8.The effect of ANXA5 down-regulation on the tumorigenicity of Hca-P in vivo was detected by instillated cell to mouse footpad.IHC was used to detect the expression of ANXA5,CRKI/II,RAC1,CD34 and VEGF-3 in solid tumors.The effect of ANXA5down-regulation on the tumorigenesis of renal carcinoma cell line ACHN in vivo was detected by subcutaneous injection into nude mice.HE staining was used to detect the lymph node metastasis;9.Co-immunoprecipitation and immunofluorescence co-localization were used to measure the interaction of ANXA5 and MYH9;10.According to MYH9 mRNA sequence,designed special siRNA sequence for MYH9,with LipofectamineTM 2000 siMYH9 and siControl were transfected into 786-O and ACHN cells.Clony formation assay,MTT assay,Transwell chamber and FITC-phalloidin cytoskeleton staining were used to detect the effect of MYH9downregulation on the proliferation,migration,invasion and cytoskeletal F-actin expression of 786-O and ACHN;11.Knockdown ANXA5 and MYH9 expression,detected the changes of proliferation,migration and invasion ability,and down regulated MYH9 expression in pcDNA3.1-V5/HisB-Anxa5 cell,to detect whether MYH9 downregulation could release the influnce of ANXA5 upregulation on proliferation and metastasis ablility of renal cell.Results:1.ANXA5 overexpression correlates with HCC and RCC progression and metastasis;CRKI/II and RAC1 are unregulated and synergistically correlated with ANXA5 in HCC progression;MYH9 and RAC1 are unregulated and synergistically correlated with ANXA5 in RCC progression;2.Obtained stable down-regulation monoclonal cell line of ANXA5 in Hca-P,786-O and ACHN cell,the expression of ANXA5 was downregulated more than 90%;2.The knockdown of ANXA5 significantly reduced the proliferation,migration,invasion ability and cytoskeleton stress fibers formation of Hca-P,786-O and ACHN cells,inhibited the in situ adhesion of Hca-P cells and enhanced their cell-cell adhesion ability;3.The results of Western blot showed that ANXA5 downregulation reduced the expression of CRKI/II/DOCK180/RAC1 and MEK/ERK/C-MYC signaling pathways in Hca-P cell;and decreased the expression of PI3K/AKT/MEK/ERK/MMP pathway and CRKL/RAC1 in renal cell carcinoma cell lines;4.ANXA5 knockdown suppresses murine tumorigenicity and lymph node metastasis induced by Hca-P and ACHN implantation.Immunohistochemistry showed that in Hca-P tumor-bearing mice,the expression of ANXA5,CRKI/II,RAC1,CD34 and VEGF-3 was significantly decreased in Hca-P cell line with ANXA5 downregulation.5.Up-regulation of ANXA5 promoted the proliferation and metastasis of 786-O and ACHN cells.6.Co-immunoprecipitation and immunofluorescence co-localization results showed that ANXA5 and MYH9 had a direct interaction in cells;7.MYH9 downregulation inhibited the proliferation and metastasis of 786-O and ACHN cells;8.Double knockdown ANXA5 and MYH9 expression,compared with knocking down ANXA5 alone,ACHN cells proliferation,migration and invasion ability was more decreased;in ACHN-pcDNA3.1-V5/HisB-Anxa5 cell,knockdown MYH9 could release the influnce of ANXA5 upregulation on proliferation and metastasis ablility of renal cell.Conclusion:1.In clinical level,ANXA5 overexpression correlates with HCC and RCC progression and metastasis;2.ANXA5 could affect the proliferation,migration and invasion abilities of Hca-P,786-O and ACHN cells in vitro and in vivo;3.ANXA5 overexpression correlates with CRKI/II and RAC1 both in HCC tissues and Hca-p cell,in addition to,also regulated Hca-P cell proliferation and metastasis via MEK-ERK/MMP9 pathway;4.ANXA5 overexpression correlates with MYH9 and RAC1 both in RCC tissues and renal cancer cell,furthermore,also regulated the proliferation,migration and invasion abilities of renal cancer cell by PI3K/AKT/MEK/ERK/MMP pathway and CRKL/RAC1.