Effect Of Knockdown ANXA11 On Malignant Behavior Of Hepatocellular Carcinoma Cell HCCLM3 In Vitro And In Vivo

Background: ANXA11 belongs to the annexin family and is involved in the regulation of various cellular functions and is associated with the development,metastasis,drug resistance and poor prognosis of various tumors.The preliminary results of this group showed that knockdown of ANXA11 was associated with in vitro migration,lymphatic metastasis and drug resistance of Hca-P,Hca-F and human hepatoma Bel7402 cells.In this paper,we further studied the effects and molecular mechanisms of knockdown of ANXA11 on the malignant behavior of human hepatocarcinoma HCCLM3 cells in vitro and in vivo.Objective:1.To detect the expression of ANXA11 in clinical samples and liver c ancer cells of liver cancer;2.To study the effects of knockdown ANXA11 on p roliferation,migration and invasion,cell adhesion,cytoskeletal formation and drug re sistance of HCCLM3 cells in vitro;The effect of ANXA11 on HCCLM3 cell tran splantation in nude mice and lymph node metastasis was knocked out,and the e xpression level of related molecules was detected.Methods:1.Western Blot was used to detect the expression level of ANXA11 in liver cancer and liver cancer cells.The appropriate cell strain was selected for f urther experiment.2.The monoclonal antibody strain shANXA11 and shCtrl whic h stably down-regulated ANXA11 were screened by limiting dilution method.The effect of knockdown ANXA11 on the proliferation of cells in vitro was detecte d by MTT assay.4.The effect of knockdown ANXA11 on cell migration and inv asion was detected by Transwell method.5.The effect of knockdown ANXA11 on the adhesion ability of extracellular matrix and lymph nodes was detected.6.FI TC marker phalloidin staining assay was used to detect the effect of knockdown ANXA11 on cytoskeleton formation.7.MTT assay combined with Hoechst33258 staining to detect the effect of knockdown ANXA11 on cell resistance;8.WB assay for knockdown ANXA11 to regulate cell malignant behavior Molecular me chanism;9.HCCLM3 cells in nude mice to detect the effect of knockdown ANX A11 on tumor growth and lymph node metastasis;10.HE and IHC experiments t o detect tumor cell metastasis and expression levels of related molecules.Results:1.23 cases of liver cancer clinical samples,22 cases of cancer tissue A NXA11 high expression,the overall level increased by 3.363 times(P=0.0329);2.Liver cancer cells HUH7,HCCLM3,HEPG2 and normal liver cells LO2,AN XA11 in HCCLM3 cells The highest expression level was 1.87 times of LO2 ce lls(P=0.0044),which was used as a cell model for subsequent experiments.3.WB detected ANXA11 expression level in monoclonal HCCLM3 cells.Compared with the shCtrl group,ANXA11 was reduced by 89% in the shANXA11 group(P<0.001);4.Compared with the shCtrl group,the in vitro proliferation ability of the s hANXA11 group was inhibited;5.Compared with the shCtrl group,the shANXA11 group had a 1.47-fold increase in in vitro migration ability(P=0.0007),and the invasive ability was increased in vitro 1.53 times(P=0.0009);6.The cell adhes ion rate of shANXA11 group was 1.48 times(P<0.001),and the number of cells adhering to lymph nodes in shANXA11 group was 4.54 times that of shCtrl gro up(P<0.001);The filamentous actin of shANXA11 group increased significantly,a nd a large number of filopodia appeared.8.Knockdown ANXA11 reduced the se nsitivity of HCCLM3 cells to cisplatin;9.Knockdown ANXA11 inhibited Erk and CRKL-Rac1 pathways,enhanced MMP2、MMP9、Cdc42 expression.10.The resu lts of tumor formation in nude mice showed that the average tumor weight in th e shCtrl group was 4.09 times that of the knockdown AMXA11 group(P=0.0026),and the average tumor volume in the shCtrl group was 3.45 times that of the sh ANXA11 group.11.The average quality of the lymph nodes in the shANXA11 group For the shCtrl group 2.78 times(P=0.0265),HE staining experiments showe d that knockdown of ANXA11 promoted cell metastasis to lymph nodes;12.IHC experiments showed that shNPXA11 group had high expression of MMP2、MMP9 and Cdc42,and low expression of CRKL.Conclusion: 1.ANXA11 is highly expressed in clinical samples of liver cancer;2.Knockdown of ANXA11,inhibiting proliferation of HCCLM3 cells in vitro,pr omoting migration and invasion of HCCLM3 cells,enhancing cell adhesion,enha ncing HCCLM3 cells resistance to cisplatin,and promoting cell filamentous The formation of actin inhibits the Erk and CRKL-Rac1 pathways and promotes the expression levels of MMP2、MMP9 and Cdc42;3.Knockdown ANXA11 to inhibi t the growth of HCCLM3 cells transplanted into nude mice and promote the tra nsfer of cells to lymph nodes.