| Hepatocellular carcinoma(HCC)is a primary liver cancer with high mortality and a highly aggressive tumor with frequent metastases.In recent years,the number of HCC patients has been increasing,but the clinical treatment effect of most patients is not satisfactory.HCC patients are mainly treated with surgical resection and liver transplantation.However,HCC patients are unable to detect changes in the body in time and lack the awareness of regular physical examinations in the initial stage of illness.Therefore,when most patients notice the abnormal body and go to the hospital for treatment,the cancer cells in the body have spread,the cancer has deteriorated and entered the middle and late stages,so currently surgical resection and local treatment alone can no longer solve the problem.Early metastasis is an important reason for the frequent recurrence and high fatality rate of HCC.Finding and elucidating the molecular mechanism of HCC is an urgent need to inhibit metastasis and improve patient survival.Insulin-like growth factor binding protein 2(IGFBP2)is one of the family of insulin-like growth factor(IGF),which can function through IGF dependent and IGF independent pathways.Mature IGFBP2 contains 289 amino acids,which is mainly expressed during embryonic development,with low or no expression in adult tissue cells,but high expression in the serum and cerebrospinal fluid of many patients with malignant tumors.In recent years,IGFBP2 has been found to be elevated in prostate cancer,breast cancer,pancreatic cancer and other malignant tumors and is closely related to the progression of tumor course.IGFBP2 is overexpressed in salivary adenoid cystic carcinoma,and in xenograft tumor models,IGFBP2 overexpression promotes lung and liver metastasis of tumor cells,and if the expression of IGFBP2 in the nucleus is reduced,the formation of lung and liver metastasis is weakened.In pancreatic cancer,increased expression of IGFBP2 is associated with lymph node metastasis and short survival time,and IGFBP2activates epithelial-mesenchymal transition(EMT)to endow cancer cells with clinical characteristics of invasion.The pathological characteristics of liver cancer show that tissues with high expression of IGFBP2 are large in size and strong in cell proliferation ability,and up-regulation of IGFBP2 enhances the migration and invasion ability of Huh7cells.EMT is a landmark event in invasion and metastasis and plays a vital role in the malignant progress of HCC.EMT was initially considered to be a developmental process in which epithelial cells transition and transform into mesenchymal cells under a specific environment,and thus the cells acquire the ability to move and transfer.In the process of cancer,the occurrence of EMT is manifested by the lack of expression of epithelial phenotype marker of E-cadherin,which is also the key step of EMT;mesenchymal phenotype markers of N-cadherin and Vimentin increased,and the expression of EMT-related transcription factors increased,such as slug,snail,Twist,zeb1,zeb2.Studies have shown that EMT can lead to tumor infiltration or malignancy,and down-regulation of these factors can inhibit malignant transformation of tumors.The activation ofβ-catenin is closely related to the development of EMT and cancer.β-catenin is the core factor of this study,and its activation is a key step in causing downstream gene transcription.When the Wnt/β-catenin signaling pathway is closed,the intracellular protein complex is stable and phosphorylatesβ-catenin,making it inactive,and finally degraded by the proteasome;But when the signal pathway is activated,the stability of the intracellular protein complex is reduced,β-catenin is dephosphorylated and released,So that intracellular expression ofβ-catenin increases,then part ofβ-catenin into the nucleus,binds to nuclear transcription factors,and initiates the transcription of target genes,including E-cadherin,c-myc,and EMT-related transcription factors Snail.LRP6 is the key receptor protein forβ-catenin activation.Also,the activation ofβ-catenin plays a key role in the normal growth and development of the liver.In HCC,theβ-catenin is usually activated and its expression level is increased,but the molecular mechanism of its activation is still unclear.In this study,we using three methods:clinical samples of tumor tissue of HCC patients,HCC cell lines with overexpression and knockdown of IGFBP2,and subcutaneous tumor models in nude mice to explore the expression levels of IGFBP2 and EMT-related molecules in HCC patient tumor tissues,the mechanism of IGFBP2mediates EMT to promote the invasion and metastasis of HCC cell lines,and the effect of IGFBP2 on subcutaneous tumors in nude mice,which will provide an important experimental basis for finding and elucidating the molecular mechanism of HCC invasion and metastasis.ObjectiveTo explore the mechanism of IGFBP2 regulates LRP6/β-catenin signaling to drive EMT and invasive character in HCC and provide experimental basis for revealing new mechanism of invasion and metastasis and strategies for treatment in HCC.MethodsDetection of clinical samples:Collect tumor tissues of HCC patients,HE staining method was used to detect the pathological features of precancerous lesions and tumor tissues in HCC patients,immunohistochemistry was used to detect the expression of IGFBP2 and EMT related proteins in precancerous lesions and tumor tissues,the co-expression of IGFBP2 and LRP6 and the expression ofβ-catenin in precancerous lesions and tumor tissues were detected by immunofluorescence.Research on the molecular mechanism of cell lines:We used transfection technology to transfect IGFBP2 plasmid and small interfering RNA into Hep G2 cells and HCC-LM3cells respectively,and the migration and invasion ability of cells were detected by transwell,the expression of IGFBP2 and EMT related proteins were detected by western blot and immunofluorescence,the expression of genes of IGFBP2 and EMT related proteins were detected by QPCR,Co-IP and immunofluorescence methods were used to detect the co-expression of IGFBP2 and LRP6 in Hep G2 cells,the expression and localization ofβ-catenin proteins in Hep G2 cells were detected by Western blot method and immunofluorescence method,and the transcriptional activity ofβ-catenin was detected by double luciferase reporter gene method.Establishment and detection of subcutaneous tumor model in nude mice:BALB/c nude mice were randomly divided into two groups,1×10~6 Hep G2/pc DNA3.1-Ctr cells and Hep G2/pc DNA3.1-IGFBP2 cells were inoculated in the subcutaneous tissue of the back of nude mice.Six weeks later,nude mice were sacrificed and the tumor tissue was removed.HE staining method was used to detect the pathological characteristics of tumor tissue,the expression of IGFBP2 and EMT related proteins in tumor tissues were detected by western blot and immunohistochemistry,the co-expression of IGFBP2 and LRP6 and the expression ofβ-catenin were detected by immunofluorescence.Results1.Detection of clinical samples1.1 Pathological features of precancerous lesions and tumor tissues in HCC patientsThe nucleus of hepatocytes was round,and it were arranged regularly in hepatic cords in precancerous lesions;in tumor tissue,the nucleus of hepatocytes was large and staining became deep,the proportion of nucleus and cytoplasm was disordered,more dense areas of the nuclear,the arrangement of cells were irregular and inflammatory cells infiltrated.1.2 Expression of IGFBP2 and EMT related proteins in precancerous lesions and tumor tissues of HCC patientsIGFBP2 protein was negative or low expression in precancerous lesions,while it was obviously high in most tumor tissues.Compared with precancerous lesions,the expression of N-cadherin and Vimentin increased,while the expression of E-cadherin decreased in tumor tissues.The positive staining of IGFBP2 and Vimentin were mainly located in the cytoplasm,and the positive staining of E-cadherin and N-cadherin were mainly located in cytomembrane.1.3 Expression of LRP6/β-catenin signaling pathway related proteins in precancerous and tumor tissues of HCC patientsThe co-expression level of IGFBP2 and LRP6 in tumor tissues of HCC patients was significantly higher than that of precancerous lesions.The expression level ofβ-catenin in tumor tissue of HCC patients was significantly higher than that of precancerous tissue,and it was mainly expressed in cytoplasm and cytomembrane.2.Research on the molecular mechanism of cell lines2.1 The effect of IGFBP2 on the migration and invasion of HCC cell linesThe immunofluorescence results showed that the expression of IGFBP2 was low in Hep G2 cells with relatively low metastatic potential,but high in HCC-LM3、SMMC-7721 cells with high metastatic potential.Transwell results showed that compared with the pc DNA3.1-Ctr group,overexpression of IGFBP2 increased the number of migration and invasion of Hep G2 cells;compared with the control group,knocking down the expression of IGFBP2 significantly reduced the number of migration and invasion of HCC-LM3 cells.2.2 Effect of IGFBP2 on the expression of EMT-related factors in HCC cell linesCompared with the pc DNA3.1-Ctr control group,overexpression of IGFBP2reduced the expression levels of gene and protein of E-cadherin and increased the expression levels of genes and proteins of N-cadherin and Vimentin;on the contrary,compared with the control group,silencing IGFBP2 up-regulate the expression of gene and protein of E-cadherin and down-regulate the expression of genes and proteins of N-cadherin and Vimentin.2.3 Effects of overexpression IGFBP2 on expression levels of LRP6/β-catenin signaling pathway related proteinsCompared with the pc DNA3.1-Ctr group,overexpression of IGFBP2 up-regulated the co-expression level of IGFBP2 and LRP6,and up-regulated the total expression and the nuclear expression levels ofβ-catenin.The results of dual luciferase reporter gene method showed that compared with the control group,250ng/ml recombinant human IGFBP2 protein and 100ng/mlβ-catenin degradation complex inhibitor CHIR-99021stimulated Hep G2 cells to up-regulate TOP/FOP Flash luciferase signal strength,which enhances the transcriptional activity ofβ-catenin.3.Establishment and detection of subcutaneous tumor model in nude mice3.1 The size,weight and pathological characteristics of tumor tissue in nude miceThe tumor tissue of nude mice in the Hep G2/pc DNA3.1-IGFBP2 group was larger,and the weight was significantly higher than that in the Hep G2/pc DNA3.1-Ctr group.The nucleus of the tumor tissue of the nude mice in the Hep G2/pc DNA3.1-Ctr group was round or oval,with multinucleated cells,less cytoplasm,less dense areas of nuclear,and inflammatory cell infiltration;The tumor tissue of the Hep G2/pc DNA3.1-IGFBP2 group of nude mice has large nuclear with darker staining,the proportion of nucleus and cytoplasm was disordered,more dense areas of the nuclear,and a large number of inflammatory cell infiltration.3.2 Expression of IGFBP2 and EMT related proteins in tumor tissues of nude miceThe expression of IGFBP2 protein in Hep G2/pc DNA3.1-IGFBP2 group was significantly higher than that in Hep G2/pc DNA3.1-Ctr group.Compared with Hep G2/pc DNA3.1-Ctr group,the expression of E-cadherin was significantly decreased,and the expression of N-cadherin and Vimentin were significantly increased in Hep G2/pc DNA3.1-IGFBP2 group.3.3 Expression of LRP6/β-catenin signaling pathway related proteins in tumor tissues of nude miceCompared with the Hep G2/pc DNA3.1-Ctr group,the co-expression level of IGFBP2 and LRP6 in the Hep G2/pc DNA3.1-IGFBP2 group increased;theβ-catenin expression level in the Hep G2/pc DNA3.1-IGFBP2 group was significantly higher than that of the Hep G2/pc DNA3.1 group-Ctr group,andβ-catenin was mainly expressed in the cytoplasm and partly in the nucleus.Conclusions1.The expression of IGFBP2 was increased in tumor tissues of HCC patients;overexpression of IGFBP2 down-regulated the expression of E-cadherin,while up-regulated the expression of N-cadherin and Vimentin in tumor tissue of nude mice.2.Overexpression of IGFBP2 enhanced the migration and invasion ability of HCC cells,and the process was produced by regulating EMT.3.Overexpression of IGFBP2 up-regulated the co-expression level of IGFBP2 and LRP6,and up-regulated the total expression and nuclear expression levels ofβ-catenin,which enhances the transcriptional activity ofβ-catenin.Suggesting that the LRP6/β-catenin signaling pathway was involved in the process of IGFBP2 regulated EMT. |
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