The Roles And Mechanism Of IGFBP2 In The Proliferation,migration And Invasion Of Hepatocellular Carcinoma Cells

The incidence of hepatocellular carcinoma(HCC)is increasing year by year and has become a major disease that threatens human life and health.The early diagnosis of HCC includes imaging(ultrasound,CT and magnetic resonance)and serological tests.Due to the high cost of imaging tests,the detection of serologically specific tumor markers has received increasing attention.Among them,a fetal protein(α-fetoprotein,AFP)is one of the most specific markers for the diagnosis of HCC.However,due to individual differences,false positive or false negative results may occur in AFP tests.Therefore,we need to seek new biomarkers and AFP for the diagnosis of HCC to improve the accuracy of diagnosis.In recent years,insulin-like growth factor(IGF)family proteins have become important factors in the development and progression of HCC.Among them,insulin-like growth factor binding protein 2(IGFBP2)is highly expressed in the serum of patients with HCC,which is related to the poor prognosis of patients.The mechanism of action in HCC is unclear.Studies have shown that IGFBP2 can mediate cell growth through an IGF-independent pathway.The C-terminus of the protein has an Arg-Gly-Asp(RGD)domain that interacts with the integrin receptor β1(Integrin β1).Under the activation of Integrin β1,FAK phosphorylation can mediate the activation of downstream signaling pathways,such as the JNK pathway,Rac1 pathway,and Mek / Erk pathway,etc.,suggesting that Integrin β1 may play a role as its receptor in HCC proliferation.In HCC,the expression of early growth response factor1(Egr-1)is closely related to cell proliferation.Studies have shown that Egr-1 can play an important role in promoting tumor growth,invasion and metastasis by regulating downstream genes in HCC.This project intends to use clinical serum and tumor samples from HCC patients,exogenous recombinant IGFBP2(r IGFBP2)to stimulate HCC cell lines in vitro as research objects,using western blot,CCK-8,Transwell,co-immunoprecipitation(Co-IP),immunofluorescence,etc.experimental technology to study the role of IGFBP2 in the proliferation,migration and invasion of HCC cells,and further explore the molecular mechanism of IGFBP2 in promoting the proliferation,migration and invasion of HCC cells.The research will provide important evidence for revealing new pathological mechanisms of HCC and finding new drug targets.Objective:1.To observe the expression of IGFBP2 in serum and liver tumor tissues of HCC patients.2.To observe the proliferation,migration and invasion ability of HCC cells under the stimulation of r IGFBP2,and clarify the role of IGFBP2 in HCC cells.3.To observe the activation of Integrin β1 and co-expression of p-FAK in HCC cells stimulated by r IGFBP2,detect the expression of p-Erk,p-AKT and Egr-1,and partially reveal the molecular mechanism of IGFBP2 promoting cell proliferation,migration and invasion.Methods:1.Collect serum and liver tumor tissue samples from HCC patients and healthy people,use protein chip method to detect the expression of IGFBP2 in the serum of HCC patients,analyze the pathological characteristics of HCC tissues and precancerous lesions(PCL)by HE staining,immunohistochemistry and western blot was used to detect the expression of IGFBP2,Integrin β1 and Egr-1 proteins in HCC tissues and PCL tissues of HCC patients.2.Select HCC cell lines Hep G2 cells and HCC-LM3 cells,use CCK-8 method and Transwell method to detect cell proliferation,migration and invasion ability,western blot and immunofluorescence method to detect the activation of Integrin β1 protein.Western blot was used to detect Integrin β1,p-FAK,Egr-1,p-Erk,p-AKT expression.Co-expression of Integrin β1 and p-FAK was detected by immunofluorescence and Co-IP.Results:1.Increased expression of IGFBP2 in serum and tumor tissues of HCC patients Serum samples were collected from the peripheral blood of clinical HCC patients and healthy people,and the expression of IGFBP2 was detected by protein chip method.The results showed that the content of IGFBP2 in the serum of HCC patients was higher than that of healthy people;the tumor tissues of surgically resected HCC patients were paraffin-embedded,Sections were observed with HE staining for histopathological features.As a result,hepatocytes were arranged in hepatic cords in PCL tissues,hepatocytes were arranged irregularly in tumor tissues and inflammatory cells infiltrated,hepatocytes were enlarged,nucleus became larger,and cytoplasmic HE Staining became lighter and pseudolobules appeared in liver tissue.The expression of IGFBP2,Integrin β1,and Egr-1 proteins in HCC tissues and PCL tissues of HCC patients were detected by immunohistochemistry and western blot.The expression is higher than that of PCL tissue.2.Exogenous r IGFBP2 promotes proliferation,migration and invasion of HCC cell lines HCC cell lines Hep G2 cells and HCC-LM3 cells were selected,and different concentrations of r IGFBP2(15.625 ng/m L,31.25 ng/m L,62.5 ng/m L,125 ng/m L,250ng/m L)were used to detect Hep G2 cells and CCK-8.The effect of HCC-LM3 cell proliferation,the results showed that r IGFBP2 stimulation at 62.5 ng/m L,125 ng/m L,250 ng/m L can significantly promote cell proliferation.Transwell experiments examined the effects of different concentrations of r IGFBP2 on the migration and invasion of Hep G2 cells and HCC-LM3 cells.The results showed that different concentrations of r IGFBP2 could significantly promote the migration and invasion of Hep G2 cells and HCC-LM3 cells.3.r IGFBP2 can activate Integrin β1,promote co-expression of Integrin β1 and p-FAK,and activate Erk and AKT signaling pathways in HCC cells,.The suboptimal concentration of 125ng/m L r IGFBP2 was used to stimulate Hep G2 cells.Western blot experiments showed that intracellular expressions of Integrin β1,Egr-1,p-FAK,p-Erk and p-AKT increased from 30 min,4 h or 12 h.Reached a peak,then gradually decreased.HCC cells were stimulated with r IGFBP2 for 30 min,the expression of activated Integrin β1 in Hep G2 cells and HCC-LM3 cells was detected by immunofluorescence method,and co-expression changes of Integrin β1 and p-FAK in Hep G2 cells were detected by immunofluorescence method and Co-IP method.The results showed that when r IGFBP2 acted on Hep G2 cells and HCC-LM3 cells for 30 minutes,Integrin β1 was activated,and co-expression with p-FAK increased,suggesting that r IGFBP2 can activate Integrin β1 in HCC cells and promote the integration of Integrin β1 and p-FAK.Co-expression,activates Erk and AKT signaling pathways.Conclusion:1.The content of IGFBP2 in the serum of HCC patients is greater than that in healthy people.The expression of IGFBP2,Integrin β1 and Egr-1 in the tumor tissues of patients is greater than that of PCL tissues,suggesting that IGFBP2 may participate in the development of HCC.2.Exogenous r IGFBP2 can promote the proliferation,migration and invasion of HCC cells,which may be related to r IGFBP2 activating Integrin β1,promoting co-expression of Integrin β1 and p-FAK,and activating Erk and AKT signaling pathways.