| Background: Primary liver cancer is the second most common cause of cancer-related death,most of which was hepatocellular carcinoma(HCC).Half of the world's HCC patients are in China,while only about 15% of them are diagnosed at an early stage that are eligible for curable intervention.For patients with advanced HCC,sorafenib,the only clinically approved first-line treatment,just only extended the median survival by 3 months.Sorafenib also cause certain drug resistance and side effects.Therefore,the limited treatment options for patients with advanced HCC increase the need to develop new treatment methods.Tumor microenvironment(TME),a complex and comprehensive system,is being gradually studied.The majority of HCC patients may infected by HBV,that the occurrence and development of HCC is closely related to immune evasion in the microenvironment,which is one of the reasons for the failure of various treatment methods.Traditional Chinese medicine(TCM)has been playing an increasingly important role in the prevention and treatment of multiple tumors with its advantages of combination and multi-target intervention.Our team summarized a recipe named Jiedu Fang(JDF)in long-term clinical practice,which was clinically used for a variety of tumors,especiallyHCC,and achieved good efficacy.Combined JDF intervention could significantly prolong the survival time of unresectable HCC patients,which may relate with improving the inflammatory microenvironment of HCC.In our previous study,we found that JDF may inactivate the M2 macrophage,which target may be insulin-like growth factor binding protein 2(IGFBP2).Recent researches also indicated that IGFBP2 may play an important role on a wide variety of tumor.However,its role in HCC and its exact molecular mechanism remain unclear.Therefore,our present study aims to explore whether JDF may inhibit proliferation,migration and invasion of HCC by targeting IGFBP2 and the molecular mechanism of this effect.Objective: 1.To observe the effect of IGFBP2 on the proliferation,migration and invasion of HCC.2.To determine the effect and mechanism of whether JDF can inhibit the proliferation,migration and invasion of HCC induced by IGFBP2.Methods: 1.The effect of JDF on the Cytokine secretion of M2-type macrophages was detected by Human XL Cytokine Array Kit,and the possible target IGFBP2 was screened.The expression of IGFBP2 in normal hepatocytes and various hepatocellular carcinoma cell lines was detected by Western Blot.2.The roles of IGFBP2 on the proliferation,migration and invasion of HCC cells were verified by transfecting IGFBP2 overexpression plasmid or silencing through transfecting IGFBP2 RNAi.3.MTT assay,plate colony formation assay and soft lipid gum colony formation assay were used to verify the effects of JDF on the proliferation and clonal formation of HCC cells.Meanwhile,the effects of IGFBP2 on the proliferation of HCC cells and the antagonistic effects of JDF were also detected.4.Transwell chamber migration and invasion experiment was used to detecte the effects of IGFBP2 on the migration and invasion of HCC,and whether JDF could antagonistic the effect of IGFBP2.5.GeneChip primeview human was used to detect the differences in gene expression between the Control Group and IGFBP2 treatment Group and Ingenuity Pathway Analysis(IPA)analysis were used to explore the possible signaling pathway of IGFBP2.6.Western Blot and ELISA were used to explore the effects of IGFBP2 on the expressions of EMT-related proteins such as E-cadherin,N-cadherin,Vimentin,Snail and Twist,and the matrix metalloproteinases MMP 2 and MMP 9.P-ERK and p-AKT signaling pathways were also explored.7.Three models including the subcutaneous HCC model,orthotopic HCC transplantation model and the metastasis to the lung model were used to further verify the role of IGFBP2 in the occurrence and development of HCC and the antagonistic effect of JDF in vivo.Results: 1.Results of Human XL Cytokine Array Kit showed that JDF may inhibit the expression of BAFF,Complement Component C5/C5 a,IGFBP2 and other genes and up-regulated the expression of GM-CSF,G-CSF and IL-4 on conditioned medium of M2-type macrophages.Combined with literature analysis,IGFBP2 may be an effective target of JDF.Western Blot results showed that IGFBP2 was poorly expressed in normal hepatocytes,and its expression was lowest in Huh 7 cells and highest in MHCC 97 H cells.2.MTT results indicated that JDF could effectively inhibit the proliferation of HCC in a time-and dose-dependent manner.When the concentration of JDF was 1.2 mg/mL,the proliferation inhibition showed statistical difference,while treated with 0.8 mg/mL JDF for 24 h,the proliferation was 94.74% with no significant difference compared to the Control Group.On the other hand,IGFBP2 could promote the proliferation of Huh 7 cells and MHCC 97 H cells in a time and dose-dependent manner.For Huh 7 cells,the maximum proliferation rates were 122.37%,125.85% and 146.20% at 24 h,48h and 72 h,respectively.For MHCC 97 H cells,the data were 111.92%,110.27% and 117.15%,respectively.Colony forming experiment of both recombinant cytokines IGFBP2 or IGFBP2 overexpression may promote Huh 7 cells' colony forming,although the number of colony formation had no significant difference when compared to the Control Group,the average size of colony were significantly larger in IGFBP2 Group than that in the Control Group.And silent IGFBP2 and JDF may antagonize the above effects.3.The results of Transwell chamber experiments suggested that the effect of different concentrations(50 ng/mL and 100 ng/mL)IGFBP2 on Huh 7 cells for 24 h could promote cell migration and invasion,and 100 ng/mL IGFBP2 was statistically significant when compared to the Control Group(P < 0.05).After the transfection of the overexpressed IGFBP2 plasmid,Huh 7 cells showed significantly enhanced migration and invasion ability compared with the Vector cells(P < 0.05).JDF can effectively antagonize the migration and invasion effects of IGFBP2.After silencing the expression of IGFBP2 in MHCC 97 H cells by lentivirus transfection,the migration and invasion ability of IGFBP2 cells was significantly decreased compared with the Vector cells(P < 0.05).4.GeneChip primeview human assay indicated that the gene expression profile of Huh 7 cells treated with IGFBP2 was mostly related to the disease of tumor,and is closely related to cell movement disorder.Oxidative Phosphorylation was the most activated pathway when analyzed by classic signaling pathways with the GeneChip data.Phosphorylation of ERK and AKT signaling pathway also played an important role.Western Blot results showed that IGFBP2 could inhibit the expression of E-cadherin while up-regulating the expression of N-cadherin,Vimentin,Snail and Twist,while JDF and silencing IGFBP2 could both antagonize the above effect.On the other hand,IGFBP2 can also activate the phosphorylation of AKT and ERK,and the effect is most obvious when the treated with IGFBP2 for 60 min.PI3 K inhibitor LY294002 and MEK inhibitor U0126 can antagonize these effects,and ultimately antagonize the process of EMT induced by IGFBP2 on Huh 7 cells.5.Results of subcutaneous tumor in nude mice established by MHCC 97 H cells show that JDF treatment may inhibit the tumor such as tumor size and weight,and down-regulate the content of serum IGFBP2 significantly than Control Group.Transfected with IGFBP2 RNAi also inhibits the tumor significantly when compared with the MHCC 97H-luc-Control Group.There are no signigicant difference between the MHCC 97H-luc-Control Group and MHCC 97H-luc Group was observed.Immunohistochemical results show that both JDF and IGFBP2 RNAi could effectively inhibit the expression of PCNA,and up-regulated the expression of E-cadherin and inhibit the expression of N-cadherin,Vimentin,Snail,MMP 2 and MMP 9.The hepatoorthotopic transplantation tumor model established by Huh 7 cells in nude mice show that the overexpression of IGFBP2 significantly increased the tumor size and net weight compared with the Vector Group,while JDF significantly antagonized the effect of IGFBP2.There was no significant difference in tumor size between the Vector Group and the Control group.Immunohistochemical results show that JDF can effectively inhibit the expression of PCNA and up-regulate the expression of E-cadherin,while inhibiting the expression of N-cadherin,Vimentin,Snail,MMP 2 and MMP 9.Overexpression of IGFBP2 shows the opposite effects.HCC metastatic model established by MHCC 97 H cells also showed that both JDF and IGFBP2 RNAi could effectively inhibit lung metastasis of HCC,and the immunohistochemical results showed that the effects were related to the inhibition of EMT,MMP 2 and MMP 9 expression.Conclusion: 1.IGFBP2 may promote the proliferation and metastasis of HCC both in vivo and in vitro,which can be inhibited by JDF.2.IGFBP2 may promote HCC EMT by activating the PI3K/AKT and MAPK/ERK signaling pathways,and JDF can inhibit the effect of IGFBP2 and play the anti-liver cancer effect ultimately. |
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