| Research Background:Hepatocellular Carcinoma(HCC)is currently the leading cause of cancer-related human death.Due to the lack of effective treatment,patients with advanced stage recurrent and metastatic liver cancer often have very poor prognosis.Clinical investigations have found that insulin-like growth factor 2(IGF2)mRNA-binding protein 1(IGF2BP1)is overexpressed in patients of HCC,promotes the activation of oncogenes,and is associated with cancer progression and poor prognosis.Therefore,targeting IGF2BP1 to inhibit the activation of oncogenes can effectively control the progression of HCC.Rhizoma Coptidis is bitter in taste and cold in nature.It has the effects of clearing away heat and purging fire,purging fire and detoxifying.The theory of traditional Chinese medicine believes that tumors are caused by the invasion of evil qi and the accumulation of poison in the body for a long time.Modern pharmacological studies have clarified that Rhizoma Coptidis has antipyretic,analgesic,bacteriostatic,anti-inflammatory,hypoglycemic and anti-tumor effects.The anti-liver cancer activity of various alkaloids in Rhizoma Coptidis was screened by cell experiments,and it was found that coptisine(COP)can inhibit the proliferation and migration of liver cancer cells Hep G2 and Hu H7,and promote cell apoptosis.However,the specific mechanism is not clear,and further in-depth research is needed to provide a theoretical basis for clarifying the anti-cancer active components of Rhizoma Coptidis and new drug research.Objective:1.To clarify the role of coptisine,an active anti-hepatocarcinoma component in Rhizoma Coptidis,in the occurrence,development and metastasis of HCC.2.Based on transcriptomic analysis,cell thermal shift(CETSA)and mass spectrometry combined analysis,etc.,to explore the key direct targets of coptisine in inhibiting the proliferation and metastasis of hepatocellular carcinoma.3.The role of IGF2BP1 in hepatocellular carcinoma was elucidated by overexpression and silencing of IGF2BP1,a key target directly acting on coptisine,in vivo and in vitro.4.To clarify the mode of action of coptisine on the key target IGF2BP1,and to clarify the molecular mechanism of the protein level reduction of IGF2BP1 after coptisine intervention.Methods and results:1.Activity evaluation of coptisine against hepatocellular carcinoma in vitro and in vivoThe results of MTT,cell colony formation and EdU staining showed that coptisine had inhibitory effect on the proliferation of Hu H7,7404 and Hep G2 cells.Hu H7 cells are more sensitive to the effect of coptisine,while normal human hepatocyte LO2 is not sensitive to the effect of coptisine;Flow cytometry results show that coptisine can induce cell cycle arrest and apoptosis of Hu H7,7404 and Hep G2 cells;Scratch healing and transwell assays show that coptisine inhibits the migration and invasion of liver cells;Taking the subcutaneous transplanted tumor of Hu H7 nude mice as a model,in nude mice,coptisine can significantly inhibit the tumor growth of Hu H7 tumor-bearing nude mice.There was no significant difference in body weight and organ index of nude mice in each group,and coptisine had no obvious toxic and side effects on nude mice.2.IGF2BP1 is a potential target of coptisine against hepatocellular carcinomaBased on the transcriptomic analysis of the changes in the transcript level of coptisine Hu H7,combined with the differential genes of liver cancer in the TCGA database,a total of 2052 intersecting genes were obtained.The 2052 genes were analyzed by GO enrichment and KEGG,and the results showed that after treatment of Hu H7 cells with coptisine,the altered differential genes were mainly related to cell cycle,proliferation,and cell migration and invasion;Combined with CETSA and LCMS/MS proteomics technology,205 proteins were obtained after mass spectrometry identification,and the key target IGF2BP1 was confirmed;Molecular docking confirmed that the binding energy of coptisine to IGF2BP1 was 3.40 kcal/mol,and it was found that coptisine could interact with lysine at positions 538 and 487 and glutamic acid at positions 489 and 520 in the KH3/4 domain of IGF2BP1;The target genes of IGF2BP1 and transcriptome difference genes have 7774 overlapping genes,and 1190 genes were obtained by intersection with the TCGA liver cancer related database.The results of GO enrichment and KEGG analysis showed that they were related to cell proliferation,cell cycle and cell migration;The expression of IGF2BP1 in hepatocellular carcinoma was analyzed through the website(http://ualcan.path.uab.edu),and the results showed that IGF2BP1 was lowly expressed in normal tissues and highly expressed in liver cancer tissues,and gradually changed with the grade of the tumor.At the same time,the high expression of IGF2BP1 is significantly correlated with the survival and prognosis of liver cancer;Western blot detection can significantly reduce the expression of IGF2BP1 after treatment with coptisine in vitro and in vivo.3.Effects of overexpression and silencing of IGF2BP1 on hepatocellularcarcinomaThe effect of IGF2BP1 on the growth of hepatoma cells in vitro was investigated by silencing and overexpressing IGF2BP1.Cell clone formation and EdU staining assays detected that silencing IGF2BP1 had an inhibitory effect on the proliferation of Hu H7 and 7404 cells,and overexpression of IGF2BP1 had a promoting effect on the proliferation of 7404 and Hep G2 cells;Flow cytometry showed that silencing of IGF2BP1 could induce cell cycle arrest and apoptosis in Hu H7 and 7404 cells,and overexpression of IGF2BP1 could alleviate the cycle arrest and apoptosis induced by coptisine.Scratch healing and transwell assays detected that silencing IGF2BP1 inhibited the migration and invasion of MHCC97-H and 7404 cells,and overexpression of IGF2BP1 could promote the migration and invasion of 7404 and Hep G2 cells,and alleviate the migration and invasion inhibition caused by coptisine;Based on Hu H7 cells,a cell line with stable overexpression and silencing of IGF2BP1 was constructed,and a subcutaneous transplanted tumor in nude mice was established as a model.Silencing of IGF2BP1 can significantly inhibit the growth of tumor-bearing nude mice,while overexpression of IGF2BP1 can significantly promote the growth of tumorbearing nude mice.4.Coptisine promotes the degradation of IGF2BP1 through ubiquitinationWestern blot was used to detect the changes in the stability of IGF2BP1 protein in liver cancer Hu H7,7404 and Hep G2 cells after treatment with coptisine.The results showed that the half-life of IGF2BP1 protein was significantly shortened and the protein stability decreased significantly after treatment with coptisine.Western blot was used to detect the changes of IGF2BP1 protein ubiquitination in human liver cancer Hu H7 cells after coptisine intervention.The results showed that IGF2BP1 could increase the ubiquitination of IGF2BP1 after direct action.The protein level of IGF2BP1,which was decreased by coptisine,was significantly increased after the intervention of proteasome inhibitor MG132 by Western blot.Combined immunoprecipitation and biological mass spectrometry to identify the proteins that interact with IGF2BP1,and find the key enzymes that cause the degradation of IGF2BP1 after the action of coptisine,including the deubiquitinase ubiquitinase ubiquitination enzyme and E3 ligase data sets,which are predicted by IGF2BP1 to intersect.protein-specific protease 10(USP10)or E3 ligase pre-mRNA processing factor 19(PRPF19).Furthermore,silencing PRPF19 or overexpressing USP10 in liver cancer cell lines could rescue the protein level of IGF2BP1 decreased by coptisine,and could slow down the shortening of IGF2BP1 protein half-life caused by coptisine.Conclusion:This study shows that coptisine can inhibit the growth of human liver cells both in vitro and in vivo.Further research found that IGF2BP1 is the direct target of coptisine against hepatocellular carcinoma.Further research found that IGF2BP1 is the direct target of coptisine against liver cancer.Coptisine has anti-cancer effects by promoting the effect of IGF2BP1 and E3 ligase PRPF19,reducing the effect of IGF2BP1 and deubiquitinating enzyme USP10,increasing the ubiquitination and degradation of IGF2BP1,and reducing the expression of IGF2BP1.It provides a new direction for the study of modern pharmacology and molecular mechanism of traditional Chinese medicine anti-tumor. |
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