The Mechanism Of EGFR Inducing The Transcriptional Regulation Of HMGA2 And Its Role In The Metastasis Of Hepatoma

Background and objective:Hepatocellular carcinoma(HCC)is a high-mortality primary liver cancer,which is the most common malignancy in a global scale,especially in Asia,Africa and southern Europe.In China,the incidence of hepatocellular carcinoma accounts for almost half of the global incidence.The symptoms of the hepatocellular carcinoma at early stage is not obvious,so when symptoms appear,it has reached the middle and late stage,or with distant metastasis.At this time,the effect of surgical treatment is limited,and the patient is quickly dying.Therefore,our study focus on the metastasis mechanism of hepatocellular carcinoma,in order to find the key molecules that mediate hepatocellular carcinoma metastasis,provides a new target for the treatment of hepatocellular carcinoma.HMGA2(high mobility group AT-hook2)is a member of the high mobility group(HMG)family.The family also includes two proteins,HMGA1 and HMGA2.Among them,HMGA1 is a structural transcription factor that synergizes with other transcription factors to coordinate the transcription of downstream target genes.The expression of HMGA1 is significantly up-regulated in various tumor tissues(such as liver cancer,breast cancer,pancreatic cancer,skin cancer,etc.),and tumor proliferation and metastasis can be promoted by regulating the transcription of various target genes.The HMGA2 protein is located in the nucleus.Although it has no transcriptional activity,it could affects the transcriptional regulation of downstream genes by changing the chromatin structure.Although the expression of HMGA2 has been detected increased in oral cancer,non-small cell lung cancer,breast cancer,colon and esophageal cancerand positively correlated with tumor metastasis and poor prognosis.There are little report about the role of HMGA2 in liver cancer.Therefore,this subject will study the role of HMGA2 in themetastasis and proliferation of liver cancer,and hope to find new targets for the treatment of liver cancer.Part Ⅰ:HMGA2 promotes metastasis of hepatocellular carcinomaPurpose:To study the role of HMGA2 in the development of hepatocellular carcinomaMethod:1)Rat liver cancer model was established by DEN.The expression of HMGA2 mRNA and protein in rat liver was detected by RT-PCR and Western Blot.2)Overexpression or knockdown of HMGA2 in hepatocarcinoma cells,MTT assay to detect the proliferation of hepatoma cells,Scratch assay and Transwell assay to detect liver cancer cell migration ability,Transwell Matrigel gel assay to detect liver cancer invasion ability.3)The role of HMGA2 in the proliferation and metastasis of hepatocarcinoma was analyzed by subcutaneous xenograft and lung metastasis in nude mice.Results:1)The mRNA and protein expression of HMGA2 in the liver cancer model rats was significantly higher than that in the control group(P<0.01).2)The expression of HMGA2 can significantly affect the ability of migration and invasion of liver cancer cell lines.High expression of HMGA2 significantly promoted the migration and invasion of HepG2 cells(P<0.05).After knockdown of HMGA2,the migration and invasion ability of MHCC-97H cells were significantly decreased(P<0.05).3))Overexpression or knockdown of HMGA2 had no significant effect on the proliferation of subcutaneous xenografts in nude mice.In the tail vein inoculation experiment,the number of nodules in the lungs of nude mice inoculated with HepG2-HMGA2 cells was significantly higher than that in the.control group(P<0.05),while the number of lung nodules in mice inoculated with MHCC-97H-shHMGA2 cells was significantly less in the control group(P<0.01).Conclusion:HMGA2 can promote the metastasis rather than the proliferation of liver cancer cells in the development of liver cancer.Part Ⅱ:Activation of EGFR promotes transcription of HMGA2 in hepatocellular carcinomaPurpose:To prove the role of EGFR in the regulation of HMGA2 in liver cancer.Method:1)Detection of EGFR and p1068-EGFR expression in liver tissue by ELISA and western Blot2)Real-time PCR and Western Blot were used to detect the expression of HMGA2 in transcription level and protein level in hepatocellular carcinoma cell lines after exogenous addition of EGF and EGFR inhibitors.3)RT-PCR and western Blot were used to detect the expression of HMGA2 in hepatoma cells after incubation with EGF and different pathway inhibitors.4)Transwell assay was used to detect the migration and invasion of HepG2 and MHCC-97H cell lines affected by exogenous EGF and ERK pathway inhibitors.Results:1)Compared with the rats in the solvent group and the blank control group,the plasma EGF content in the liver cancer model group was significantly up-regulated(P<0.01);the expression of EGFR was also up-regulated in the liver cancer tissues(P<0.01);p1068-EGFR The expression was also significantly higher than that of the solvent group and the blank control group(P<0.01).2)Exogenous EGF incubation can significantly increase the expression of HMGA2 mRNA and protein in HepG2 cells and MHCC-97H cells;EGFR tyrosinase inhibitor Gefitinib can significantly inhibit EGF-induced up-regulation of HMGA2 mRNA and protein(P<0.01).3)By adding three inhibitors(JAK inhibitor AG490,ERK inhibitor PD98059 and PI3K inhibitor LY294002)to EGFR regulation of HMGA2,it was found that only ERK inhibitor PD98059 was evident in HepG2 and MHCC-97H.Inhibition of EGF-induced upregulation of HMGA2 mRNA and protein levels(P<0.01).4)Transwell chamber experiments showed that EGF-induced migration and invasion of HepG2 and MHCC-97H cells were significantly decreased after adding exogenous ERK pathway inhibitor PD98059 compared with EGF alone(P<0.01).Conclusion:EGFR can regulate the expression of HMGA2 in liver cancer tissues through the MEK-ERK pathway.Part Ⅲ:ERK/ELK-1 promotes the transcription of HMGA2Purpose:To investigate whether ERK/ELK-1 is involved in the transcriptional mechanism of EGF-induced HMGA2 geneMethod:1)Western Blot analysis the phosphorylation of ELK1 serine 383 induced by EGF2)RT-PCR and Western Blot were used to detect the regulation of transcription of HMGA2 by ELK1 knockdown and the mutation of Ser 383.3)Dual luciferase assay and chromatin immunoprecipitation assay to analyze the regulation ability and binding site of transcriptionof HMGA2 by ELK1 in hepatoma cell linesResult:1)Western Blot analysis showed that the expression of pS383-ELK1 was up-regulated in HepG2 and MHCC-97H cells induced by EGF,but the expression of total protein of ELK1 was not significantly changed,and the expression of ELK1 in the nucleus was up-regulated.Gefitinib and PD98059 can significantly inhibit EGF-induced phosphorylation of the ELK1 serine 383 site.The total protein expression of ERK1/2 and phosphorylated protein in liver tissue of rat cancer model was completely consistent with that in cell experiments.These results indicated that EGF is involved in the induction of phosphorylation of ELK 1 serine 383.2)After ELK1 knockdown,the expression level of HMGA2 in mRNA and protein of HepG2 and MHCC-97H cells were significantly down-regulated.After transferred with mutated ELK1Ser 383,the expression level of HMGA2 in mRNA and protein of HepG2 and MHCC-97H cells were significantly higher than those of the empty vector pcDNA3.1 group,ELK1 WT group and ELK1 S383A group,which demonstrated that the phosphorylation of ELK1 serine 383 is involved in the transcriptional regulation of HMGA2.3)Double luciferase results showed that luciferase activity in group of p-1958-HMGA2-luc,p-1247-HMGA2-luc and p-867-HMGA2-luc had higher fluorescein than that of transfected p-GL3 basic group,while the luciferase activity of p-132-HMGA2-luc group was significantly weaker compared with the above three groups.The result of co-immunoprecipitation indicated that the level of ELK1 in the promoter region of HMGA2 was significantly increased after EGF incubation.ELK1bind at the site of 217-221 upstream of the promoter region of HMGA2.Conclusion:EGF is involved in the regulation of transcription and expression of HMGA2 by inducing the phosphorylation of ELK1 serine 383.