| Objective:Liver cancer is one of the most common cancers around the world and its incidence has been increasing in many countries for many years.In China,its morbidity in men arises to top five.Surgical resection,liver transplantation,and local radiotherapy have been identified as efficient curative therapies for liver cancer.However,liver cancer is also known as its high rate of recurrence and poor prognosis after curative resection or local therapy within 5 years.Such characteristics of liver cancer are related to many factors.In recent years,with the growth of patients of diabetes,the numbers of patients with liver cancer combined with diabetes are increasing.Metabolic factors,such as obesity and diabetes,closely have been closely identified as risk factors for several other types of cancer.Glycemic Control is also considered as a risk factor for the aggravation and prognosis in liver cancer.However,related studies about the regulatory network of high glucose concentration on the function of liver cancer cells and the relationship between RNA and protein are rare.This study started with the changes of the endoplasmic reticulum protein through proteomics technology,and screen candidate proteins for further researching.Then we will explore the mechanisms affecting the expression of the protein under different glucose concentration conditions.We want to initially establish a regulatory pathway,which explained how high glucose influence the development and function of Hepatocellular Carcinoma Cells at Gene and Protein Levels.Methods:1? Analysis of Effects of High Glucose on Endoplasmic Reticulum Proteins of Human Hepatocellular Carcinoma Cell Line(HepG2)by using Label-free ProteomicsHuman liver hepatocellular carcinoma cell lines(HepG2)were maintained in low glucose DMEM medium with 10% FBS under the condition of 37? and 5% CO2.HepG2 cells were divided into Control Group(DMEM medium with low glucose,5.6mmol/L)and High Glucose Group(DMEM medium with high glucose,25mmol/L).The isolation and identification of organelles in HepG2 cells were carried by using the differentia ultracentrifugation and density gradient centrifugation.These separate ER preparations were analyzed by using Label-free quantification,which is a method in mass spectrometry that aims to determine the relative amount of proteins in two cell samples.2? We made use of bioinformatics to find candidate proteins for further research.Endoplasmic reticulum resident protein 29(ERP29)was one of the candidates.Using Western Blot was utilized to verify the effect of high glucose on ERP29 expression at the cell level.Then we pick out some paraffin sections,which are divided into three groups(Control Groups,Hepatocellular Carcinoma with or without diabetes).Immunohistochemistry is used to measure the expression levels of ERP29.3?Analysis of ERP29 on the biological functions in Hepatoma Carcinoma cells(HepG2):HepG2 cells were divided into Control Group(DMEM medium with low glucose,5.6mmol/L),High Glucose Group(DMEM medium with high glucose,25mmol/L),High Glucose-overexpression Group and High Glucose-siRNA Group.Cell functions of proliferation and migration were measured by CCK-8 and Wound-Healing experiment.Protein expressions associated with epithelial-mesenchymal transition were detected by western blot.4? The relationship between selected miRNA and ERP29.We predicted possible binding miRNAs related to ERP29 by using the miRNA databases(Tarbase v8,TargetScanHuman Release 7.1,miRTargetLink Human et al.).Possible miRNAs were selected and verified by qRT-PCR in control group and high glucose groups for further research.HepG2 cells were divided into Control Group,High Glucose Group,High glucose miRNA mimics Group and High glucose miRNA inhibitor Group.After transfection,the expressions miR-483-3p were examined by qRT-PCR.Western Blot was used to test the expression levels of ERP29.Cell Functions of HepG2 were measured by CCK-8 and Wound Healing Scratch Test.5?The relationship between selected LncRNA and miR-483-3pAnd then we predicted possible binding LncRNA relevant to miR-483-3p by using the LncRNA database(LncBase v.2,Starbase v2.0).Possible LncRNAs were selected and verified by qRT-PCR in control group and high glucose groups for further research.HepG2 cells were divided into Control Group,High Glucose Group,High glucose MEG3 Group and High glucose vector Group.After transfection,the expressions MEG3 and miR-483-3p were examined by qRT-PCR.Western Blot was used to test the expression levels of ERP29.Cell Functions of HepG2 were measured by CCK-8 and Wound Healing Scratch Test.Results:1? We succeeded in isolating endoplasmic reticulum from HepG2 cell lines,and we selected endoplasmic reticulum protein 29(ERP29)as a possible protein through Label-free method for further research.2? HepG2 were divided into Control Group and High Glucose Group and were cultivated for 24 h,48h,and 72 h.Compared with Control Group,High glucose can inhibit the expression of ERP29 in 72h(p<0.05).The expression of ERP29 in 24 h and 48 h didn't change in both groups.3? Immunohistochemistry was used to measure the expression of ERP29 in hepatic tissues.Compared with Control tissue,the expression of ERP29 decreased in the group of hepatocellular carcinomas with or without diabetes.And the expression of ERP29 in hepatocellular carcinoma with diabetes decreased more.4? ERP29 were overexpressed or knockdown in vitro.After transfection,CCK-8 and Wound Healing experiment indicated that overexpression of ERP29 inhibited cell proliferation and migration and knockdown of ERP29 improved cell proliferation and migration.The level of E-cadherin decreased and the level of Vimentin and N-cadherin increased in the overexpression group(p<0.05).Also,the level of E-cadherin increased and the level of Vimentin and N-cadherin decreased in the knockdown group(p<0.05).These three proteins represent Epithelial-mesenchymal transition.5? miR-483-3p overexpression suppressed ERP29 protein expression(p<0.05),and miR-483-3p inhibition improved the expression of ERP29 protein(p<0.05).CCK-8 and Wound healing test indicated that miR-483-3p overexpression improved cell proliferation and migration and miR-483-3p inhibition decreased cell proliferation and migration(p<0.05).6? LncRNA MEG3 overexpression suppressed the expression level of miR-483-3p,and the expression of ERP29 increased compared with control group(p<0.05).CCK-8 and Wound healing test indicated that MEG3 overexpression inhibited cell proliferation and migration(p<0.05).However,after knockout the miR-483-3p,MEG3 overexpression didn't change the level of ERP29(p>0.05).We preliminarily established the network: LncRNA MEG3: miR-483-3p: ERP29.High glucose mediates the expression LncRNA MEG3 which regulate the expression of ERP29 through miR-483-3p to change the HepG2 function.The influence of ERP29 on cell function may be related to epithelial-mesenchymal transition,which lead to the loss of polarity in liver cancer cells and increase the ability of migration and mobility. |
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