Effect Of MicroRNA-3178 On The Biological Behaviors Of Hepatocellular Carcinoma Tumor Endothelial Cells And Underlying Mechanism

Hepatocellular carcinoma (HCC) is a kind of solid tumors with rich blood supply.Tumor vessels is an important source of nutritional support,tumor invasion and migration of hepatocellular carcinoma. Antiangiogenesis therapy has become importa nt treatment of patients with advanced hepatocellular carcinoma which has lost the best operation opportunity.In recent years, tumor blood vessel blocking treatment such as transcatheter arterial chemoembolization (TACE) had been widely used in clinic in the treatment of HCC and and benefits.However,after traditional clinical blood vessel blocking treatment or anti-tumor angiogenesis treatment,the disease of some HCC patients had not been relieved.To avoid the interference of liver cancer cells, macrophages and other cells, this study directly choose hepatocellular carcinoma tumor endothelial cells (HCC TECs) as the research objecct, and flow cytometry was used to detect the expression of cell-surface molecules CD 105 and CD31 to determine purity of HCC TECs.Small RNA (microRNAs (miRNA)) widely exists in eukaryotic organisms, although not directly encoded proteins,but when combination with the target gene, miRNAs can degrade mRNA or inhibit translation and plays an important role in the molecular biology behavior such as groth,development,proliferation,differentiation and cell apoptosis.miRNAs has a good application prospectin in gene function research, early diagnosis and target gene therapy of tumor. At present the hot topics of research mainly concentrated in the role of miRNAs on tumor occurrence and development,a growing number of studies have found abnormal expression of miRNAs in HCC,the abnormal expression of miRNAs may be involved in the occurrence and development of HCC.Studies of these abnormal expression of miRNAs may help early diagnosis and treatment of HCC.However,the research of communication between miRNAs and hepatocellular carcinoma tumor endothelial cells rarely reported.If we can find the abnormal expression of miRNAs in hepatocellular carcinoma tumor endothelial cells and regulate its expression,then we can regulate generation and metastasis of hepatocellular carcinoma tumor endothelial cells,thereby regulating angiogenesis of HCC,eventually inhibit the growth and metastasis of hepatocellular carcinoma.This study screening the result of differential expression of miRNAs between hepatic sinusoidal endothelial cells (HSECs) and HCC TECs in our previous HmiOA v4 Human miRNA OneArray(?) Chip expression spectrum,miR-3178 was lower expression in HCC TECs (option standard log2|a Fold change|acuity 0.585 and P< 0.05) and was not reported in other studies,thus,miR-3178 was selected for further research.Explore the malignant biological behaviors of HCC TECs and its molecular mechanism caused by miR-3178 is expected to provide experimental basis and new targets for HCC TECs targeted therapy. This study was divided into three parts as follow.Part ? Screening and validation of differentially expression of miRNAs between hepatic sinusoidal endothelial cells and hepatocellular carcinoma tumor endothelial cells and synthetic miRNAs mimic or inhibitor and transfection hepatocellular carcinoma tumor endothelial cellsObjective Screening and validation of differentially expression of miR-3178 between hepatic sinusoidal endothelial cells and hepatocellular carcinoma tumor endothelial cells and hepatocellular carcinoma tumor endothelial cells were transfected with miRNA-3178 mimic/inhibitor or their respective negative sequence.Methods Flow cytometry was used to detect cell surface molecules CD105, CD31 expression levels of HCC TECs to validate purity of HCC TECs. Screening the results of our previous HmiOA v4 Human miRNA OneArray(?) Chip,miR-3178 was significantly lower expression in HCC TECs and was selected for further research. Real-time polymerase chain reaction (RT-PCR) was performed to detect the differential expression of miR-3178 in HSECs and HCC TECs.Hepatocellular carcinoma tumor endothelial cells were transfected with miRNA-3178 mimic/ inhibitor, (negative-control mimic) Nc-mimic or (negative-control mimic) Nc- inhib itor.According to the different methods of processing, HCC TECs were divided into five experimental groups:(1) the blank control group, namely the untreated HCC TECs group (CON group);(2) the miR-3178 up-regulation group, namely the group transfected with 2'-O modified miR-3178 mimic group (mimic group);(3)the miR-3178 negative-control mimic group, namely the group transfected with 2'-O modified sequence of negative-control mimic(Nc-mimic group);(4)the miR-3178 down-reg ulation group,namely the group transfected with 2'-O modified miR-3178 inhibitor (inhibitor group); (5) the miR-3178 negative-control inhibitor group, namely the group transfected with 2'-O modified sequence of negative-control inhibitor (Nc-inhibitor group).Real-time quantitative polymerase chain reaction was used to detect expression of miR-3178.Green fluorescent tags was observed and photographed under fluorescence microscopy.Results The results of flow cytometry showed that the HCC TECs cell surface molecule CD105 expression rate was 100% and the CD31 expression rate was 97.9%, excluding the contamination of hepatoma cells,macrophages and fibroblasts.miR-3178 was significantly lower expression in HCC TECs and was selected for further research(the standard to choose miR-3178:log2|Fold change|?0.585 and P<0.05).RT-PCR was used to validation differentially expressioin of miR-3178 between HSECs and HCC TECs,the result showed that the expression of miR-3178 was significantly lower in HCC TECs (0.31±0.03) than in HSECs (1.04±0.08) (P<0.001),the result proves the accuracy of the result of gene chip.RT-PCR was used to detect the expressioin of miR-3178 in each experimental group,the result showed that no significant difference in miR-3178 expression was detected between the CON group (1.04±0.14) and the Nc-mimic group(0.99±0.09) or the Nc-inhibitor group (1.02± 0.11),the mimic group (365.98±49.49) showed significantly higher miR-3178 express ion than the other groups (P<0.001),whereas the inhibitor group (0.19±0.04) showed significantly lower miR-3178 expression than the other groups (P<0.001).Green fluorescent tags was observed and photographed under fluorescence microscopy,no green fluorescent was observed in CON group, obvious green fluores cence was observed in mimic group,Nc-mimic group,inhibitor groups and Nc-inhibitor group,the transfection efficiency in HCC TECs were higher than 90%.Conculsion miR-3178 expression is significantly reduced in HCC TECs.Transfection of miR-3178 mimic significantly up-regulated miR-3178 expression, and transfection of miR-3178 inhibitor significantly down-regulated miR-3178 expression,the transfect ion efficiency in HCC TECs were higher than 90%.Part II The effect of changes in miR-3178 expression on biological behavior of HCC TECsObjective Study the effect of up-regulate or down-regulate the expression of miR-3178 on malignant biological behavior such as proliferation,apoptosis,cell cycle, invasion, migration and tube formation of HCC TECs.Methods MTT(Methyl thiazol tetrazolium) assay was used to detect HCC TECs proliferation in each experimental group.Flow cytometry was used for detecting HCC TECs cell apoptosis and cell cycle in each experimental group. Trans well invasion and migration assay was used to detect invasion and migration ability of HCC TECs in each experimental group.Tube formation assay was used to detect HCC TECs tube fomation in each experimental group.Results MTT assay showed that transfection after 24,36,48 and 72 h, no significant difference in the proliferation rate of HCC TECs was observed between the CON group and the Nc-mimic or the Nc-inhibitor group.The proliferation rate of HCC TECs was lower in the mimic group than in the other groups at corresponding time points after transfection and achieved the lowest proliferation rate at 72 h.The proliferation rate of HCC TECs was higher in the inhibitor group than in the other groups at corresponding time points after transfection and achieved the highest proliferation rate at 72 h.The result of flow cytometry showed that no significant difference in the apoptotic rate of HCC TECs was detected between the CON group and the Nc-mimic group or the Nc-inhibitor group.The mimic group showed significantly higher apoptotic rate than the other groups, whereas the inhibitor group showed significantly lower apoptotic rate than the other groups.The result of flow cytometry cell cycle assay showed that no significant difference in the cell population at the G0/G1 phase and S phase was observed between the CON group and the Nc-mimic or Nc-inhibitor group.The mimic group showed significantly higher G0/G1 phase cell population than the other groups.The inhibitor group showed significantly lower G0/G1 cell population than the other groups.The mimic group showed significantly lower S phase cell population than the other groups,whereas the inhibitor group showed significantly higher S phase cell population than the other groups.The result of transwell invasion and migration assay showed that no significant difference in the number of invaded and migrated cells was detected between the CON group and the Nc-mimic group or the Nc-inhibitor group.The mimic group showed significantly lesser number of invaded and migrated cells than the other groups, whereas the inhibitor group showed significantly greater number of invaded and migrated cells than the other groups. The result of tube formation assay showed that no significant difference in the number of capillary-like structures was found between the CON group and the Nc-mimic group or the Nc-inhibitor group.The mimic group showed significantly lesser number of capillary-like structures than the other groups, whereas the inhibitor group showed significantly greater number of capillary-like structures than the other groups.Conculsion miR-3178 may be act as a tumor suppressor gene involved in the regulation of biological behavior of HCC TECs,up-regulation of miR-3178 expression can inhibit HCC TECs proliferation,invasion,migration,tube formation and promote apoptosis and cell cycle G0/G1 phase arrest of HCC TECs.Part III Mechanism of the effect of miR-3178 on biological behavior of HCC TECsObjective To further investigate the mechanism of the effect of miR-3178 on biological behavior such as proliferation,apoptosis,cell cycle,invasion,migration and tube formation of HCC TECs.Observe the effect of regulating miR-3178 expression on mRNA expression and protein expression of potential target genes to verify the target gene.Methods Because the important role of the 3'UTR region in miRNA target genes, which could affect post-transcriptional expression of this gene.TargetScan,PicTar,Miranda and RNAhybrid target gene prediction software were used to predict the target genes of miR-3178.Western Blot was used to detect target gene protein expression in HCC TECs. RT-PCR was used to detect target gene mRNA expression in HCC TECs.Results Target gene prediction software showed EGR3 as a possible candidate target. Western blot showed that no significant difference in EGR3 protein expression was detected between the CON group(0.77±0.08)and the Nc-mimic group(0.73±0.06) or Nc-inhibitor group(0.79±0.07)(P>0.05).The mimic group(0.51±0.04) showed signi ficantly lower EGR3 protein expression than the other groups(P<0.01),whereas the inhibitor group(1.27±0.12) showed significantly higher EGR3 protein expression than the other groups(P<0.01).RT-PCT showed that no significant difference in EGR3 mRNA expression were detected between the CON group (1.02±0.10)and the Nc-mimic (1.01 ±0.09) group or Nc-inhibitor group(0.99±0.07)(P>0.05).The mimic group (0.69±0.05) showed significantly lower EGR3 mRNA expression than the other groups(P<0.01), whereas the inhibitor group(1.31±0.11) showed significantly higher EGR3 mRNA expression than the other groups(P<0.01).Conculsion EGR3 may be one of the target genes of miR-3178.The up-regulation of miR-3178 expression could inhibit EGR3 gene and protein expression in inhibiting cell proliferation,invasion,migration, angiogenesis and promoting apoptosis, G0/G1 phase arrest of HCC TECs.