Growth Inhibition Of Hepatocellular Carcinoma Tumor Endothelial Cells By MiR-204-3p And Its Underlying Mechanism

Hepatocellular carcinoma (HCC) is a solid tumor with a rich blood supply. Its growth, invasion, and metastasis are all closely associated with angiogenesis, and regulation of tumor angiogenesis can therefore control tumor growth. During the progression of tumor development from the avascular stage to the vascular stage, angiogenesis is regulated by angiogenesis factors and inhibitors, and once the balance is disturbed, angiogenesis can be accelerated. Although significant progress has been achieved in studies focusing on HCC treatment, effective treatment methods are still missing, and the prognosis remains poor. Anti-angiogenic therapy targeting tumor neovasculature is a new route for HCC treatment. Therefore, the identification of specific molecular markers and target genes of tumor vascular endothelial cells can provide new bases for the diagnosis and targeted therapy of HCC. miRNAs are non-coding, small RNAs that regulate gene expression at the post-transcriptional level. These RNAs regulate the expression of downstream target genes at the protein level, thus playing important regulatory roles in cellular pathways. Abnormal expression of miRNA target genes is associated with many diseases, such as cancer and cardiovascular disorders. Current studies on miRNAs are mostly focused on tumor cells. Thus far, no studies have described the miRNAs of the tumor endothelial cells (TECs) of human HCC. Therefore, this study first employed a microarray to detect the differentially expressed miRNAs in HCC TECs as compared to normal hepatic sinusoidal endothelial cells (HSECs) with the goal of identifying specific miRNAs that play important roles in the angiogenesis of HCC. Of the differentially expressed miRNAs, miRNA-204-3p had the most significant decrease in expression and was used as an indicator to investigate its mechanism in the growth of TECs of HCC. Fibronectin1(FN1), a target gene of miR-204-3p, might participate in the miR-204-3p-mediated regulation of TEC growth. FN1is an important component of the extracellular matrix and a multi-functional, glycoprotein macromolecule that participates in the processes of cellular adhesion, migration, and damage repair; it also plays important roles in resistance to infection and the maintenance of micro vascular integrity. In addition, FN1binds to the cell surface integrin receptor to initiate cell adhesion-mediated drug resistance (CAMDR). This study transduced TECs with a lentiviral vector to over-express miR-204-3p and showed that the expression of FN1protein was significantly inhibited. Our study proved that FN1is a potential target gene of miR-204-3p, suggesting that FN1regulates the growth of HCC TECs via the miR-204-3p/FN1signaling pathway. The related underlying mechanism of the signaling pathway was also investigated to provide new targets and theoretical bases for the anti-angiogenic gene therapy of HCC. Part I The differentially expressed miRNAs in HCC TECs as compared to normal HSECs were examined using the HmiOA v4Human miRNA OneArray(?) microarrayObjective To examine the differentially expressed miRNAs in tumor endothelial cells (TECs) of human hepatocellular carcinoma (HCC) as compared to normal hepatic sinusoidal endothelial cells (HSECs) using microarray analysis and to investigate the mechanism by which miRNA and its target gene fibronectin1(FN1) inhibit the growth of TECs.Methods Flow cytometric detection of the expression of the TEC cell surface molecules CD105and CD31. Quality control of RNA samples before microarray hybridization. Detection of microarray expression profile using the HmiOA v4Human miRNA OneArray(?) Chip.Results Expression of the TEC cell surface molecules CD105and CD31:Currently, the main molecular markers used for sorting vascular endothelial cells include CD31and CD105. However, CD31can cross-react with hematopoietic cells, and therefore CD105becomes the best choice for sorting HCC TECs. Flow cytometry results showed that the CD105expression rate was100%and the CD31expression rate was98.7%, which excluded the possibility of hepatoma cell, macrophage, and fibroblast contamination. Number of Differentially Expressed Genes: The number of differentially expressed genes for each comparison is shown in the below table. Standard selection criteria to identify differentially expressed genes are established at log2|Fold change|≥0.585and P<0.05.Conculsion Fifteen differentially expressed miRNAs were identified that may participate in the proliferation and metastasis of HCC TECs. PartⅡ Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3pObjective miR-204-3p showed the most significant decrease in expression and was selected as an indicator. Over-expression of miR-204-3p was achieved using lentiviral transduction into TECs of HCC. The biological changes in HCC TECs before and after transduction were detected using MTT and apoptosis assays.Methods Construction,packaging, and titer detection of the miR-204-3p overexpressing lentiviral vector. Infection of TECs by lentiviruses:The experimental cells were divided into3groups: the CON group consisted of normal TECs without lentiviral infection, the NC group consisted of normal TECs infected with negative control viruses, and the micro-up group included normal TECs infected with the miR-204-3p-up virus containing the target gene. Detection of TEC proliferation before and after transduction using the MTT assay. Flow cytometric detection of TEC apoptosis before and after transfection using the annexin V-APC single staining method.Results Determination of lentivirus titers after infection of TECs: Three days after infection, the expression of GFP was observed under a fluorescence microscope. The results showed that the CON group had no green fluorescence, the NC group had green fluorescence that was not specific to the target gene, and the micro-up group contained green fluorescence as granules of the target gene; the fluorescence rate was over90%. Detection of TEC proliferation before and after transduction using the MTT assay:After5days of continuous observation, it was found that cell proliferation was significantly inhibited in the micro-up group, while proliferation of the CON and NC groups was not affected. On day4, compared with the other2control groups, the difference in proliferation inhibition of the micro-up group (3.61±0.29) was the largest (p<0.01). There were no significant differences (p>0.05) between the CON group (6.31±0.14) and the NC group (6.11±0.31).Flow cytometric detection of apoptosis in each group after transduction: Four days after transduction, the confluence of each group of cells was approximately90%. There were no significant differences between the CON group (1.54±0.11%) and the NC group (1.61±0.25%), while the micro-up group (4.09±0.16%) showed significant apoptosis compared to the other groups (p<0.01). Conculsion The results from this study, however, confirmed that miR-204-3p was also involved in regulating the growth of TECs, and miR-204-3p inhibited the proliferation and promoted the apoptosis of TECs of HCC through the regulation of specific target genes. PartⅢ Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3p and its underlying mechanismObjective To further investigate the mechanism of miR-204-3p, we showed that FN1was one of its target genes using authoritative target gene prediction software. FN1plays important roles in the proliferation, apoptosis, and metastasis of tumor cells. And to investigate the mechanism by which miR-204-3p and its target gene fibronectin1(FN1) inhibit the growth of TECs.Methods Construction of plasmids over-expressing the wild type version of the target gene FN1(FN1-WT) and the mutant FN1(FN1-MUT). Luciferase analysis after plasmid transfection. Detection of FN1protein expression in TECs using western blot analysis.Results Identification of FN1as a target gene of miR-204-3p using the dual luciferase activity assay: The luciferase activity in the FN1-WT group (0.52±0.01) was significantly lower than that in the FN1-MUT group (0.71±0.02), and this difference was statistically significant (p<0.05). This evidence indicated that miR-204-3p had an inhibitory effect on FN1expression, and the inhibitory effect was achieved through miR-204-3p binding to the FN13’UTR region, suggesting that FN1is a target gene of miR-204-3p. Silencing effect of miR-204-3p on the expression of FN1in TECs:After infection, the expression of FN1protein in TECs was significantly reduced, and there were significant differences between the groups (p<0.01). In contrast, the FN1protein expression in the CON and NC groups did not change significantly.Conculsion The over-expression of miR-204-3p significantly inhibited the expression of fibronectin protein, which further verified the target gene prediction results and elucidated the mechanism that miR-204-3p inhibits the proliferation and promotes the apoptosis of HCC TECs through the silencing of its target gene, FN1.