The Mechanism Of MicroRNA-301a In Promating Tumor Proliferation, Invasion And Metastasis By Targeting GAX In Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC), one of the most common cancers worldwide, is the third leading cause of cancer-related death worldwide and accounts for about1million deaths each year. Although treatments for HCC detected in the early stages include surgical resection, percutaneous ablation, liver transplantation, and the administration of the multikinase inhibitor sorafenib, the incidence of tumor recurrence and metastasis is still high. This high mortality disease is considered to be a complex genetic disease. So far Several signaling pathways and growthfactors, oneogenes and tumor suppressor genes are potentially involved in HCC development and progression. Therefore, making clear the molecular events underlied the pathogenesis of HCC are important for its sereening, prevention, alidtreatlnent.In recent years, microRNAs (miRNAs) have been recognized as one of the many types of noncoding small RNAs that are involved in several biological processes, such as development, proliferation, differentiation and apoptosis. It has been shown that miRNAs can silence post-transcriptional genes through imperfect base pairing with the3’-untranslated region of target mRNAs, resulting in mRNA degradation or translational repression. Previous studies have suggested that the deregulation of miRNAs may contribute to the development of many of the human cancers including breast cancer, lung cancer, liver cancer, brain cancer, gastric cancer, and colorectal cancer. Thus thorough research in miRNAs expression and function will contribute to understand the pathogenesis of tumor and might profit in the diagnosis, treatment and prognosis of cancer. The growth arrest-specific homeobox (GAX, also known as MEOX2), a member of homeobox gene family, encodes a homeodomain-containing transcription factor and is expressed both in vascular smooth muscle (VSMCs) and vascular endothelial cells (ECs). It has been previously demonstrated that GAX is significantly induced in various types of tumors. However, little is known about the correlation of its expression with clinical features and prognosis in HCC.ObjectiveThis study aims to investigated the expression of miR-301a in human HCC and explore the function and mechanism of miR-301a in tumor invasion and metastasis of HCC. We also analyzed the relationship between miR-301a target gene GAX and HCC clinicopathologic significance. Methods1. Investigating the expression of miR-301a by real-time PCR in25primary HCC tumors and adjacent matched tissues, and in6adjacent liver tissues from Hepatic hemangiomas. These tissues had been collected from liver resection and all were diagnosed by Xiangya Hospital in Central South University during2011.02-2011.05. GAX mRNA and protein expression were detected in these tissues by real-time PCR and western blot. Three human HCC cell lines (HepG2, Huh7, and LM3) and a normal liver cell line (LO2) were detected miR-301a expression.2. Hsa-miR-301a inhibitor were transfected into HepG2cells by use of Lipofectamine2000. Cells transfected with no significant homology with negative group and blank group were treated in parallel. Real-time PCR were performed to detect GAX expression after transfection. Real-Time PCR and western blot were using to detect downstream target gene GAX. In vitro proliferation was evaluated by the MTT assay. The migration and invasion assays were performed using the Transwell assay. Apoptosis was demonstrated by Flow Cytometry assay.3. Three bioinformatics softwares (miRbase, TargetScan, and miRanda) were utilized to predict GAX maybe downstream target gene and Luciferase Reporter Assay proved it. The GAX vector pEGFP-C1- GAX were transfected into HepG2cells and detected expression of target gene NF-kB.4. For GAX immunohistochemical assays,94pairs of paraffin-embedded HCC samples and adjacent non-tumor tissues were obtained from patients who underwent curative resection from2006to2007at Xiangya Hospital. The relationships between GAX expression and clinicopathologic parameters were analyzed. Survival curves were obtained using Kaplan-Meier curves and log-rank tests.Results1. Real-time PCR analysis showed the levels of miR-301a expression in the HCC sections(0.05494±0.03649) were higher than in the adjacent tumors(0.02681±0.02056) and normal tissue(0.01306±0.00613, P<0.01). In advanced liver cancers at stages III and IV, miR-301a expression was significantly higher than in the early stage I and II tumors (0.06839±0.04112vs.0.03783±0.02054, P<0.05). We also found the mature form of miR-301a was overexpressed distinctly in in the HCC cell lines compared with in L02cell (P<0.01).2. Real-time PCR analysis showed that miR-301a expression was decreased by transfecting the miR-301a inhibitor. MiR-301a inhibitor reduced cell proliferation24-72h after transfection compared with the negative control and the blank control (P<0.05).Anti-miR-301a reduced the migratory ability of HepG2cells to40%that of the NC cells (P<0.01). The invasive capacity was also suppressed by nearly60%in the anti-miR-301a transfectants compared with the NC cells (P<0.01).3. GAX is a target of miR-301a in HCC-derived cells through bioinformatics and dual-luciferase reporter assay system. Both real-time PCR and western blot analysis showed that the expression levels of GAX increased after miR-301a inhibitor transfection compared with in the negative control. Western blot analysis results showed that in the HepG2cells transfected with both miR-301a inhibitor and GAX vector, there was a decrease in NF-κB expression compared with the negative control. miR-301a may indirectly affect NF-kB expression by repressing GAX mRNA transcription.4. Real-time PCR analysis showed the levels of GAX expression in the HCC sections(0.00568±0.00308) were lower than in the adjacent tumors(0.00792±0.00386) and normal tissue(0.01164±0.00314, P<0.05). Western blot analysis showed that the GAX protein expression in HCC was significantly lower than that in adjacent or normal liver tissues. In early liver cancers at stages I and II, GAX expression was significantly higher than in the advanced stage III and IV tumors (0.00715±0.00315vs.0.00452±0.00198, P<0.05). The correlation analysis showed that there was a negative correlation between miR-301a and GAX in hepatocellular carcinoma tissues (r=-0.498, P<0.05). 5. GAX positive staining was predominantly observed in the nucleus. GAX were mainly negative expression in HCC but mainly positive expression in adjacent and normal tissues. Furthermore, there were significant differences in GAX expression associated with Edmondson stage, capsule invasion, and vascular invasion. Patients with negative GAX expression had a shorter overall survival and disease-free survival than GAX-positive patients.Conclusions1.The levels of miR-301a expression in the HCC sections were higher than in the adjacent tumors and normal tissues. Downregulation of miR-301a by miR-301a inhibitor could obviously inhibit the growth and promote the apoptosis of HepG2cells. It also obviously inhibited their migration and invasion ability. miR-301a may serve as a potential target for treatment of HCC.2. GAX was a target of miR-301a in HCC-derived cells through bioinformatics and dual-luciferase reporter assay system. Downregulation of miR-301a can promote the expression of GAX but inhibit NF-κB expression in HepG2cells. All of them maybe involve in the regulation of HCC signaling pathways.3. GAX protein expression in HCC was significantly lower than that in adjacent or normal liver tissue. There were significant differences in GAX expression associated with Edmondson stage, capsule invasion, and vascular invasion. Lower GAX expression represented shorter overall survival and disease-free survival time. GAX might associated with HCC development and progress.