MiR-141Inhibits Tumor Proliferation And Invasion By Targeting Hepatocyte Nuclear Factor-3β In Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies with the fifth most common cause of cancer-related mortality in the world. About600000patients diagnosed as HCC annual worldwide. In China, the fatality rate of HCC is the third common in malignant tumors, just behind gastric cancer and esophageal cancer. The pathogenesis of HCC is complex and still not entirely clear. Early stage of HCC with preserved liver function can be effectively treated by resection, liver transplantation, or percutaneous and with a more ideal5-year survival rate. However, HCC is often diagnosed at an advanced stage which has a dismal prognosis due to lacking of conspicuous early clinical symptoms and specific biochemical indicators. And HCC often show symptom such as liver pain, anaemic, angular, jaundice and ascites at the late stage. Therefore, studying the molecular basis of HCC is vital for exploration a new therapeutic agents. Generally, HCC progression can be defined by a decrease in differentiation, an extinction of tissue-specific gene expression, acceleration of cell proliferation, and ultimately metastasis. Patients with HCC often exhibit tumor cell invasion and metastasis before conventional diagnosis. Thus, it has important clinical value to studying the differentiation and gene regulation characteristics of HCC. Maintenance of hepatocyte differentiation and control of liver-specific gene expression is attributed in large part to hepatocyte nuclear factor3(HNF-3). The HNF-3/forkhead family of transcription factor in mammals includes three genes designated as Foxa-1(HNF-3a), Foxa-2(HNF-3β), and Foxa-3(HNF-3y), which share homology in the winged-helix DNA binding domain. The HNF-3β gene is located in chromosome20p11.21and there have been reported apoptotic injury was associated with a downregulation of HNF-3β. HNF-3β expressing plasmids decreased apoptosis, whereas siRNA silencing of HNF-3β increased apoptosis in HepG2cells. However, the mechanisms of HNF-3β, as well as the clinical and prognostic significance of HNF-3β expression have never been thoroughly studied in HCC. Recently, several studies have shown that HNF-3β expression and activity are regulated at post-transcriptional level. HNF-3β protein but not mRNA levels are regulated in insulinoma cells by microRNAs (miRNA).miRNAs are non-coding small endogenous RNAs of～22nucleotides (nts) in length widespread in a variety of biological, which regulate target genes expression at the post-transcription level. Mature miRNA may cause either inhibition of translation of the targeted mRNAs or induce their degradation by interacting preferentially with the3’-untranslated regions (3’-UTRs) of target mRNAs. Recent studies demonstrate that abnormal miRNA expression plays an important role in the formation of a wide variety of tumors, and is also directly involved in the occurrence, development, diagnosis and staging of HCC.Although the deregulation of miRNAs and HNF-3β play important roles in HCC or hepatic tumor cell lines, no correlation between HNF-3β and its potential target miRNA---miR-141has been reported. In this study, we first tested the expression level of HNF-3β in HCC tissues and corresponding adjacent tissue. Subsequently, we found miR-141as a potential target miRNA of HNF-3β through bioinformatics methods. Through the qPCR and luciferase reporter experiments, the target relation and control patterns of miR-141and HNF-3p were then verified. In addition, based on the studies mentioned above, we explored the effects of miR-141targeted HNF-3β on HCC cell lines, including cell proliferation, apoptosis and invasion.Our study is divided into two parts as below:Part oneValidation of HNF-3p as a direct target of miR-141OBJECTIVEWe test and analysis the expression of HNF-3β in human HCC tissues. Next, we explore the potential effects of miR-141targeting on HNF-3β, as well as the probable mechanism of HCC genesis and development.METHODS1. Cell cultureHuman HepG2cell culture:Cells taken from liquid nitrogen were put in37℃water bath to melt completely. Then cells were infused in centrifuge tube which contains9ml DEME medium (FBS free). Supernatant the cells with low sugar DEME medium which containing10%FBS after centrifuged5min in1000g. Cells were cultured in75cm flask at37℃in a humidified5%CO2incubator, and were given fresh media24h later. HepG2cells were subculture every2-3days with trypsin digestion.Cell transfection:Cells were seeding in6or12pore plate according to the experiment. When each hole of pate fulls50%-60%cells at the next day, the same mount of pre-miR-141, siRNA, HNF-3β plasmid or negative control RNA was transfected with Lipofectamine2000. After24h or48h, cells were harvest to extract RNA for real-time PCR or extract protein for western blot.2. Human HCC samples and tissue dissection All tissues sample and clinical information were collected at the Liver and Gallbladder Surgery, Nanfang Hospital, SMU, Guangzhou.3. Establish and application of luciferase report systemConstruct the plasmid:Firstly, real-time PCR to amplification HNF-3β3’-UTR using the whole genome DNA as a template. Then, insert the PCR products to the3’-UTR of luciferase report gene p-MIR-report, and using sequencing detection to ensure the correction of insert fragment.Luciferase assay:HepG2cell was seeded in12pore plate, and co-transfected Lipofectamine2000with1ug luciferase report plasmid or0.5ug β-galactose glucoside enzyme expression plasmids. The β-galactose glucoside enzyme expression plasmid was used as a contrast of worm luciferase report plasmid.24h after transfection, fluorescent values of all components were analyzed using luciferase analysis kit.4. RNA isolation and Real-time PCR analysis RNA isolation and real-time PCR were performed to examine the expression differences of miR-141and HNF-3β mRNA in HCC tissues and HepG2cell lines.5. Western blot analysis Western blotting was performed to examine the expression of HNF-3β protein and quantitative analysis was carried out through Image J software.RESULTS1. The expression of HNF-3β in human HCC tissues Results of Western blotting showed that the expression of HNF-3β protein were significantly higher in tumor tissues compared with the matched normal tissues. However, qRT-PCR showed that HNF-3β mRNA appeared to be irregular in tumor than in normal tissue of specimens, but overall the HNF-3β mRNA expression between HCC tissues and the matched normal tissues was not statistically difference. These results indicate that HNF-3β expression and activity are regulated at post-transcriptional level.2. The expression of miR-141and HNF-3β in human HCC tissues is inversely correlated With the help of the publicly available databases, we found that miR-141was predicted target the HNF-3β mRNA transcript. We examined the expression of miR-141using qRT-PCR and founding that miR-141was significantly lower in human HCC tissues compared with the adjacent normal tissues. The correlation analysis results shown that miR-141has a higher correlation with HNF-3β protein than with HNF-3β mRNA. Those results further evidence that the expression of miR-141and HNF-3β in human HCC tissues is inversely correlated.3. Identification of direct miR-141targets by luciferase reporter screening The expression of HNF-3β protein was significantly reduced by the transfection of pre-miR-141, which proved the assumptions we put forward before. However, transcript-based expression analysis and computational predictions alone cannot directly measure the actual miRNA-target interaction. Thus we use luciferase analysis to prove that miR-141direct targets HNF-3β. Results shown that relative luciferase activity was significantly lower in HepG2cells after cotransfection with pmiR-HNF-3β-wt and pre-miR-141, whereas no statistically significant difference was observed between cells co-transfected with pmiR-HNF-3β-mut and pre-miR-141. These results indicated that the binding site strongly contributes to the miRNA.CONCLUSIONIn this study, we found the expression of HNF-3β was significantly higher in human hepatocellular carcinoma tissues compared with normal liver tissues. In addition, for the first time, we discovered and proved that miR-141has direct effects on HNF-3β3’-UTR. miR-141is a direct target miRNA of HNF-3p, could downregulation the expression of HNF-3β.Part twoEffects of miR-141targeting HNF-3β on hepatoma carcinoma cells proliferation, invasion and apoptosisOBGECTIVEExplore the effects of miR-141targeted HNF-3β on cell proliferation, invasion or apoptosis of hepatocellular carcinoma cells, which providing a treatment ideal for hepatocellular carcinoma.METHODS1. Cell culture HepG2cell line was purchased from the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. Cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with10%fetal bovine serum and1%Penicillin-Streptomycin within a humidified atmosphere containing5%CO2at37℃32. Cell proliferation assay The proliferation of HepG2cells were monitored using Cell Counting Kit-8, according to the manufacturer’s instructions. Briefly, HepG2cells were plated at1.0×104cells per well in96-well plated and incubated overnight in DMEM medium supplemented with10%FBS. Cells viability was documented at12,24,36,48h after transfected with equal doses of scrambled pre-miR-control, pre-miR-141, control siRNA, HNF-3β siRNA, control plasmid or HNF-3β plasmid in HepG2cells,10ul CCK-8liquid was added to the test well and incubated for1h. Absorbance (A) was then measured at a wavelength of450nm. 3. Cell Transwell assay200ul Cell suspension with HepG2cells (4×104) were placed in the upper compartment, and500ul DEME culture media containing10%fetal calf serum was added to the lower compartment. The tanswell plates were incubated at37"Cfor12-16h in a humidified atmosphere with5%CO2. Then the chamber was fixed on30min in paraformaldehyde and stained with10%crystal violet. Migration cells were defined as cells that had moved into the lower surface of the membrane, and the non-migrating cells which were retained on the upper surface of the membrane were removed by a cotton swab.4. Flow-cytometric analysis of apoptosis24h after transfection with pre-miR-control, pre-miR-141, control siRNA or HNF-30siRNA, HepG2cells were treated with200uM hydrogen peroxide (H2O2) for30min to induce apoptosis. As per the manufacturer’s instructions of the Annxi V-FITC/PI staining kit, the cells were then washed twice with cold PBS and resuspended in1×106cells/ml. Cells were transferred to a5ml culture tube, and Alexa Fluor488Annexin V and propidium iodide (PI) were added. The cells weres incubated for15min at room temperature in the dark and were analyzed by flow cytometry.RESULTS1. Effect of miR-141targeting HNF-3P on hepatoma carcinoma cells proliferation Cells transfected with pre-miR-141proliferated at a significantly lower rate compared with pre-miR-control. Meanwhile, the similar significant difference was observed in the proliferation rates between the cells transfected with HNF-3β siRNA and control siRNA. In addition, Cells transfected with the HNF-3β plasmid alone showed more expression level of HNF-3β compared to cells transfected with an empty plasmid control or control plasmid. Whereas compared to cells transfected with pre-miR-141, the cells co-transfected with pre-miR-141and the HNF-3β plasmid exhibited significantly higher levels of HNF-3β.Consequently, overexpression of HNF-3β rescued miR-141mediated downregulation of the proliferation rates of HepG2cells. These results suggest that miR-141might inhibit cell proliferation by silencing HNF-3β.2. Effect of miR-141targeting HNF-3β on hepatoma carcinoma cells invasion The chamber assays showed that downregulation of HNF-3β was able to inhibit the invasion of HepG2cells relative to control cells. However, cell invasion efficiency were dramatically attenuated when simultaneously transpected with pre-miR-141and HNF-3β plasmid than the invasion induced by miR-141.3. Effect of miR-141targeting HNF-3β on hepatoma carcinoma cells apoptosis When compared to cells transfected with pre-miR-control, the percentage o apoptotic cells in the mimics transfection group was significantly higher. When compared to cells transfected with control siRNA, transfection with HNF-3β siRNA increased the percentage of apoptotic cells. Moreover, compared with cells transfected with pre-miR-141and HNF-3β plasmid, the cells co-transfected with pre-miR-141and the HNF-3P overexpression plasmid exhibited significantly lower apoptosis rates, suggesting that HNF-3β can attenuate the apoptosis effect of miR-141.CONCLUSIONThis study provide evidence that miR-141might inhibits tumor proliferation and invasion, and enhanced cells apoptosis by silencing HNF-3β.