| Background and Objection:Hepatocellular carcinoma (HCC) is the most common malignant tumor. It is the third leading cause of cancer deaths in the world and the second leading cause of cancer deaths in China. Surgical resection is generally accepted as the first choice of treatment for HCC; however, at the time of diagnosis, more than 80% patients are found to be ineligible for surgical resection because they are in an advanced stage and/or have associated liver diseases. Although curative surgical resection offers a 5-year survival rate of 50-70%, the rate of tumor recurrence or intrahepatic metastasis is very high. Therefore, there is an urgent need to identify molecular markers that are prognostic predictors and are potential targets in HCC patients who are unable to tolerate curative resection or in whom the cancer recurs after surgery.MicroRNAs (miRNAs) are a class of small, endogenous, noncoding RNA molecules that are about 22-nucleotide long and are responsible for posttranscriptional regulation of target genes. miRNAs are encoded in the genome and are generally transcribed by RNA polymeraseⅡin the nucleus. The primary transcripts are subsequently cleaved by the nuclear RNaseⅢenzyme Drosha to produce-70-nucleotide-long stem-loop-structured precursor molecules (pre-miRNAs). The pre-miRNAs are then exported to the cytoplasm, where the RNase III enzyme Dicer further processes them into mature miRNAs (-22-nucleotide long). Like short interfering RNAs (siRNAs), miRNAs control gene expression at the posttranscriptional level by inhibiting translation or inducing target mRNA cleavage. Emerging evidence suggests aberrant expression of miRNAs in cancer. miRNAs have been shown to be involved in several processes, including development, differentiation, apoptosis, and cell proliferation. They may function as tumor suppressors or oncogenes by targeting oncogenes or tumor suppressor genes. In this regard, tumor-suppressive miRNAs are usually underexpressed in tumors. miR-199b has been reported to be downregulated in HCC, suggesting that it is associated with hepatocarcinogenesis.Hypoxia is commonly observed in many malignant neoplasms. Tumor hypoxia has been shown to negate the therapeutic effects of chemicals and radiation. Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in mediating essential adaptive responses to hypoxia by stimulating new blood vessel formation and changing glucose/energy metabolism in cells. HIF-1 is a heterodimer composed of HIF-1 a and HIF-1β(arylhydrocarbon receptor nuclear translocator). HIF-1βis constitutively expressed, while the expression of HIF-1αis precisely regulated by the levels of cellular oxygen. HIF-1αis induced under hypoxic conditions, following which there is an exponential increase in its expression and transcriptional activity. Some previous studies have shown that the downregulation of HIF-1 may reduce Hif-1-induced transcriptional activation of genes, inhibit cell proliferation, reduce angiogenesis, and reverse hypoxia-induced resistance to chemotherapy and radiotherapy.It has been shown that Hif-1αprotein expression is upregulated by underexpressed miR-199a in cardiac myocytes during hypoxia and by miR-17-92 miRNA cluster in lung cancer. However, Hif-la is repressed by miR-17-92 only under a normoxic condition, whereas it was robustly induced under hypoxia, regardless of the level of miR-17-92 expression; this suggests that Hif1αmight be expressed with other miRNAs targeting Hif1α. By performing quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found that miR-199b is downregulated in about 77% of HCCs compared to its level in matched cirrhosis tissues. A search for miR-199b regulatory targets using bioinformatics tools led to the identification of Hif1αas a possible target of miR-199b, but no definitive evidence has been reported in HCC. Although various miRNA targets have been identified in particular types of human cancer, mechanisms through which miRNAs are involved in tumorigenesis still remain unclear.In this study, we aimed to investigate the role of miR-199b in the regulation of Hif1αin HCC and to explore its effect on the progression and prognosis of HCC.Materials and methods1. The expression of miR-199b in HCC tissues and its clinical significanceTo investigate whether miR-199b is differentially expressed between HCC and cirrhosis, we analyzed its expression levels in 35 cases of matched HCC/LC using real-time TaqMan MicroRNA assay. Of the 35 matched normal and cancer tissues,27 cancer tissues underexpressed miR-199b in comparison with the matched normal tissues (P=0.006). Chi-square test was used to analyze the relationship between miR-199b expression and clinicopathological characteristics. Survival curves were plotted by using the Kaplan-Meier method and compared by the log-rank test.2. miRNA target analysisThe potential miR-199b targets were determined using the algorithms TargetScan 5.1 (http://targetscan.org/), PicTar (http://pictar.mdc-berlin.de/), and miRanda (http://microRNA.org). To identify the genes commonly predicted by the 3 different algorithms, results of the predicted targets were intersected using miRGen (http://www.diana.pcbi.upenn.edu/miRGen/v3/miRGen.html) website tools. HepG2 and Huh-7 cells from HCC-derived cell lines were transfected with 100 nM of miR-199b or negative control 1 in 6-well plates. After transfection, cells were cultured under hypoxic (1% O2) conditions at 37℃, and intermediate samples at 24 and 48 h were collected and analyzed by western blot to assess Hifla expression.HepG2 cells were transfected with 0.1μg of pGL3-Hifla along with 0.01μg of pRL-TK vector (Promega) containing Renilla luciferase and 30 pmol of miR-199b or negative control 1 oligonucleotides. After 24 h, the cells were lysed, and luciferase activity was measured.3. Cell proliferation assay and Clone formation assayThe cellular proliferation of transfected cells was measured using the MTT assay. After transfection with miR-199b precursor or negative control 1 precursor, HepG2 cells were seeded in 96-well plates at a density of 20 000 cells/well. To each well,10μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide) (5 mg/mL) (Sigma) was added with a final volume of 100μL of culture medium containing viable cells. After an additional incubation for 4 h, the resulting formazan was dissolved in 100μL of isopropanol with 40 mM hydrochloric acid. Spectrophotometric absorbance was measured at 570 nm (for formazan dye), and absorbance at 630 nm was used as a reference.Clonogenic survival is the ability of cells to maintain clonogenic capacity and to form colonies. To determine the clonogenic ability of HepG2 cells after exposure to radiation,200-10 000 cells transfected with miR-199b precursor or negative control 1 precursor were seeded in 90-mm dishes and exposed to 0,1.0,2.0,4.0,6.0, or 8.0 Gy of irradiation under hypoxic or normoxic condition. After 10 days of incubation, the colonies were stained with crystal violet (Sigma) and counted. Cells were irradiated using a 6-MV 2100C linear accelerator (Varian, Palo Alto, CA, USA) with a dose rate of 200 cGy/min.Results1. MiR-199b is downregulated in HCC tissuesTo investigate whether miR-199b is differentially expressed between HCC and cirrhosis, we analyzed its expression levels in 35 cases of matched HCC/LC using real-time TaqMan MicroRNA assay. Of the 35 matched normal and cancer tissues,27 cancer tissues underexpressed miR-199b in comparison with the matched normal tissues (P=0.006).2. Low miR-199b expression is correlated with poor prognosis in HCC patientsThe association between miR-199b expression and clinic characteristics of tumors was elucidated. As summarized in Table 2, there was no significant correlation between miR-199b expression and gender, age, or a-fetoprotein (AFP) level (P> 0.05). However, miR-199b expression was found to be negatively correlated with clinical TNM stage (UICC,2002) (P=0.039). More advanced tumors expressed lower levels of miR-199b.MiR-199b expression in HCC was significantly correlated with progression-free survival (P=0.031, Kaplan-Meier method). The survival time was significantly different between the groups with low and high expression of miR-199b, indicating that low level of miR-199b was associated with poor prognosis as per the Kaplan-Meier log-rank test. miR-199b might be a useful prognostic biomarker for HCC patients.3. Hifla is a target of miR-199bThe role of miR-199b in human liver tumorigenesis remains unclear. To determine the potential role of miR-199b in hepatocarcinogenesis, we identified putative target genes by using microRNA, TargetScan, and PicTar algorithms. At least 2 of the algorithms showed that Hif1αis a potential miR-199b target gene.HepG2 and Huh-7 cells, both expressing very low intrinsic levels of miR-199b and high levels of Hifla protein, were used to determine whether miR-199b downregulated Hifla expression. In comparison with negative control 1 precursor miRNAs, transfection of miR-199b into Huh-7 cells and HepG2 caused a 23% and 44% decrease of Hif1αprotein levels, respectively. This finding suggests that Hif1αis downregulated by miR-199b in HCC cells.To determine whether the 3’untranslated region of Hifla mRNA was a functional target of miR-199b, we cloned a 260-bp Hifla 3’-UTR segment, which includes a potential target site for miR-199b, downstream of the pGL3 luciferase reporter gene to generate the pGL3-hif1αvector. This vector was cotransfected into HepG2 cells along with miR-199b or negative control 1. A Renilla luciferase vector (pRL-TK) was used to normalize the differences in the transfection efficiency. Luciferase activity in the HepG2 cells cotransfected with pGL3-hif1αvector and miR-199b decreased by 59% compared to that in the negative control 1 (P<0.001, t-test). Taken together, immunoassay and luciferase results provided strong indications that Hifla is a target of miR-199b in HCC cells.4. MiR-199b influences cell proliferation and radiosensitivity of HCC-derived cell linesTo test whether miR-199b affects cell growth, we transiently transfected miR-199b into HepG2 cells. We found that miR-199b restrained cell growth in a time-dependent manner under both hypoxic and normoxic conditions. Compared with the negative control 1 precursor molecules, miR-199b overexpression inhibited HepG2 cell growth under hypoxic (P=0.000) and normoxic conditions (P=0.000), respectively.The cloning efficiency of HepG2 was about 50-60%. The radiobiological parameters of HepG2 cells treated with radiation and miR-199b under hypoxia were DO=1.87, Dq=0.24, N=1.14,α/β=21.56, and SF2=0.41 (2 Gy survival fraction, SF2), while those of radiation and negative control treated HepG2 cells were DO= 2.15, Dq=1.19, N=1.74,α/β=4.87, SF2=0.61. In the present study, sensitive enhancement ratio (SER)=DO (radiation+negative control group)/D0 (radiation+ miR-199b group)=1.15. The SER in miR-199b-treated cells indicated that treatment with miR-199b significantly improved the biological effect of irradiation. Taken together, the data suggested that miR-199b could obviously enhance the sensitizing effect of radiation in HepG2 cells under hypoxia. There was no radiosensitizing effect in HepG2 cells under normoxia (data not shown).5. Hifla shows an inverse correlation with miR-199bThe Hifla protein level was determined by western blot analysis using matched HCC and cirrhosis tissues. Of the 35 cases,24 exhibited Hifla protein overexpression in HCCs with regard to matched cirrhosis. When Hifla protein level was plotted against miR-199b expression, an inverse correlation (2-tailed Pearson’s test, r=-0.923; P< 0.001) was observed. However, we could not detect a significant correlation between miR-199b and Hifla mRNA (data not shown). No association was found between miR-199b levels and the underlying causes of liver disease, particularly with viral infections, gender, and AFP levels (data not shown).Conclusion:1. Our results suggest that miR-199b, which is underexpressed in HCCs, acts as a tumor suppressor and exerts a radiosensitizing effect via the upregulation of Hif1α.2. miR-199b expression is inversely correlated with survival and directly correlated with the malignant status of HCC. Innovations of this study:1. We first confirmed that Hifla 3’-UTR is a target of miR-199b in HCCs.2. We found that miR-199b expression is related with the progress, prognosis of HCC and response to radiotherapy-related. |
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