| Purpose:MicroRNAs are essential for the regulation of cell proliferation and had been implicated in tumorigenesis. However, the roles and molecular mechanisms of aberrant microRNAs in pancreatic cancer remain unclear. In this study,we investigate the expression levels of miR-494in pancreatic cancer tissues and pancreatic cancer cells, and significance of SIRT1in human pancreatic carcinoma and their association with the clinical pathologic characters, and evaluate the relationship between miR-494expression levels and clinic characteristics. After restoration of miR-494, biological behaviours of Aspc-1and PANC-1cells were investigated such as cellular growth, senescence, cycle progression, apoptosis and chemosensitivity etc. We also explore the mechanism of the antitumor effects of miR-494. To confirm the potential targets of miR-494, we prove the direct interaction between miR-494and its targets c-Myc and SIRT1. To elucidate the antitumor mechanisms of miR-494, we silenced c-Myc/SIRT1expression through the introduction of siRNA-c-Myc/into PANC-1cells. After inhibition of miR-494, biological behaviours were investigated. We hypothesized that miR-494might be involved in pancreatic cancer progression through the modulation of c-Myc and SIRT1.Experimental Design:The expression levels of miR-494were detected in of pancreatic cancer tissues and pancreatic cacer cells(AsPC-1, BxPC-3, PANC-1, SW1990and Miapaca-2) using qRT-PCR. We analyze the correlations among the variety expression levels of SIRT1and pancreatic cancer clinical pathologic characters. To explore the roles of miR-494in pancreatic cancer progression, AsPC-1and PANC-1cells were transfected with miR-494mimics, miR-494inhibitor and their respective negative controls (NC). After transfection, the expression of miR-494in AsPC-1and PANC-1cells was detected by qRT-PCR. MTT was used to measure the proliferation of AsPC-1and PANC-1cells after transfection with miR-494mimics/inhibitor. To further investigate the mechanisms of miR-494in proliferation inhibition, apoptosis and cell cycle were analyzed using flow cytometry while cell senescence was analyzed using β-Galactosidase (β-Gal) staining. Moreover, several associated proteins, including BAX, Bcl-2, acetylated p53, p21and CyclinDl were detected by western blot analysis. The effects of miR-494on the migration and invasive ability of pancreatic cancer cells were investigated using Transwell analysis after transfection with miR-494. We also observed whether miR-494could sensitize pancreatic cancer cells to5-FU and GEM. Immunohistochemistry (IHC)was used to detect the expression of c-Myc and SIRT1in86cancer tissues (PC) and41normal pancreatic tissues (NP). Next, we determined the expression levels of c-Myc and SIRT1mRNAs in PC and NP tissues, and analysis the correlation between miR-494levels and c-Myc and SIRT1mRNA expression in pancreatic cancer tissues. The putative targets of miR-494were revealed by bioinformatic analyses, includingmiRanda, miRDB and TargetScan algorithms. The expression of c-Myc and SIRT1was detected qRT-PCR and western blot results after transfection of miR-494mimics/inhibitor. After that, the correlation between miR-494levels and c-Myc and SIRT1mRNA expression was analysised. Furthermore, we cloned3’-UTR sequences that contained the predicted target sites (wild type, WT) or mutated sequences (mutant type, MUT) of c-Myc and SIRT1into the pGL3vector. A luciferase reporter assay was then used to prove the direct interaction between miR-494and its targets. To confirm the antitumor mechanisms of miR-494, we silenced c-Myc/SIRTl expression through the introduction of siRNA-c-Myc/SIRT1into PANC-1cells. Cell biology behaviors were investigated after inhibition of c-Myc/SIRT1. Further, restoration of miR-494was evaluated in nude mice to confirm whether our findings shown above in vitro would be reproducible in vivo. Results:The mean expression level of miR-494in PC tissues was significantly lower than that of NP tissues (0.567±0.049vs1.000±0.104, P<0.001). We also found that miR-494was significantly down-regulated in four different pancreatic cancer cell lines (AsPC-1, BxPC-3, PANC-1and SW1990) compared with NP tissues, especially in AsPC-1(0.171±0.023vs1.060±0.131, p=0.0026) and PANC-1cells (0.270±0.024vs1.060±0.131, p=0.004). Low miR-494expression was found to be significantly correlated with age (p=0.0438), tumor size (p=0.0041), TNM stage (p=0.0003), lymphatic invasion (p=0.0010) and distant metastasis (p=0.0362). However, there was no obvious correlation between miR-494levels and other clinic features. Kaplan-Meier survival curves showed that patients with high miR-494expression levels had significantly longer survival time than patients with low miR-494levels (P=0.0366). After restoration of miR-494, the proliferation of AsPC-1and PANC-1cells was significantly inhibited by45.13±5.38%and41.42±3.95%, respectively (F<0.01). While after transfection with miR-494inhibitor, the proliferation rate increased by23.15±2.18%and16.59±3.23%(P<0.05). Moreover, AsPC-1and PANC-1cells transfected with miR-494mimics displayed fewer and smaller colonies than negative control. The number of apoptotic cells were markedly increased in AsPC-1(12.3±0.5%vs19.5±0.6%, P<0.05) and PANC-1cells (8.9±0.8%vs17.1±0.3%, P<0.05) transfected with miR-494. Furthermore, overexpression of miR-494notably increased the proportion of cells in the G1phase in AsPC-1(63.3±3.1%vs73.7±2.5%, P<0.05) and PANC-1cells (40.5.6±4.2%vs58.6±3.7%, P<0.01). Analysis of β-Gal staining indicated that miR-494significantly induced senescence of AsPC-1(17.5±2.1%vs40.7±1.7%, P<0.01) and PANC-1cells (14.6±3.3%vs60.9±3.8%, P<0.01). Bcl-2and CyclinD1were significantly down-regulated, whereas acetylated p53, BAX and p21were obviously up-regulated. The migration and invasive ability of pancreatic cancer cells were significantly decreased after transfection with miR-494. After restoration of miR-494, the IC50of AsPC-1cells exposed to5-FU (7.93±1.05vs27.56±2.89ug/mL, P<0.01) and GEM (17.5±1.39vs82.56±4.27ug/mL， P<0.01) were significantly decreased. Moreover, the IC50of PANC-1cells exposed to5-FU (9.41±1vs1.63vs42.62±3.16ug/mL, P<0.01) and GEM (25.33±1.75vs63.25±5.36ug/mL, P<0.01) were also decreased. Compared to the NP group, the percentage of cells that were positive for c-Myc (52.74±1.534%vs26.49±1.56%, P<0.001)and SIRT1(48.35±1.51%vs20.51±1.34%, P<0.001) were significantly increased in the PC group. The expression levels of c-Myc (1.379±0.086vs0.940±0.070, P=0.0017) and SIRT1(1.583±0.0.110vs1.044±0.092, P=0.0022) mRNAs were also significantly up-regulated in the PC group. A significant inverse correlation between miR-494levels and c-Myc(r=-0.6799, P<0.001) and SIRT1(r=-0.5293, P<0.001) mRNA expression was observed in pancreatic cancer tissues. The qRT-PCR and western blot results confirmed that miR-494restoration led to the down-regulation of c-Myc and SIRT1expression, whereas the inhibition of miR-494expression increased c-Myc and SIRT1expression. In addition, we found that miR-494expression was negatively correlated with c-Myc (r=-0.6374, P=0.0142) and SIRTl(r=-0.5973, P=0.0239) expression. Luciferase reporter assay proved that miR-494targeted c-Myc and SIRT1. Specific knockdown of c-Myc by siRNA significantly inhibited the proliferation and invasion of PANC-1cells and induced cell cycle arrest and senescence. The down-regulation of c-Myc did not lead to obvious apoptosis, which implied that the effects of miR-494on pancreatic cancer do not rely exclusively on targeting c-Myc. After co-transfection of PANC-1cells with c-Myc-RNAi and SIRTl-RNAi further inhibited proliferation, invasion and cell cycle arrest. Moreover, the co-transfection induced obvious apoptosis in PANC-1cells (12.65±0.55%vs.16.85±0.75%, P=0.0457), which was similar to the effect of miR-494induction. After co-transfection, the associated proteins BAX, Bcl-2, acetylated p53, p21and CyclinDl expression levels were consistent with induction of miR-494. Restoration of miR-494inhibited the growth of pancreatic cancer cells in vivo (0.83±0.18vs0.32±0.07g, P<0.05). Western blot analysis demonstrated that the expression levels of c-Myc and SIRT1appeared to be significantly down-regulated in the pancreatic cancer xenografts.Conclusions:1. MiR-494was down-regulated in pancreatic cancer tissues and cell lines. Low miR-494expression was significantly correlated with age, tumor proliferation, and could participate in invasion and metastasis of pancreatic cancer. The expression of miR-494may be viewed as a biomarker of malignancy degree and prognosis of pancreatic cacer.2. Overexpression of miR-494in pancreatic cancer cells could significantly inhibit cell proliferation by inducing senescence, G1phase arrest and apoptosis. Moreover, restoration of miR-494expression also inhibited migration and the invasive ability of pancreatic cancer cells. The induction of miR-494enhanced the chemosensitivity of pancreatic cancer cells to5-FU and Gemcitabine. These results indicated that miR-494might be involved in the carcinogenesis of pancreatic cancer and that its low expression levels could be considered as a novel diagnostic and prognostic indicator.3. MiR-494down-regulated c-Myc and SIRT1expression by directly binding to the3’-UTR of c-Myc and SIRT1mRNA. Inhibition of miR-494increased expression of c-Myc and SIRT1. Furthermore, the inverse correlation between miR-494levels and c-Myc or SIRT1expression was observed in pancreatic cancer tissues as well as cell lines. Therefore, these results confirmed that miR-494directly targeted c-Myc and SIRT1.4. After co-transfection of PANC-1cells with c-Myc-RNAi and SIRT1-RNAi, the biological behaviours were similar to the effect of miR-494induction. Howover, the down-regulation of c-Myc did not lead to obvious apoptosis, which implied that the effects of miR-494on pancreatic cancer do not rely exclusively on targeting c-Myc. |
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