| BackgroundThe main obstacles in the field of pancreatic cancer research, which we must face, are difficulty in early diagnosis, chemoresistance and lack of effective biomarkers for prognosis. Identification of new biomarkers for early detection and predicting prognosis, disclosing the mechanisms of tumorigenesis and development, and reversal of multidrug resistance are effective strategies to improve overall survival of patients with pancreatic cancer.MicroRNAs (miRNAs) are short, single-stranded RNA molecules with22nucleotides, which involve in tumorigenesis, cancer development, proliferation, metastasis and chemoresistance. Our previous study showed miR-497was significant downregulation in gemcitabine resistant cell lines, compared with that in parent cell lines, which meant that miR-497might play an important role in the regulation of drug resistance in pancreatic cancer. MiR-497decrease has been illustrated in breast, colorectal and cervical cancers and contributes to the malignancy of multiple tumors. However, the expression levels and regulatory roles of miR-497in pancreatic cancer are still unclear. It is necessary to investigate the regulatory roles and mechanisms of miR-497in pancreatic cancer and assess its diagnostic and prognostic values.ObjectiveThis study aimed to investigate the effects of miR-497on regulating proliferation, apoptosis, cell cycle, migration and invasion, chemosensitivity, biomarkers of cancer stem cells in pancreatic cancer cells. Then we studied the mechanisms of miR-497in regulating the progression of pancreatic cancer. We also investigated the regulatory roles and mechanisms of Pim-1, which was a target of miR-497. In addition, we detected the expression levels of miR-497and Pim-1in tissues and plasma, and then assessed their diagnostic and prognostic values. MethodsFor the in vitro experiments, proliferation was analyzed using a cell count kit (CCK-8). The effects on the chemosensitivity of gemcitabine and erlotinib were evaluated by CCK-8assay. Cell-cycle and apoptosis analysis were performed by fluorescence-activated cell sorting (FACS) flow-cytometry. Real-time PCR was used to detect the expression levels of miRNA and mRNA. Protein levels were detected by western blot. For the in vivo experiment, SW1990cells steadily expressing miR-497were established. Subcutaneous transplantation models of pancreatic cancer in nude mice were established to observe the tumor growth. Luciferase reporter assays were used to verify the candidate genes. The expression levels of miR-497and Pim-1in tissues were detected by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. Enzyme linked immunosorbent assay (ELISA) were used to detect the levels of plasma Pim-1. The prognostic values of miR-497and Pim-1were assessed by univariate and multivariate survival analysis. The diagnostic values of plasma pim-1levels were evaluated using receiver operating characteristic curve (ROC curve).Results1. The effects of miR-497on the malignant phenotype of pancreatic cancer cellsUpregulation of miR-497in SW1990and MiaPaCa-2cells significantly suppressed proliferation, increased sensitivity to gemcitabine and erlotinib, promoted expression of cleaved caspase-3and PARP activated by gemcitabine, inhibited migration and invasion capacities, decreased the percentage of S phase cells, and attenuated the expression levels of ABCG2and EZH2mRNA, which were biomarkers of pancreatic cancer stem cells. Opposite effects were observed after downregulation of miR-497. For the in vivo experiment, overexpression of miR-497significantly suppressed tumor growth, compared with control group.(P<0.05).2. The mechanisms of miR-497in regulating the progression of pancreatic cancer. Dual-luciferase reporter assays were performed to confirm the targets of miR-497. We found that luciferase activities were significantly decreased after co-transfection of miR-497mimics and vectors expressing wild type target sequence in293A cells, compared with that in cells co-transfected with mimics and vectors expressing mutated target sequence (P<0.05), which meant IGF-1R, FGF2, FGFR1, Kras and Pim-1were direct targets of miR-497. We also detected proteins levels, which showed upregulation of miR-497significantly decreased the expression of IGF-1R, FGF2, FGFR1, Kras and Pim-1. On the contrary, inhibition of miR-497increased the levels of these proteins. Additionally, we investigated the levels of the key molecules in the important signaling pathways. We observed that the expression levels of p-AKT and p-ERK decreased significantly after overexpression of miR-497. On the other hand, inhibition of miR-497increased the expression levels of p-AKT and p-ERK.(P<0.05).3. The effects of Pim-1on the malignant phenotype of pancreatic cancer cellsAs a target of miR-497, the regulatory roles of Pim-1in pancreatic cancer were still unknown. We showed that decrease of Pim-1levels by siRNA significantly suppressed proliferation, induced apoptosis, increased sensitivity to gemcitabine and erlotinib, inhibited migration and invasion capacities, decreased the percentage of S phase cells, and attenuated the expression levels of ABCG2and EZH2mRNA. Similar effects were observed after blocking Pim-1activity by SGI-1776. In addition, we investigated the mechanisms. We found that Pim-1and EGFR pathway formed a feedback loop.Knockdown of Pim-1by siRNA decreased the expression of EGFR and p-EGFR. Whereas blocking EGFR pathway by erlotinib or EGFR siRNA suppressed the expression of Pim-1.(P<0.05).4. The expression levels and prognostic values of miR-497and Pim-1in tissuesThe expression levels of miR-497were significantly decreased in pancreatic cancer tissues, compared with that In tumor-adjacent tissues (P=0.000).Whereas Pim-1protein levels were significantly increased in cancer tissues, compared with that in tumor-adjacent tissues (P=0.041). There was a negative correlation between the expression levels of miR-497and Pim-1protein in pancreatic cancer tissues (P=0.006). There was no association between the expression levels of miR-497/Pim-1and clinicopathological parameters, including sex, age, tumor locations, differential degree, TNM staging, diabetes, and perineuronal invasion. Univariate analysis showed TNM staging, Pim-1levels and miR-497levels were the potential prognostic factors of pancreatic cancer (P=0.027, P=0.039, and P=0.027, respectively). Multivariate analysis indicated that TNM staging (Ⅱ/Ⅲ/Ⅳ) and miR-497expression (Low) were the independent adverse prognostic factor (P=0.016, HR=1.97,95%CI:1.13-3.42, P=0.01, HR=2.76,95%CI:1.159-6.58, respectively).5. The expression levels and diagnostic values of miR-497and Pim-1in plasma78plasma samples were collected for detection of miR-497. Depressingly, miR-497could not be effectively detected for its extremely low expression in plasma. The plasma levels of Pim-1in patients with pancreatic cancer (29.8±47.7ng/ml) were significantly higher than that in healthy volunteers (0.21±0.31ng/ml), chronic pancreatitis (3.11±5.2ng/ml), other pancreatic tumors (8.75±6.6ng/ml), pancreatic neuroendocrine pancreatic patients (PNET,15.7±8.9ng/ml)(P=0.000; P=0.000; P=0.000; P=0.01, respectivley). In addition, the plasma levels of Pim-1in patients with pancreatic cancer were significantly higher than that in all controls (29.8±47.7ng/ml VS7.21±8.3ng/ml,P=0.000).The plasma levels of Pim-1displayed diagnostic values for distinguishing patients with pancreatic cancers from healthy volunteers, chronic pancreatitis, other pancreatic tumors patients and all controls (AUC=0.984,95%CI:0.963～1.006, P=0.000; AUC=0.895,95%CI:0.812～0.977, P=0.000; AUC=0.706,95%CI:0.611～0.802, P=0.001, AUC=0.768,95%CI:0.701～0.836, P=0.000, respectively). The sensitivity and specificity for distinguishing pancreatic cancer from healthy volunteers, chronic pancreatitis, other pancreatic tumors patients and all controls were95.6%and100%,87.8%and77.8%,51.1%and86.2%,73.3%and66.7%, respectively.6. The correlation between plasma Pim-1levels and clinicopathological parameters and survival analysisPlasma Pim-1levels were associated with age, tumor locations and TNM staging (P=0.031,P=0.000, and P=0.013, respectively). Patients with high levels of Pim-1had advanced tumors. Univariate analysis demonstrated that M staging, TNM staging, R status and Pim-1levels were the potential prognostic factors of pancreatic cancer (P=0.000; P=0.000; P=0.002; P=0.026). Multivariate analysis indicated that TNM staging (advanced) and plasma Pim-1expression (High) were the independent adverse prognostic factor (P=0.000, HR=1.88,95%CI:1.17-2.57, P=0.037, HR=1.87,95%CI:1.04-3.35, respectively).ConclusionMiR-497involved in regulation of proliferation, apoptosis, cell cycle, migration and invasion, chemosensitivity, biomarkers of cancer stem cells in pancreatic cancer cells through negatively regulating the expression levels of IGF-1R, FGF2, FGFR1, Kras and Pim-1. As a target of miR-497, Pim-1contributed to the malignancy of pancreatic cancer. MiR-497and Pim-1could be new therapeutic targets in pancreatic cancer. The levels of miR-497and Pim-1in tissues and the plasma levels of pim-1displayed prognostic values, and could be new biomarkers in predicting the prognosis of pancreatic cancer. Plasma Pim-1levels presented a potential value for distinguishing pancreatic lesions, and could be also a new plasma biomarker for early detection of pancreatic cancer. |
PDF Full Text Request