Bthe Expression And Function Of MiR-193b In Pancreatic Cancer

BackgroundPancreatic cancer (PC) is one of the most lethal solid malignancies and the8th most frequent cause of cancer death worldwide. The dismal prognosis of pancreatic cancer is attributed to its late presentation, lack of accurate biomarkers for early diagnosis for the possibility of curative resection, the propensity of early metastasis as well as the limited effect and toxicity of standard chemotherapy and radiotherapy. The mechanism of PC development is not clear yet. Therefore, identification of novel targets, which are differentially expressed in PC and normal pancreatic tissues, is extremly important for early diagnosis and treatment.MicroRNAs (miRNAs) are non-coding RNAs (19-24nucleotides) that regulate gene expression by targeting mRNAs for translational repression, degradation, or both. Increasing lines of evidence indicate aberrant expression of microRNAs linked to cancer, and those miRNAs are thought to function as tumor suppressors, or oncogenes. Thus thorough research in miRNAs expression and function will contribute to understand the pathogenesis of tumor and might benefit in the diagnosis, treatment and prognosis of cancer. Hsa-miR-193b is located in16p13.12. It has been demonstrated that miR-193b is deregulated in a variety of cancers and significantly correlated with the development of these malignacies, such as cervical neoplasm, breast cancer, hepatocellular carcinoma, prostate cancer, acute myeloid leukemia etc. Ikeda and colleagues also suggested that miR-193b is a MAPK-associated miRNA and exogenous overexpression of miR-193b had the most notably inhibitory effect on the proliferation of cultured pancreatic cancer cells. However, little is known about the expression of miR-193b in pancreatic cancer tissue and the exact role of miR-193b in pancreatic cancer.ObjectiveThis study aims to investigate the expression of miR-193b in human PC and explore the function and mechanism of miR-193b in tumor growth and metastasis of PC. We also analyze the relationship between miR-193b and STMN1. Furthermore, we analyze the relationship between STMN1and PC clinicopathologic features and investigate the functional and mechanism role of STMN1on PC cells.Methods1. Real-time PCR assay was used to detect the expression level of miR-193b and STMN1mRNA in fresh PC tissues, paired non-tumor tissues, normal pancreatic tissues and PC cell lines. Western blot assay was used to detect the expression level of STMN1protien in fresh PC tissues and PC cell lines. Furthermore, STMN1protein expression was assessed by immunohistochemical analysis of paraffin-embedded specimens of PC and normal pancreatic tissues. Correlations between STMN1and clinicopathologic features and prognosis were also tested.2. Hsa-miR-193b mimics were transfected into Panc-1cells by use of LipofectamineTM2000. Cells transfected with no significant homology with negative group and blank group were treated in parallel. Real-time PCR were performed to detect miR-193b expression after transfection. 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. Western blot assay was performed to detect STMN1protein expression after transfection. Luciferase reporter assay was performed to detect whether the STMN1was directly regulated by miR-193b.3. A specific small interfering RNA of STMN1(STMN1-siRNA) was bought from Santa Cruz and transfected into Panc-1cells by use of LipofectamineTM2000. Cell transfected with no significant homology with human gene sequences (negative group) and without transfection treatment (blank group) were treated in parallel. Real time-PCR and Western blot were performed to determine the effects of STMN-siRNA on STMN1expression. In vitro proliferation was assessed by MTT assay. Apoptosis was demonstrated by flow cytometry. Migration and invasive ability were determined by use of the Transwell assay. Western blot were performed to detect the amount of acetylated a-tubulin and a-tubulin in Panc-1cells after transfection of STMN1-siRNA into Panc-1cells.Results1. The expression of miR-193b was downregulated in PC tissues in comparision to the paried non-tumor tissues and normal pancreatic tissues. In advanced pancreatic cancers at stages â…¢ and IV, miR-193b expression was significantly lower than in the early stage I and â…¡ tumors. Similarly, the expression of miR-193b was lower in PC cell lines than in normal pancreatic cells.2. STMN1may be a target of miR-193b through bioinformatics analysis.3. The mRNA and protein expression of STMN1was deregulated in PC tissues compared with paried non-tumor tissues. The mRNA and protein expression of STMN1was higher in PC cell line than in normal pancreatic cells.4. STMN1positive staining was predominantly observed in the cytoplasm. STMN1were mainly positive expression in PC but negative expression in normal pancreatic tissues. Furthermore, there were significant differences in STMN1expression associated with histological differentiation, lymphatic metastasis and the TNM stage. Patients underwent radical surgery of PC in positive STMN1expression group had a shorter overall survival than patients in STMN1negative expression group. Univariate and Multivariate analysis indicated that STMN1expression was an independent prognostic factor of patients who undergone radical surgery of PC.5. The correlation analysis showed that there was a negative correlation between miR-193b and STMN1mRNA in PC tissues.6. With upregulation of endogenous miR-193b through transfected miR-193b mimics, there was a significant downregulation of STMN1protein expression level in Panc-1cells. The luciferase reporter assay confirmed that STMN1was a direct target of miR-193b.7. Overexpression of miR-193b inhibited the proliferation, migration, and invasion of PC cells and increased cell apoptosis.8. Real time PCR and Western blot showed that STMN1-siRNA significantly inhibited STMN1expression. MTT assay revealed that the STMNl-siRNA group significantly reduced cell proliferation compared with the negative group or blank group cells. The Transwell assay revealed that Panc-1cells transfected with STMN1-siRNA had much lower migratory ability and invasive activity than the negative group or blank group cells. FCM revealed that apoptosis was significantly increased in STMN1-siRNA transfected cells compared with negative group or blank group cells. Western blot analysis showed that the protein levels of acetylated a-tubulin in Panc-1cells transfected with STMN1-siRNA were significantly increased compared with the negative group or blank group cells. Conclusions1. miR-193b was significantly downregulated in PC.2. STMN1was significantly upregulated in PC. Overexpressed STMN1was associated with histological differentiation, lymphatic metastasis, the TNM stage and poor prognosis of pantients who undergone radical surgery of PC. STMN1expression was an independent prognostic factor of patients who undergone radical surgery of PC.3. The expression of miR-193b and STMN1mRNA in PC tissues were correlated.4. miR-193b could directly inhibit expression of STMN1protein in Panc-1cells and inhibit the proliferation, migration and invasion and induce apoptosis of Panc-1cells.5. STMN1could downregulated the acetylation level of a-tubulin in Panc-1cells, enhance the depolymerization of tubulin, inhibit the proliferation, migration and invasion Panc-1cells and induce apoptosis of Panc-1cells.