Study Of Screening And Mechanisms Of Pancreatic Cancer-specific Micrornas

Background Pancreatic cancer is one of the most aggressive tumor and continues to be a major unsolved health problem. It is the second most frequent gastrointestinal malignancy with poor prognosis, and the fourth leading cause of cancer deaths in America and has the lowest 5-year survival rate among all malignancies. The high mortality rate for pancreatic cancer is primarily due to diagnosis mostly at advanced stage no sensitive and specific tools of early detection. Furthermore, the conventional treatment approaches, such as surgery, chemotherapy, or combinations of both, have had little impact on the natural course of this aggressive neoplasm. Although surgical resection remains the only curative treatment, around 15-20% of patients have resectable disease, only around 20% of them survive to 5 years. For locally advanced, unresectable, and metastatic disease, chemotherapy seems to hold the greatest promise, but the dismal prognosis is partly due to its intrinsic and extrinsic drug resistance characteristics. Pancreatic cancer remain a challenging problem during the 21st century. However, improvements in early detection and increasing the drug sensitive could be novel strategies to prolong substantially rvival of patients with this disease.The microRNAs (miRNAs) are a group of small RNAs, which are single-stranded and consist of 18-25 nucleotides (～22 nt). They do not code for any protein or peptide; however, they regulate gene expression by multiple mechanisms. It is commonly accepted that mature miRNAs regulate gene expression by binding to the 3'-untranslated region (3'-UTR) of target mRNA, causing degradation of mRNA or inhibition of their translation to functional proteins. miRNAs play important roles in many normal biological processes; however, the aberrant miRNA expression and its correlation with the development and progression of cancers is an emerging field.Recently, miRNAs have received increasing attention in the field of cancer research. miRNA alterations are involved in the initiation and progression of human cancer. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target proteincoding genes involved in cancer. miRNAs could be used as biomarkers for diagnosis of cancer, prediction of prognosis and to constitute a novel target for cancer treatment. Importantly, some miRNAs could regulate the formation of cancer stem cells and the acquisition of epithelial-mesenchymal transition, which are critically associated with drug resistance. Moreover, some miRNAs could target genes related to drug sensitivity, resulting in the altered sensitivity of cancer cells to anti-cancer drugs. Therefore, the study of miRNAs holds much promise for improving diagnosis and treatment of pancreatic cancer.Objective 1,To indentify and validate the expression patterns of miRNAs associated pancreatic cancer's diagnosis and drug resistance.2,To validate targeting of miR-155 by hMLHl induced cancer advance.Methods 1.Detection of Human pancreatic cancer based on measurement of a pancreatic cancer-expressed miRNAs in Plasma①To investigate the feasibility of identifing specific circulating miRNAs as Potential markers for diagnosis of pancreas-related disease and to establish the approaches of blood-based miRNAs cancer detection by using Real-time PCR;②Using qPCR-based expression profiling (TaqMan low density arrays, TLDA) followed by Real-Time quantitative Polymerase Cycle Reaction (RT-qPCR) validation, we compared the levels of circulating miRNAs in plasma samples from 20 pancreatic cancer patients,6 Pancreatitis patients and 5 matched healthy controls.③To further explore the potential roles of these circulating miRNAs, let-7b,miR-miR-363,miR-942,miR-645,miR-190b, using qPCR between pancreatic cancer and healthy control.2. To investigate the involvement of miRNAs in drug-resistant of pancreatic cancer cell lines①The drug resistance of SW1990/ ADM, SW1990/ 5Fu and SW1990/ GEM cells was evaluated using CCK-8 assay.②③High-throughput analysis of the miRNAs profile in a panel of adriamycin- (SW1990/ADM), 5-fluorouracil- (SW1990/5Fu) and gemcitabine resistant (SW1990/GEM) cells was assessed using a microarray platform and subsequent validation with qPCR.3.To confirm the possible mechanisms of inducing mortality in pancreatic cance①To explore the miR-155 target gene hMLH1 using dual luciferase reporter assay validation, PCR and Western Blot.②A total of 43 patients with pancreatic cancer underwent surgical resection were included. Expression of hMLH1 in tumor and para-tumor tissues was determined by immunohistochemistry. The relationship between hMLH1 expression and clinicopathological and prognostic variables were evaluated.Results 1.①We established a novel approach to test the miRNAs in plasma.②We established a list of likely blood-based miRNAs biomarker candidates for pancreatic cancer and Pancreatitis patients. The data indicated that a total of 188 miRNAs could be detected. Compared the levels of circulating miRNAs from pancreatic cancer and Pancreatitis group or from pancreatic and healthy group, there are 7 and 19 miRNAs dysregulating significantly respective (P<0.01)③We chose to analyze 5 candidates in a case-control cohort of plasma samples collected from 20 individuals with pancreatic cancer and 22healthy age-matched control individuals. Of all of the candidates, miR-942 showed the greatest differential expression in the pancreatic cancer compared with the healthy control (P=0.023)2.①The drug resistance index of SW1990/ADM, SW1990/5Fu, SW1990/ GEM ells relative to the SW1990 cells was 12.57,133.68 and 438.96, respectively.②The microarray indicated that 59,47,58 miRNAs were differentially expressed in ADM,5Fu, GEM resistant cells, respectively, with a>2-fold change. Seven miRNAs (hsa-miR-33a,33b,1285,486-5p,99b*,125a-3p,200c*) were always diversely expressed in all the resistant cell lines. Another seven miRNAs (miR-34a,424,449a,497,451,21,101), inducing drug resistance in other tumors, were diversely expressed in resistant cell lines with a>1-fold change.③To further validate our results, miR-486-5p,miR-99b*,miR-125a-3p, four of the most differentially expressed miRNAs, performed frequent deregulation as same as the microarray.3.①Dual luciferase reporter assay was carried out and showed that Hmlh1 is regulated by miR-155directly.②hMLH1 induced the development and associated with the prognosis in pancreatic cancer.Conclusion 1. We established a novel approach to test the miRNAs in plasma and established a list of likely blood-based miRNAs biomarker candidates for pancreatic cancer and pancreatitis patients. has-mir-942 identified as a candidate biomarker of pancreatic neoplasia. In summary, we show the feasibility of developing plasma miRNAs profiling as a sensitive and specific blood-based biomarker assay for pancreatic cancer that has the potential of translation to the clinic with additional improvements in the future.2. Drug resistance cell lines showed a different miRNAs expression profile from its parental SW1990 cells, suggesting the involvement of miRNAs in tumor cells drug resistance. Four miRNAs, miR-486-5p, miR-99b*, miR-125a-3p and miR-1285, identified as a potential biomarker of drug resistance in pancreatic cancer. This finding provides a experimental basis for further study of mechanism underlying the drug resistance of pancreatic cancer.3. hMLH1 was confirmed as the target of miR-155 in pancreatic cancer cell line. The result of modulation of hMLH1 by miR-155 induced mortality of pancreatic cancer. miR-155 is an potential treatment target to prolong the life of patients with pancreatic cancer.