| Pancreatic cancer is a highly malignant tumor, lack of typical symptoms in early stage. Most pancreatic cancer patients were discovered with tissue infiltration of peripancreatic structures and distant metastasis at the beginning leading to poor prognosis. If the diameter of pancreatic tumor greater than 30mm, 5-year survival of pancreatic cancer patients after resection is about 10% to 20%. If the diameter of pancreatic tumor less than 20mm, 5-year survival of pancreatic cancer patients after resection is about 30% to 60%. If the resection surgery was carried out when the the diameter of pancreatic tumor less than 10mm, 5-year survival of pancreatic cancer patients after resection is up to 75%. Therefore, improving the diagnosis leverl of early pancreatic cancer is the key strategy to improve the resection rate and prolong survival time. But the sensitivity of current diagnostic marker for early stage pancreatic cancer is very low, which greatly limits the level of diagnosis and treatment of pancreatic cancer. Therefore, the diagnosis level will be elevated if new biomarkers of pancreatic cancer were found and their regulatory mechanisms were unveiled.Pancreatic cancer development is a process from atypical hyperplasia to carcinoma in situ endothelium to invasive tumor. The process is multi-stage and multi-step, requiring consecutive accumulation of multiple genetic alterations. In this process, the control of cell differentiation, proliferation and apoptosis of multiple genes appeared abnormal, including mutations or over-expression in oncogenes and inactivation of tumor suppressor gene. MiRNAs were found participating in the pathogenesis of pancreatic cancer. Therefore, pancreatic cancer is fundamentally a genetic disease.MiRNAs are endogenous 18~25-nucleotide non-coding single-stranded RNAs that can regulate the translation of target mRNA molecules in a sequence-specific manner. MiRNAs have dual role with tumor suppressor gene and oncogene, and play an important role in cell proliferation, differentiation and apoptosis. Usually a miRNA can regulate multiple target genes, a target gene can be regulated by multiple miRNAs. Post-transcriptional regulation of miRNAs target genes is very important. If miRNA is fully complementary to its target, the mRNA is cleaved and degraded. However, if base pairing is partially complementary, translation is repressed. MiRNAs expression profiles are associated with tumor type, different types of tumors have different expression profiles of miRNAs, which suggest that miRNAs can be used as diagnostic marker for cancer, and can be used for cancer treatment and prognosis.Studies have shown that miRNAs have a close correlate with the biological characteristics of pancreatic cancer. Bloomston et al. used miRNA microarray and quantitative real-time PCR method in 8 pancreatic cancer samples and 8 matched benign pancreatic tissue controls for miR-21, miR-221, miR-222, miR-18la, miR-181b, miR-181d, and miR-l55. All of these miRNAs were overexpressed in tumor samples relative to benign pancreatic tissue.Lee et al. found that miR-221, miR-100, miR-125b, miR-21 is highly expressed and miR-375 is lower expressed in pancreatic cancer than that in normal tissue by miRNA microarray. Though miR-34 was down regulated in pancreatic cancer cell lines, it was highly expressed in two untransformed pancreatic ductal cell lines such as HPNE and HPDE. Moriyama et al. found miR-21 was significantly higher expression in human pancreatic cancer cell lines and pancreatic cancer tissues. Overexpressing miR-21 in pancreatic cancer cell lines can enhance the proliferation, invasion and drug resistance of pancreatic cancer cells lines. Inhibition the expression of miR-21 gained the opposite. MiR-21 could increased tumor invasion-related genes and vascular endothelial growth factor, and enhanced the degree of malignancy of pancreatic cancer cells by targeting PTEN gene. Moreover, the expression level of miR-21 in pancreatic cancer tissue is associated with the prognosis and survival rate of pancreatic cancer patients.Research has shown that, in some solid tumors such as colorectal cancer, ovarian cancer, breast cancer, non-small cell lung cancer, prostate cancer and pancreatic cancer, peripheral blood miRNAs can be used as potential diagnostic or prognostic markers and can be used to determine the origin of tumor. Abnormal expression of miRNAs in plasma may reflect tumor progression and may be used for diagnosis because of its tumor-specific. But the diagnostic value of circulating miRNAs is not clear.About 10 species of miRNAs are high expressed in pancreatic cancer in the known human miRNA currently. Previous studies reported that high expression of miR-196a in plasma and pancreatic cancer tissue is associated with the poor prognosis of pancreatic cancer. However, the control mechanism and targeted gene of miR-196a in pancreatic cancer carcinogenesis is unclear.Based on the above situation, we produced the expression profiles of miRNAs in plasma of pancreatic cancer by miRNAs microarray. Further validation of these miRNAs differential expressed in pancreatic cancer plasma was carried out in pancreatic cancer cell lines and pancreatic cancer tissue. The relative abundances of miRNAs in pancreatic cancer and chronic panreatitis patients were detected to investigate whether plasma miRNAs can be used as diagnostic biomarkers for pancreatic cancer. Deep research of miR-196a was carried out to explore its role in the biological characteristics such as proliferation and apoptosis in pancreatic cancer cell by genetic intervention. This study included four parts, as described below.1. The expression profile of miRNAs in plasma of pancreatic cancer patients.Aim: To find the differentially expressed and pancreatic cancer-specific miRNAs in plasma of pancreatic cancer patients.Methods: Produced the expression profile of miRNAs in pancreatic cancer plasma by Agilent microarrays to find the the different miRNAs in plasma among the three groups (pancreatic cancer and chronic pancreatitis patients and healthy volunteers). All the targeted gene regulated by these miRNAs were acquired in the Sanger microRNA database for target gene Pathway analysis and GO analysis.Results: 29 plasma samples (9 cases of pancreatic cancer, 11 cases of chronic pancreatitis and 9 healthy volunteers) were carried out miRNAs microarray analysis and screened 146 differentially expressed miRNAs in three groups. 31 miRNAs were selected after further trend analysis. GO analysis and Pathway Analysis showed that target genes regulated by these selected miRNAs significantly associated with cancer signaling pathways, such as Pathways in cancer, MAPK signaling pathway, Focal adhesion, Wnt signaling pathway and Pancreatic cancer and many other signaling pathways. These selected miRNAs were pancreatic cancer associated.Conclusion: Differentially expressed and pancreatic cancer-specific miRNAs in plasma screened by microarray analysis could be obtained by trend analysis of gene expression and functional analysis.2. The expression level of miRNAs in pancreatic cancer cell lines and pancreatic cancer tissuesAim: To verify the relative abundances of those miRNAs screened by microarray in pancreatic cancer cell lines and in pancreatic cancer tissues and confirm the correlations between those miRNAs and pancreatic cancer.Methods: The RNA of pancreatic cancer cell lines and fresh specimens after pancreatic surgery (paired cancer tissues and adjacent tissues) were extracted for further study. The relative abundances of 17 miRNAs (miR-16, miR-155, miR -181a, miR-181b, miR-196a, miR-21, miR-210, miR-20a, miR-26a, miR-142-3p, miR-223, miR-939, miR-1202, miR-1207-5p , miR-1228, miR-1825, miR-1915) in pancreatic cancer cell lines, pancreatic cancer tissues and adjacent tissues were detected by Real-time PCR. The relationship between the expression level of miRNAs and the clinical features of pancreatic cancer patients was analyzed. Results: Compared with normal pancreatic tissue, miR-155, miR-196a, miR-1202, miR-1207-5p, miR-1228, miR-1825, and miR-1915 were high expressed in SWl990, PaTu8988s, ASPC-1, BxPC-3, CFPAC-1, CPANC-1, and PANC-l pancreatic cancer cell lines. And all 17 miRNAs were significantly overexpressed in PaTu8988s cell line (P<0.05). The expression level of miR-16, miR-155, miR-181a, miR-181b, miR-196a, miR-21, miR-210, and miR-142-3p was significantly higher in pancreatic cancer tissues than in adjacent tissues (P<0.05), while miR-223 and miR-939 were significantly lower expressed in pancreatic cancer tissues than in the adjacent tissues (P<0.05). MiR-155, miR-196a were significantly higher expressed in pancreatic cancer cell lines and pancreatic tissues. There was no correlation between the relative abundance of miRNAs in pancreatic cancer tissues and adjacent tissues with pancreatic cancer patients'gender, age and the level of serum CA19-9. There was a significant positive correlation between the expression level of miR-26a in pancreatic cancer tissues and the maximum tumor diameter of pancreatic tumor. While the expression level of miR-223 was significantly negatively correlated with the maximum tumor diameter. The expression level of miR-196a, miR-142-3p, and miR-1228 in cancer tissues was significant positive correlated with TNM staging (P<0.05). The expression level of miR-223 in cancer-adjacent tissues was significantly negatively correlated with the maximum tumor diameter. And the expression level of miR-181a, miR-21, miR-210, miR-20a, miR-939, miR-1202, miR-1207-5p, miR-1228, miR-1825, and miR-1915 in cancer-adjacent tissues was significantly positive correlated with TNM staging (P <0.05).Conclusions: MiRNAs in plasma screened by microarray were also highly expressed in pancreatic cancer cell lines and tumor tissues. The expression level of miRNAs in pancreatic cancer tissues and adjacent tissues was no significant correlation with patients'age, sex, and serum CA19-9. MiR-223 expression level was negatively correlated with the maximum tumor diameter and tumor stage, which suggested that miRNAs may change with the occurrence and progress of pancreatic cancer.3. Combined diagnostic value of plasma miRNAs in pancreatic cancerAim: To assess diagnostic value of the relative expression of miRNAs in plasma in pancreatic cancer.Methods: 140 pancreatic cancer patients, 111 chronic pancreatitis (CP) patients, and 68 normal controls were enrolled. RNA was extracted from the plasma and seven miRNAs (miR-16, 21, 155, 181a, 181b, 196a, 210) were measured by real-time PCR. The diagnostic utility and inter-relationships between the miRNAs in PCa patients were analyzed.Results: The relative abundances (RAs) of all 7 miRNAs were significantly higher in the PCa group compared with CP and normal groups (P<0.001). However, only miR-16 and miR-196a independently discriminated PCa from normal and CP. Furthermore, when miRNAs were considered in addition to serum CA19-9, the diagnostic utility was significantly improved. To discriminate PCa from normal, the combination of miR-16, miR-196a and CA19-9 was superior with AUC-ROC = 0.979 (95% CI: 0.962 - 0.996) compared with CA19-19 alone (AUC-ROC = 0.903, 95% CI: 0.860 - 0.946) and the miRNA panel of miR-16 and miR-196a (AUC-ROC = 0.895, 95% CI: 0.850 - 0.939). The sensitivity and specificity of the combination were 92.0% and 95.6%, respectively. To discriminate PCa from CP, the combination of miR-16, miR-196a and CA19-9 also performed well with AUC-ROC = 0.956 (95% CI: 0.932 - 0.981) compared with CA19-19 alone (AUC-ROC = 0.897, 95% CI: 0.855 - 0.939) and the miRNA panel of miR-16 and miR-196a (AUC-ROC = 0.790, 95% CI: 0.735, - 0.845). The sensitivity and specificity of the combination were 88.4% and 96.3%, respectively. ROC analyses showed that using the miRNA panel in combination with CA19-9 could significantly improve the diagnostic value of CA19-9 alone in discriminating PCa from CP and normal. Importantly, in stage 1 PCa, the sensitivity of the combination of the miRNA panel and CA19-9 achieved 85.2%, which was significantly higher than CA19-9 alone.Conclusions: Plasma miRNAs are new biomarkers for PCa diagnosis. Plasma miR-16 and miRNA-196a combinated with serum CA19-9 could enhance the diagnostic sensitivity for early-stage PCa.4. The regulatory mechanism of miR-196a in pancreatic cancer by genetic interventionAim: To explore the role of miR-196a on the biological behavior of pancreatic cancer cell lines and regulatory mechanism in pancreatic cancer.Methods: To establish two pancreatic cancer cell lines with high and low expression of miR-196a by miRNA precursors and inhibitors transient transfection and lentivirus transfection in PANC-1 cell line. And investigate the role of miR-196a in pancreatic cancer cell proliferation and apoptosis by quantitative PCR, Western Blot, cell proliferation assays, and flow cytometry. Meanwhile, the regulatory mechanism of HOXB8 targeted by miR-196a was analyzed.Results: HOXB8 mRNA expression and protein expression in PANC-1 cell line transfected with miR-196a inhibitor was significantly higher than those transfected with miR-196a precursor (P <0.05). MiR-196a precursor transfection could enhance the proliferation ability of pancreatic cancer cell than miR-196a inhibitor transiently transfection and normal control. The apoptosis rate of pancreatic cancer cell line in miR-196a inhibitor group was significantly higher than that in miR-196a precursor group (P <0.05). And the invasive ability of pancreatic cancer cell line in miR-196a inhibitor group was significantly higher than that in miR-196a precursor group.Conclusion: MiR-196a can promote the proliferation of cell line and inhibit the apoptosis of pancreatic cancer cell lines. And miR-196a could regulate its targeted gene HOXB8.Based on the above experiments, the final conclusions were as followings:1. Differentially expressed and pancreatic cancer-specific miRNAs in plasma screened by microarray analysis could be obtained by trend analysis of gene expression and functional analysis.2. The miRNAs screened by microarray were pancreatic cancer specific. The expression level of miRNAs in pancreatic cancer tissues and adjacent tissues was no significant correlation with patients'age, sex, and serum CA19-9 level.3. Plasma miRNAs could be biomarkers for pancreatic cancer diagnosis. The combination of plasma miRNAs (miR-16 and miR-196a) and serum CA19-9 could significanctly enhance the diagnosis sensitivity of early stage pancreatic cancer.4. MiR-196a can promote the proliferation of cell line and inhibit the apoptosis of pancreatic cancer cell lines. And miR-196a could regulate its targeted gene HOXB8.In summary, new pancreatic cancer-related miRNAs in plasma could be found by microarray screening and validation in large sample. MiRNAs in plasma could be used as a new molecular marker for early diagnosis of pancreatic cancer, and miRNAs may participate in the regulation of the biological behavior of pancreatic cancer cells.
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