| 【Background】Pancreatic cancer (PC) is a highly malignant tumor type，and pancreatic ductaladenocarcinoma (PDAC) is the most common type of PC, involving more than90%ofPCs. The vast majority of PDAC patients can not been performed radical surgery becauseof metastasis and/or invasion of other organs when they were diagnosed. In addition,PDAC is not sensitive to radiotherapy and chemotherapy. Therefore, PDAC has anextremely poor prognosis, its median survival time is less than6months and its5-yearsurvival rate does not exceed5%. Its incidence and mortality is increasing year by year inChina and Western countries. With the rapid development of biotechnology, themolecular biology research of PDAC is important to clarify its pathogenesis, improve theprognosis of patients.MicroRNAs (miRNAs) are a class of small non-coding RNAs, rangingapproximately from17to25nucleotides, which regulate gene expression via complementarity with the3’-untranslated region (3’-UTR) of their target mRNAs. Thus,miRNAs regulate gene expression either by target mRNA degradation or repression of itstranslation into protein. Accumulating studies have shown that miRNAs play animportant role in many biological processes and dysregulation of miRNAs is linked to avariety of diseases. In the field of cancer research, miRNAs have tissue-specific andinvolved in tumorigenesis, progression, metabolism, drug resistance, etc. They can act aseither tumor suppressors or oncogenes via the down-regulation of oncogenes orup-regulation of tumor suppressors. Research on miRNAs in PDAC includes the cellproliferation, apoptosis, differentiation, invasion and metastasis, and drug resistance, etc.Through the analysis of PDAC cell lines, clinical PDAC samples, plasma and animalmodels, it has been found that, relative to normal control, some miRNAs is upregulatedin PDAC, such as miR-21，miR-155，miR-10b，miR-196，miR-221，miR-301，miR-95etc., while some is downregulated, including miR-217，miR-216，miR-375，miR-203andso on. These dysregulated miRNAs’ target genes include KRAS，PTEN，PDCD4，ZEB1，BCL-2，TIMP3and so on. But there are still a lot of unclarified issues in this field.Building the regulatory networks of miRNAs and its target genes and their mechanismsin PDAC need further research. miR-135a has recently been reported in several types oftumors, which suggested that it could inhibit cancer cell proliferation, promote cancercell apoptosis. However, no studies have been performed to assess the significance ofmiR-135a in PDAC.Bmi1(B-ceIl-specific Moloney murine leukemia virus integration site1), belongs topolycomb gene family, plays a critical role in cell cycle regulation, cell immortalization,and senescence. In recent years, numerous studies have showed that Bmi1is involved inthe regulation of self-renewal and differentiation of stem cells. Additional, Bmi1isgenerally considered as an important oncogene. Dysregulated expression of Bmi1isrelated to the tumorigenesis and progression of a variety of tumors such as lung cancer,breast cancer, leukemia, lymphoma, hepatocellular carcinoma and PDAC. Bybioinformatics analysis, we speculated that Bmi1may be one of the target genes ofmiR-135a in PDAC. However，it is unclear that whether miR-135a is involved in PDAC proliferation andapoptosis through targeting Bmi1, and whether therapeutic intervention on miR-135aexpression could effectively reverse the proliferation phenotype of PDAC cells.Therefore, further study is need to be explored the function and mechanism of miR-135ain tumorigenesis and progression of PDAC.【Objectives】To investigate aberrantly expressed miRNAs involved in PDAC by comparingmiRNA expression profiles in PDAC cell lines with a normal pancreas cell line usingmicroarrays. To determine the function and mechanism of miR-135a in tumorigenesisand progression of PDAC, and offer a new tools for the early diagnosis and treatment ofPDAC.【Methods】1．Agilent Human microRNA array kit (V18.0) was used to investigate aberrantlyexpressed miRNAs involved in PDAC by comparing miRNA expression profiles in3PDAC cell lines（PANC-1, BxPC-3and ASPC-1）with a normal pancreas cell line（HPDE6c7）. The microarray results were validated by qRT-PCR in PDAC tissues,paired adjacent normal pancreatic tissues, PDAC cell lines, and a normal pancreas cellline. Among these dysregulated miRNAs, we chose miR-135a for further investigation.2．The miR-135a precursor was cloned to lentivirus vectors and was infected intoPDAC cancer cells to build stable expressed cell lines. CCK-8assay, colony formationassay, Edu incorporation assay and flow cytometry were used to analyze the effect ofmiR-135a on PDAC cell proliferative ability in vitro. Transwell assay and Matrigel assaywere used to analyze the effect of miR-135a on PDAC cell invasion and migration abilityin vitro. Subcutaneous tumor formative assay in nude mice was used to examine theeffect of miR-135a on proliferative ability in vivo.3．To screen the potential target genes of miR-135a by utilizing several predictiontools: miRanda, Pita and RNAhybrid. After a comprehensive analysis, we chose Bmi1from predicted target genes for further study. qRT-PCR and western blotting were used toexamine the mRNA and protein expression of Bmi1respectively in PDAC cells which were transfected with miR-135a mimics or inhibitor. Luciferase activity assay was usedto verify miR-135a directly regulated Bmi1expression. Western blotting analysis wasperformed to detect cell cycle-and survival-related proteins in transfected cells andcontrol cells.【Results】1．The microarray results revealed that the expression of66miRNAs significantlydiffered between the PDAC and normal pancreatic ductal epithelial cells. Compared tothe normal pancreatic ductal epithelial cells,23miRNAs were significantlyoverexpressed (fold change>2), whereas43miRNAs were significantly underexpressed(fold change <0.5). Some of the significantly dysregulated miRNAs were analyzed byhierarchical clustering. Of the dysregulated miRNAs, miR-205-3p, miR-203, miR-630,and miR-135a were the most up-regulated miRNAs in the normal pancreatic ductalepithelial cells, whereas miR-193a-5p, miR-10a-5p, miR-10b-5p, and miR-301a-3p werenoted for the largest decrease in expression. miR-135a has recently been reported inseveral types of tumors, such as renal cell carcinoma, gastric cancer, lung cancer,Hodgkin lymphoma, and colorectal cancer, suggesting that the dysregulation ofmiR-135a may play an essential role in tumorigenesis. However, no studies have beenperformed to assess the significance of miR-135a in PDAC. Therefore, we chosemiR-135a for further investigation. Consistent with the microarray data, miR-135aexpression was down-regulated in all three human PDAC cell lines (PANC-1, BxPC-3,and ASPC-1) compared to the normal pancreatic ductal epithelial cell line HPDE6c7(P<0.01). We also found that the miR-135a levels were significantly decreased in9ofthe11clinical PDAC tissues relative to the adjacent nontumorous pancreas tissues2．qRT-PCR analysis demonstrated that the lentivirus vectors construction andinfection was effective. CCK-8assays, colony formation and EdU cell proliferationassays showed that the enforced expression of miR-135a resulted in a decrease in cellgrowth of the PANC-1and ASPC-1. Cell cycle assay showed that miR-135a transfectionincreased the percentage of cells in the G0G1phase compared to the negative control inPDAC cells. Cell apoptosis analyses demonstrated that miR-135a overexpression increased the number of apoptotic cells in PDAC cells. Furthermore, miR-135aoverexpression decreased the tumorigenesis ability in vivo.3．Bioinformatics predicted that Bmi1might be a target of miR-135a. We measuredthe Bmi1proterin levels in PDAC tissues and cell lines, the results showed that Bmi1expression were inversely associated with that of miR-135a. qRT-PCR and westernblotting analysis showed that miR-135a overexpression did not result in degradation ofBmi1mRNA but did drastically decrease its protein levels. Luciferase activity assayindicated that miR-135a overexpression suppressed the expression of luciferase from avector containing the wild-type miR-135a binding site, but had no effect on the mutantand negative control vectors.4．To further investigate the mechanism by which miR-135a inhibits PDAC growth,we examined several related molecules downstream of Bmi1. Our western blotting datademonstrated that the overexpression of miR-135a significantly reduced the expressionof cyclin D1, Cdk2, and Cdk4in PANC-1and ASPC-1cells; in contrast, the expressionof cyclin-dependent kinase inhibitor (p21) was up-regulated. Moreover, co-transfectionwith the miR-135a mimics and pcDNA-Bmi1(mutant3’-UTR for miR-135a) increasedthe expression of cyclin D1, Ckd2, Cdk4and suppressed that of p21at protein levels. Inaddition, following the restoration of miR-135a in PANC-1and ASPC-1cells, theexpression of phospho-Akt and Bcl-2was inhibited, whereas the level of Bax waselevated. But the expression levels of phospho-Akt and Bcl-2proteins were increasedand that of Bax was suppressed after co-transfection with miR-135a mimics andpcDNA-Bmi1(mutant3’-UTR for miR-135a).【Conclusion】1． High-throughput microassay and qRT-PCR verified that miR-135a wasdownregulated in PDAC cell lines and tissues, which indicated that miR-135a acts astumor suppressors in PDAC.2．The overexpression of miR-135a in PDAC cells could inhibit PDAC cellproliferation and also induced G0G1arrest and apoptosis. These data indicated thatmiR-135a plays an important role in tumorigenesis and progression of PDAC. 3．miR-135a inhibits the proliferation of PDAC cells, at least in part via theregulation of Bmi1. The identification of miR-135a as a regulator of BMI1may provideinsight for the establishment of new strategies and useful miRNA-based therapies for thetreatment of PDAC. |
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