Post-transcriptional Regulation Of DPC4/SMAD4 Expression By MicroRNA And Its Molecular Mechanism

Pancreatic cancer is an aggressive malignancy with one of the worst mortality. Despite the extensive research efforts, the prognosis for pancreatic cancer is the worst among all cancers due to minimal improvement in its prevention and treatment and the 5-year relative survival rate is only 5% for all stages of this disease over the last decades. Therefore, the quest for new associated factors and novel therapeutic targets for pancreatic cancer remains an imperative clinical issue.In recent years, there have been important advances in understanding of the molecular biology of pancreatic cancer and genetic analysis showed that the genetic basis of this dismal disease is extremely complex and heterogeneous. It has been suggested that pancreatic cancer is associated with the successive accumulation of gene mutations such as oncogene KRAS2 and tumor-suppressor genes INK4A, TP53 and DPC4/Smad4.Recently, microRNAs (miRNA) have emerged as a critical class of negative regulators of gene expression. miRNAs are endogenous small 21-23 nucleotide noncoding RNAs that possess remarkable evolutionary conservation. These small molecules elicit their regulatory effects by base-pairing to partially complementary mRNAs and function by at least two mechanisms: degradation of target mRNA transcripts and inhibition of mRNA translation. Emerging evidence indicates that miRNAs play critical roles in the regulation of various biological and pathologic processes, involving cell proliferation, differentiation, apoptosis, and stress resistance. More importantly, it has been suggested that the development and progression of cancer are associated with aberrant upregulation or downregulation of specific miRNAs and their targets in various types of cancer. And certain cancer histotypes can be classified based on miRNA expression profiles. In pancreatic cancer a multitude of misexpressed miRNAs have been reported, such as'onco(genic)-miRs', miR-21 and miR-155, and'tumor suppressor miRs', miR-29b and the miR-34 and let-7 families. Moreover, it was recently found that miR-96 is a tumor-suppressor gene in pancreatic cancer by repressing KRAS expression. However, it is less clear whether there is any other miRNA targeting these key regulatory genes in pancreatic cancer.In this study, using a miRNA array-based differential analysis, we explore the function and mechanism of miRNAs relate in the post transcriptional gene regulation of pancreatic cancerr-specific tumor suppressor gene DPC4/Smad4 to find a new target point for the treatment of pancreatic cancer by a variety of research methods.一,Screening and identification of differential expression of miRNAs in pancreatic cancerTo search for miRNAs which may be involved in the development of pancreatic cancer especially among Chinese patients, we collected three different human clinical specimens and then used miRCURY LNA? miRNA-array analysis to screen differentially expressed miRNAs between pancreatic cancer tissues and respective side normal tissues. 88 miRNAs are overexpressed in pancreatic cancer, such as miR-21,135b,199a-3p,221,222,29a,31, whereas 101 miRNAs are down-regulated,such as miR-126,103,132,141,148a.We next use q-PCR methods to validate that The most significantly overexpressed miRNA in pancreatic were miR-762, miR-17-92 cluster, miR-106-363 cluster and miR-23a/b; whereas miR-200c, miR-486, miR-199a, miR-143, miR-145, miR-125b, miR-31, miR-26,miR-100 and let-7 were among the most down-modulated miRNA.二,The expression and function of microRNA 483-3p in pancreatic cancermiRNAs elicit their regulatory effects by base-pairing to partially complementary mRNAs and function by at least two mechanisms: degradation of target mRNA transcripts and inhibition of mRNA translation. It has been suggested that pancreatic cancer is associated with the successive accumulation of gene mutations. DPC4/Smad4, a critical effector in TGF-βsignaling pathway, was a regulatory factor in pancreatic cancer progression and approximately 55% cases of pancreatic adenocarcinomas involve inactivation or lost expression of the DPC4/Smad4 gene .To explore the mechanism(s) by which miR-483-3p executes its function in pancreatic cancer, we firstly applied common used bioinformatic algorithms (TargetScan, PicTar and miRanda) to identify its potential target genes. Among these many candidates, DPC4/Smad4 was selected for further analysis. DPC4/Smad4, a critical effector in TGF-β signaling pathway, was a regulatory factor in pancreatic cancer progression and approximately 55% cases of pancreatic adenocarcinomas involve inactivation or lost expression of the DPC4/Smad4 gene.To further confirm the miRNA array results, Real-time PCR analysis showed that miR-483-3p was increased significantly in cancer tissues compared with matched side tissues, suggesting that miR-483-3p may be a novel factor associated with the development of pancreatic cancer.we next tried to examine whether miR-483-3p has oncogenic functions in pancreatic cancer cells. The MTT proliferation assay showed that miR-483-3p mimics significantly promoted the proliferation of both SW1990 and PANC1 cells. Consistent with that, the colony formation analysis also showed that miR-483-3p stimulated SW1990 and PANC1 cells to grow more and larger colonies. Conversely, anti-miR-483-3p transfection in SW1990 and PANC1 cells significantly reduced cell proliferation and resulted in fewer SW1990 or PANC1 colonies. These results suggest that miR-483-3p promotes the proliferation and anchorage-independent growth of pancreatic cancer cells in vitro.We overexpressed miR-483-3p mimics in SW1990 cells and Western blot assays, and they indicated that the level of DPC4/Smad4 protein was reduced significantly by miR-483-3p overexpression compared with control treatment. The same results were observed using another human pancreatic cancer cell line PANC1. As Smad4 is the common-Smad protein for the transduction of TGF-βand BMP signals, the repression of DPC4/Smad4 by miR-483-3p should impair these two signaling pathways. Next, we examined the effect of miR-483-3p on the expression of these pathways'transcriptional targets in PANC1 cells. Real-time PCR analysis showed that the expression levels of TGF-βsignaling target genes PAI and p21, as well as BMP signaling targets Msx2, were suppressed by miR-483-3p overexpression, suggesting that miR-483-3p can regulate DPC4/Smad4-regulated signaling pathways. It is known that DPC4/Smad4 is involved in TGF-β-induced epithelial-mesenchymal transition (EMT) , and that pancreatic cancers with wild type DPC4/Smad4 show frequent EMT. To test the possibility that miR-483-3p could regulate the DPC4/Smad4-mediated EMT process, we transfected PANC1 cells with miR-483-3p mimics or scramble oligos and simultaneously treated them with TGF-β. Western blot analysis showed that compared with TGF-βtreatment alone, transfection of miR-483-3p mimics increased E-cadherin levels while decreasing fibronectin expression, suggesting that miR-483-3p could antagonize DPC4/Smad4-mediated TGF-βinduction of EMT. Taken together, these data suggest that miR-483-3p may exert its biological function through regulating the expression of its direct target DPC4/Smad4.To test the hypothesis that DPC4/Smad4 might be a target of miR-483-3p, we constructed three reporters by putting the wild-type fragments covering position 1-2050, 2201-4105 or 4200-6000 from the 3'-UTR region of DPC4/Smad4 to the downstream of the luciferase coding region. Oligonucleotides of miR-483-3p mimics or scramble were cotransfected with these reporter plasmids into human pancreatic cancer cells SW1990 and PANC1. Results showed higher luciferase activities than the respective wild-type ones, suggesting that they are indeed regulated by endogenous miR-483-3p. Moreover, transfection of miR-483-3p mimics had no interference with activities of the two reporters. These results suggest that DPC4/Smad4 may be a target of miR-483-3p for its regulatory function in pancreatic cancer.Taken together, our results suggest that the upregulated miR-483-3p targets tumor-suppressor gene DPC4/Smad4 in pancreatic cancer and that miR-483-3p may be a potential target for pancreatic cancer therapy.三,The expression and function of microRNA 421 in pancreatic cancerEach miRNA can have multiple target genes, while a number of miRNAs can also regulate the same gene. This complex regulatory network either through a miRNA to regulate the expression of multiple genes, miRNAs can also be a combination of several fine regulation of the expression of a gene. MiRNA regulation of gene expression with the gradual deepening of the research will help us understand the genome of higher eukaryotes, the complexity and complex regulation of gene expression networks.we have found that miR-483-3p is involved in pancreatic cancer by repressing DPC4/Smad4. However, it is still unknown that whether there is other miRNAs regulating DPC4/Smad4 expression in pancreatic cancer.In our previous miRNA array, we compared differentially expressed miRNAs between pancreatic cancer tissues and respective side normal tissues. We determined that miR-421, which had not been suggested to be involved in pancreatic cancer in previous studies, harbored four predicted binding sites in the 3'-UTR of DPC4/Smad4 mRNA and was aberrantly upregulated in malignant tissues. To further confirm this array result, Real-time PCR analysis showed that expression levels of miR-421 were increased significantly in cancer tissues compared with matched side tissues, while DPC4/Smad4 expression was pronouncedly inhibited in pancreatic cancer tissues.This finding led us to examine whether miR-421 overexpression could reduce endogenous DPC4/Smad4 protein levels in human pancreatic cancer cell lines. We ectopically expressed miR-421 mimics in SW1990 cells.Using Western blot assays, we found that the level of DPC4/Smad4 protein was reduced significantly by miR-421 overexpression compared with control treatment. The same results were observed in another human pancreatic cancer cell line PANC1. As DPC4/Smad4 is the common Smad protein for the transduction of TGF-βand BMP signals, the repression of DPC4/Smad4 by miR-421 should impair activation of downstream target genes of the two signaling pathways. We then examined the effect of miR-421 on the expression of these pathway's transcriptional targets in PANC1 cells. Real-time PCR analysis showed that the expression levels of the TGF-βtarget genes p21and p15, as well as the BMP target gene Id3, were suppressed by miR-421 overexpression, suggesting that miR-421 can regulate DPC4/Smad4-mediated signaling pathways. Collectively, these data suggest that miR-421 indeed directly targets DPC4/Smad4 to exert its biological function in pancreatic cancer.We then tried to examine whether or not miR-421 has oncogenic functions in pancreatic cancer cells. The MTT proliferation assay showed that miR-421 mimics significantly promoted the proliferation of both SW1990 and PANC1 cells. Colony formation analysis consistently showed that miR-421 increased the quantity and size of SW1990 and PANC1 cell colonies. Conversely, the miR-421 inhibitor significantly reduced cell proliferation and resulted in fewer SW1990 and PANC1 colonies. These results suggest that miR-421 promotes the proliferation and anchorage-independent growth of pancreatic cancer cells in vitro.To test the above hypothesis, we sought to confirm the possible regulation of DPC4/Smad4 by miRNA-421. Based on the bioinformatics prediction that there are four potential positions, 438-444, 722-728, 2799-2785 and 5252-5258, in the 3'-UTR of DPC4/Smad4 mRNA targeted by miRNA-421, Mimics or scramble oligonucleotides of miR-421 were cotransfected with these reporter plasmids into the human pancreatic cancer cell lines SW1990 and PANC1, both of which contain the wild-type DPC4/Smad4 gene. Remarkably, luciferase analyses showed that miR-421 mimics repressed the activities of pGL3-UTR1-WT, pGL3-UTR2-WT and pGL3-UTR3-WT, all of which harbor miR-421 binding sites. In contrast, pGL3-UTR1-MUT, pGL3-UTR2-MUT and pGL3-UTR3-MUT, in which the putative binding sites of miR-421 are mutated, showed higher luciferase activities than their respective wild-type constructs, suggesting that they are indeed regulated by endogenous miR-421. Moreover, transfection of miR-421 mimics had no suppression or activation of the three reporters. These results suggest that DPC4/Smad4 may targeted by miR-421 for their regulatory functions in pancreatic cancer.In continuation of our previous study, we found that DPC4/Smad4 is regulated by miR-421 in pancreatic tumors and their expression levels are inversely correlated. There are four predicted binding sites of miR-421 in the 3'-UTR of DPC4/Smad4, and ectopic expression of miR-421 represses DPC4/Smad4 protein level. Besides, its overexpression inhibits downstream targets of DPC4/Smad4, such as p21, p15 and Id3 . Altogether, miR-421 may be used as a novel biomarker and therapeutic target for pancreatic and other human adult cancers.