The Expression And Function Of Hsa-miR-30b In Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of all cancers. Its characteristics of late diagnosis, highly aggressive and resistance to chemotherapy cause difficult clinical diagnosis and treatment. Although there are many studies focusing on the pathogenesis and treatment of PDAC for the past decades, the5-year survival rate is still less than5%. Less than20%patients are qualified for operation.Patients’response to either single or combination chemotherapy is poor. So it is of great significance to reveal the development mechanism and biomarkers in PDAC.MicroRNAs is a research hotspot in recent years, which were first identified as a key regulator in the developmental timing of Caenorhabditis elegans. The size of mature microRNAs ranges from18to25nucleotides, and was evolutionarily conserved in many organisms. MicroRNAs modulate the expression of genes at the post-transcriptional level through canonical base pairing between the seed sequence of the microRNAs (nucleotides2-8at its5’end) and its complementary seed match sequence (which is present in the3’UTR of target mRNAs). Mature microRNAs can be incorporated in the RNA-induced silencing complex (RISC) causing inhibition of translation or degradation of target mRNAs.More and more studies suggested that microRNAs play a critical role in tumorigenesis processes.Previously, we have studied the microRNAs expression in pancreatic cancer and found that miRNAs are differentially expressed in normal and tumor tissues.Using models in vitro and in vivo we uncovered microRNAs functions in pancreatic cancer. This study is to explore the function of miR-30b in pancreatic cancer based on our previous experimental results.Firstly,the level of miR-30b was detected using real-time quantitative PCR (qRT-PCR) in human pancreatic cancer cell lines PANC-1,MIA PaCa-2,BxPC-3and AsPC-1. The result showed that miR-30b was downregulated in all four human pancreatic cancer cell lines compared with normal pancreatic tissue sample.PANC-1and MIA PaCa-2had lower expression of miR-30b compared with BxPC-3and AsPC-l.So these two cell lines were selected as study models.The expression level of miR-30b was also validated in carcinoma and normal pancreatic tissue samples.The data from miRNA qRT-PCR analysis and results confirmed that miR-30b was downregulated in all the cancerous pancreatic tissues. The downregulation of miR-30b in pancreatic cancer suggested this microRNAs played a role as a tumor suppressor.Secondly,we then sought to determine whether downregulation of miR-30b has effect on the viability,cell cycle,migratory or invasion properties of pancreatic cancer cells.PANC-1and MIA PaCa-2cells were transfected with miR-30b mimics or microRNAs-negative control using lipofectamin-2000. Overexpression of miR-30b could be detected in cells transfected with miR-30b mimics.Cell Counting Kit-8(CCK-8) was used to examine the viability of pancreatic cancer cells(PANC-l and MIA PaCa-2).The proliferation assay showed that cell growth was reduced in PANC-1and MIA PaCa-2cells overexpressing of miR-30b compared with cells transfected with microRNAs-negative control.The soft agar colony formation experiment was performed to evaluate the growth capacity of pancreatic cancer cell lines (PANC-1and MIA PaCa-2). As expected,miR-30b-transfected cells displayed fewer and smaller colonies compared with microRNAs-negative control-transfected cells. EdU immunofluorescence assay was applied to PANC-1and MIA PaCa-2cells treated with miR-30b mimics or microRNAs-negative control. A significant decrease in cell proliferation was observed over time in PANC-1and MIA PaCa-2cells expressing miR-30b compared with cells expressing microRNAs-negative control.Fluorescence-activated cell sorting (FACS) analysis for apoptosis was conducted using Annexin V-FITC kit. The percentage of early apoptotic cells was increased in response to miR-30b transfection compared with microRNAs-negative control.Also FACS analysis revealed that reexpression of miR-30b leads to a significant increase in the number of cells in the GO/G1phase of the cell cycle whereas the S-phase population decreased,suggesting that miR-30b causes a GO/G1arrest in miR-30b-transfected PANC-1and MIA PaCa-2cells compared with a nonspecific mciroRNA-negative control.To determine whether miR-30b affects pancreatic cancer cells migration or invasiveness,wound healing and transwell invasion assays were conducted. miR-30b-overexpressing PANC-1and MIA PaCa-2cells showed a significant reduction of cell migration compared with microRNAs-negative control-transfected cells in closing an artificial wound created over a confluent monolayer. miR-30b overexpression also significantly reduced the invasiveness of pancreatic cancer cells.Thirdly, to fully understand the mechanisms by which miR-30b execute their biological function,we investigated the underlying molecular mechanism of miR-30b in pancreatic cancer.The target prediction programs TargetScan,PicTar,miRanda,RNA22,DIANA,miRDB and starBase were used to search for predicted direct target genes of miR-30b. Among the hundreds targets of miR-30b, KRAS gene was one of the earliest and most universal genetic alterations observed in pancreatic cancer which was predicted in multiple programs.For luciferase assays,wild-type or mutant KRAS3’UTR containing the miR-30b binding sites was cloned downstream the luciferase cDNA in the3’UTR/pmirGLO plasmid.Co-transfection of the luciferase reporter and miR-30b mimics into HEK293T cells produced lower luciferase activity than in cells co-transfected with the microRNAs-negative control. Mutation of the seed sequence in the miR-30b binding site rescued the luciferase activity. We next determined whether overexpression of miR-30b leads to downregulation of endogenous KRAS expression in human pancreatic cancer cells. Western blot assays showed reduced KRAS protein level in PANC-1and MIA PaCa-2cells overexpressing miR-30b compared with the control.Using dual luciferase reporter assay and western blot assays, we indicated that the oncogene KRAS was an evolutionarily conserved target of miR-30b.To determine whether KRAS is a functionally important target of miR-30b in pancreatic cancer, we further measured protein levels of downstream signaling pathways.Western blot analysis showed that miR-30b transfection downregulated the protein level of pMEK. These data indicated that miR-30b targets KRAS resulting in suppression of the RAS/RAF/MEK/ERK pathway.Finally,we carried out to determine whether microRNAs has cooperative effects on their target genes. Other studies have demonstrated the coordinate regulation of a single mRNA transcript by multiple microRNAs. In our previous study, miR-217was reported to suppress KRAS in pancreatic cancer. So we hypothesized that KRAS gene may be coordinately regulated by miR-30b and miR-217.To assess the combinatorial effect of multiple microRNAs sites in the KRAS3’UTR, a dual luciferase reporter containing a target site encompassing both active sections (miR-30b seed match sequence and miR-217seed match sequence) was created.Luciferase activity was measured in HEK293T cells. When both sites were combined, they conferred a somewhat greater repression than each site alone,suggesting that KRAS is regulated by the coordinate action of miR-30b and miR-217. The coordinate regulation was confirmed using the CCK-8proliferation assay.Taken together, our results indicated that miR-30b was downregulated in human pancreatic cancer. Reexpression of miR-30b leads to significant reduction in pancreatic cancer cells proliferation and clonal growth.Overexpression of miR-30b in pancreatic cancer cells results in induction of GO/G1cell-cycle arrest and apoptosis.miR-30b also inhibits tumor cells migration and invasion in pancreatic cancer. miR-30b may function as a tumor suppressor by targeting KRAS. miR-30b and miR-217may coordinately regulate KRAS gene in pancreatic cancer.