MicroRNA-1285 Targets YAP1 And Inhibits Cell Growth, G1/S Transition, Chemoresistance, Migration And Invasion, As Well As Promotes Apoptosis In Human Pancreatic Cancer Cells

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths in the world, with a 5-year survival rate less than 7%. Owing to a lack of specific symptoms and no accessible precursor lesions, primary diagnosis is commonly delayed, resulting in only 15%-20% of patients with potentially curable disease. MiRNAs are-21-23-nucleotide endogenous single-stranded RNAs. Recent work supports a role for microRNAs in the initiation and progression of human malignancies. It is well established that miR-1285 can function as tumor suppressors in hepatocellular carcinoma and renal carcinoma, while its roles in pancreatic cancer is unclear. In this research, we will mainly focus on the biological functions of miR-1285 as well as searching for its target genes.The transcriptional co-activator Yes-associated protein 1(YAP1) is a major effect factor of Hippo signal pathway, which takes an important part in organ development and tumorigenesis.Objective1. To investigate the impact of miR-1285 on the biological functions of pancreatic cancer cells.2. To identify the target genes.3. To investigate the downstream pathways.MethodsHuman pancreatic cancer cell lines T3M4 and SU86.86 were cultured in DMEM-high glucose and RPMI-1640 with FBS, respectively. We examined miR-1285 levels in each cell lines by Real Time PCR (qRT-PCR). Transfection was accomplished by oligonucleotides with Lipofectamine 2000. Cell proliferation and chemosensitivity were evaluated by CCK-8 assay 24 hours after transfection. Cell apoptosis and cell cycle analysis were performed by flow-cytometer 48 hours after transfection. Cell migration and invasion were tested by transwell assay 24 hours after transfection. Target mRNAs were predicted by public databases. Then, we extracted total RNA and protein 48 hours after transfection, with TRIZOL assay and RIPA Lysis Buffer, respectively. YAP1 mRNA level was examined by Real Time PCR (qRT-PCR) while protein levels were detected by western blot.Graphpad Prism 6 was used for statistical analysis.Results1. MiR-1285 was down-regulated in the Gemcitabine-resistant cell line.2. Compared with the negative control, down-regulate the expression level of miR-1285 in SU86.86 cells significantly promoted the proliferation, G1-S transition, migration and invasion, while inhibited the chemosensitivity to Gemcitabine and cell apoptosis in vitro. Up-regulate miR-1285 level in T3M4 cells acquired the opposite result.3. MiR-1285 decreased the protein level of YAP1 in both T3M4 and SU86.86 cells, while didn’t change the mRNA levels.4. Western blot shows that EGFR,β-catenin, Bim and PTEN level are changed according to the levels of miR-1285 and YAP1.ConclusionsMiR-1285 functions as a tumor suppressor in pancreatic cancer by targeting YAP1, probably by attenuating its translation, which may further regulate downstream proteins such as EGFR, β-catenin, Bim and PTEN.