Indentifcation Of The MiR-148a/b Targeted Gene-DNMT1 In Pancreatic Cancer Cell Line AsPC-1.

Objectiveusing dual fluorescent protein reporter assay system, and identify target gene of miR-148a/b in pancreatic cancer cell AsPC-1 was DNMT1. To provide the basis of theoretical support for the further research of the function of miR-148a/b in pancreatic cancer and pancreatic cancer pathogenesis.Methods1. Combination of bioinformatics and using a variety of target gene prediction software predicted target gene of miR-148a/b was DNMT1.2. Useing gene extracted from human whole blood as a template, amplification of pri-miR-148a/b sequence.by PCR which length of 225bp,279bp.Products were cloned into pcDNA3.1 (+) vector,and obtained pcDNA3.1/pri-miR-148a, pcDNA3.1/pri-miR-148b gene by restriction enzyme digestion and sequencing.3. Using the RT products of Hela cell RNA as template, DNMT1,mRNA-3'UTR area cloned by PCR amplification which length of 268bp and the pcDNA3/EGFP plasmid which expressing green fluorescence were connected. Through sequencing identification,obtained pcDNA3/EGFP/DNMT1 expression vector.4. A group(pcDNA3.1/pri-miR-148a) and B group(pcDNA3.1/pri-miR-148b) were cotransfected with pcDNA3/EGFP/DNMT1 into AsPC-1 cells of pancreatic cancer respectively.And added to the pDsRed-C1 0.1ug plasmid which expressing red fluorescent as internal reference.Also set C group (only transfection pcDNA3/EGFP/DNMT1) and D group (co-transfected pcDNA3/EGFP/DNMT1 and pcDNA3.1 (+)) as a control.Established three holes each group,and repeat 3 times, take the average of the 3 times experimental results as finally experimental results..After transfection,used fluorescent expression by fluorescencemicroscopy observated to identify the transfection rate of DNMT1,and used real-time quantitative fluorescent PCR to identify the transfection rate of pri-miR-148a, pri-miR-148b.After 48h, fluorescent protein expression were observed under a fluorescence microscope and fluorescence determined by fluorescence spectrophotometer.Results1-Expression vector pcDNA3.1/pri-miR-148a,pcDNA3.1/pri-miR-148b and pcDNA3/EGFP/DNMT1 was successfully constructed,sequencing verified the correct sequence.2-After transfection 48h, miR-148a expression of A group was significantly higher than D group (6.7569±0.3495:1.0047±0.5396, P<0.05),and miR-148b expression of B group was also significantly higher than D group ((3.2810±0.0802:1.0167±0.349, P<0.05).These prove that miR-148a/b successfully expressed by pcDNA3.1 (+) vector into the cells.3. After transfection 48h, fluorescent protein expression of A group and B group observed under a fluorescence microscope were significantly lower than C group and D group.Fluorescence expression of A group (1.1814±0.0733) and B group (1.3501±0.0669) were significantly lower than C group(1.9025±0.0507) and D group(1.9472±0.0445),which proved that fluorescent protein expression levels of A group and B group were lower than C group and D group,resulting in significant differences (P<0.05).Conclusions:miR-148a/b and DNMT1,mRNA 3'-UTR can complementary conbine in pancreatic cancer cells AsPC-1 by dual fluorescent protein reporter assay system, which prove that DNMT1 is a target gene of miR-148a/b.