Effects Of MiR-148b Silence With MicroRNA Sponge On The Aberrant Glycosylation IgA1 Molecule

Objective Glucocorticoids and immune inhibitors are the main drug of treatment of IgA nephropathy.These drugs have many side effects and poor treatment effect.The new medicine for precision treatment of IgA nephropathy is one of the main research direction in kidney disease.Studies of IgA nephropathy,both GWAS and non-coding RNA research,have won a major breakthrough.But there are few researches about these target-related gene therapy at present.The aim of this investigation is to design an an adenovirus sponge to diminish aberrant glycosylation in IgA nephropathy.Methods According to the nucleotide sequence of miR-148b which can be retrieved in miRbase database,we design an antisence sequence with a bulged site at the 9-12 position.The whole sequence of hsa-mir-148b-3p sponge with four repeated sequence was connected with the pMIR carrier and then transfect it into the pAD5/F35 carrier.The positive clone carrier was screened out and then transfected into the DAKIKI cell in vitro to study the mechanism of the Gd-IgAl molecule in the IgA nephropathy.The experiment was divided into three groups:control group,control-GFP group and miR-148b sponge group.Real-time PCR was used to detect the mRNA expression of C1GALT1 after Adenovirus transfection In 24,48,72 hours respectively.Western blot was used to detect the protein expression of C1GALT1 after Adenovirus transfection In 72 hours.ELISA was used to detect the Gd-IgA1 expression in the the supernatant of the cell.Results(1)The adenovirus sponge for miR-148b was successfully constructed and identified.(2)The high transfection efficiency was detected by fluorescence microscope after the miR-148b sponge transfected into DAKIKI cell.(3)The miR-148b expression level was diminished in the miR-148b sponge group Compared with the miR-148b scramble group.(4)The C1GALT1 protein expression level was up-regulated in the miR-148b sponge group Compared with the miR-148b scramble group.(5)The Gd-IgA1 expression level was down-regulated in the miR-148b sponge group Compared with the miR-148b scramble groupConclusions The miR-148b Sponge was constructed successfully.The miR-148b sponge can be used to diminish the aberrant glycosylation in IgA nephropathy and used as a new research tool to study the pathogenesis of IgA nephropathy.