Preliminary Study Of Bta-miR-145Function In Bovine Mammary Epithelial Cells

Mammary gland is the only organ undergoes proliferation and differentiation, apoptosis in adultanimal. Research on bovine mammary development and lactation mechanism is very important forimproving production performance and milk quality of dairy cows. MicroRNAs are a class of shortnon-coding RNAs, which regulate gene expression at post-transcription level. Study on the regulation ofbovine mammary gland development and lactation mechanism from the perspective of microRNA is arelatively new field. This experiment selected bta-miR-145as research object, which was differentiallyexpressed before and after parturition of dairy cows. By bioinformatics analysis, references comparison,3’RACE sequencing, cells transfection, RT-qPCR, Western Blotting, and dual fluorescence analysis,preliminary study of bta-miR-145function was carried out in bovine mammary epithelial cells. Themain research contents are as follows:1. In order to dig more information related to bta-miR-145, bioinformatics analysis of bta-miR-145was made. Phylogenetic analys is of bta-mir-145was performed by using MEGA5.05software.MicroRNA-145of cow had the closer homologous relationship with pig and human. Validated targets ofmiR-145were collected by miR TarBase database. It was found that miR-145targets IRS1in humanand mouse. Six hundred and forty eight targets of bta-miR-145predicted by Target Scan6.2were putinto DAVID database for analysis of KEGG signal pathway. And most of target genes were located inMAPK s ignaling pathway, including IRS1. Several softwares predicted miR-145targeting IRS1,including PicTar, miRanda, miRBase and miRGen. And Target Scan6.2predicted bta-miR-145targeting CSN2. For binding sites of IRS1and CSN2for bta-miR-145, the minimum free energy (MFE)was analyzed through RNAhybrid database. Values of MFE for biding sites were all less than-15kcal/mol. These results suggested that bta-miR-145might target IRS1and CSN2, and regulate MAPKsignaling pathway.2. In order to further determine the target relationship between IRS1/CSN2and bta-miR-145,3’UTR sequence of two genes was analyzed in NCBI database and Ensemble database. Results showedthat bovine IRS1is a predicted gene. Thus,3’ rapid amplification of cDNA end (RACE) of bovine IRS1was carried out and amplified fragments were spliced by Sequencher software. Further analysis showedthat the probable binding site for bta-miR-145remained on the3’UTR of IRS1. There were somedifferences in CSN23’UTR sequence in two databases. But the predicted binding site of CSN2forbta-miR-145all existed in both databases.3. In order to obtain experimental model of this study, highly proliferative primary bovinemammary epithelial cells were successfully cultured by mechanical crushing method. Then, the cellswere purified. The expression of cytokeratin-18was detectable in the purified cells and the cells oftwenty passages after purification. Different hormones and cytokines were used to induce mammaryepithelial cells. It was found that100ng/mL IGF-1significantly increased gene expression of betacasein in mammary epithelial cells. These provided a cell model for subsequent experiment. 4. In order to explore the optimal transfection conditions, inoculation density of cells, adding doseof RNAi and liposome, and over-expression and down-expression of bta-miR-145were studied. Resultsshowed that5×104-1×105/mL of cell density could meet the requirement of50%of confluent rate.Combination of RNAi and liposome2000(30)(1.5)(24hole plate) could achieve the optimaltransfection efficiency. And150p mol/hole mimic could achieve the effect of bta-miR-145over-expression, and300p mol/hole inhibitor could restrain the expression of bta-miR-145.(6holeplate).5. In order to study the effect of bta-miR-145on proliferation of bovine mammary epithelial cells,bovine mammary epithelial cells were cultured in96well plates, and were detected after48h by CCK-8test kit. Results showed that bta-miR-145had a regulatory effect on the growth of bovine mammaryepithelial cells.6. In order to study whether over-expression or down-expression of bta-miR-145could influencethe expression of other microRNAs or not, bta-miR-214, bta-miR-181a and bta-miR-21was detected byRT-qPCR. It was found that bta-miR-181a had the same trend with bta-miR-145; bta-miR-214showedthe opposite trend with bta-miR-145; and bta-miR-21was not affected by bta-miR-145over-expressionor down-expression.7. To investigate the effect of bta-miR-145on gene and protein expression of IRS1and CSN2,bta-miR-145mimic or inhibitor was transfected into bovine mammary epithelial cells. Expressionchanges of mRNA and protein of IRS1and CSN2were detected by RT-qPCR and Western blotting.Results showed that bta-miR-145affected protein expression of bovine IRS1and CSN2, but not theirmRNA expression.8. To study whether there were interactions between bta-miR-145and IRS1/CSN23’UTR,recombinant vectors containing IRS13’UTR and CSN23’ UTR, respectively, were constructed by dualfluorescence reporter vector pmiR-RB-REPORTTM. Recombinant plasmid was transfected into293Tcells with bta-miR145mimic. The results showed that bta-miR-145mimic did not lead to obviousreductions of dual fluorescence ratio of IRS1and CSN2, compared with cel-miR-67. Therefore,bta-miR-145had no obvious interactions with IRS1/CSN23’UTR.In conclusion, bta-miR-145had a certain regulation effect on bovine mammary development andlactation. It could adjust bovine mammary epithelial cell proliferation, and regulate IRS1and CSN2protein expression, but not their gene expression. Expression change of a kind of microRNA couldcause changes of a series of microRNAs. The view of systems biology should be adopted to explain therelated biological phenomenon.