Microrna-449b Reduces Methylation and Apoptosis-related Gene Expression in Fibroblast Cells Containing Human Insulin Gene

Background: MicroRNA-449b (miR-449b) is a ~22-base pair long nucleotide, that post-transcriptionally affects processes like proliferation, differentiation and apoptosis by directly targeting the HDAC and DNMTs family genes that regulate DNA methylation. Methods: This study aimed to investigate the effect of miR-449b mimic, inhibitor and scrambled sequence on buffalo fibroblast cells containing the human insulin (hINS) gene. A transgenic buffalo cell line containing human insulin gene (hINS) was treated with 40 nM miR-449b mimic and inhibitor by transfection method (lipofectamine-3000). Result: miR-449b mimic treated cells showed a significant decrease (P less than 0.05) in the expression of epigenetic-related genes and found a significant reduction (P less than 0.05) in the expression of HDAC1, DNMT3A and DNMT3B genes. In contrast, DNMT1 showed no significant change (P less than 0.05) in their expression level as compared to control whereas miR-449b inhibitor-treated cells found significantly increased (P less than 0.05) expression levels of HDAC1, DNMT 3A, DNMT 3B and DNMT1 genes as compared to control. In apoptosis-related genes, miR-449b mimic significantly increased (P less than 0.05) the expression of BCL-XL and MCL-1, while miR-449b inhibitor significantly increased (P less than 0.05) the expression of MCL-1 gene whereas there was no significant change (P less than 0.05) in the expression of BCL-XL. These findings indicate that treatment of miR-449b mimic on transgenic cells reduces the DNA methylation and apoptosis levels. This reduced methylation level helps in the nuclear reprogramming of transgenic cells. Thus, these cells can be a better choice as donor somatic cells for efficient handmade transgenic cloned embryo production.