MiR-126-3p inhibits apoptosis and promotes proliferation by targeting phosphatidylinositol 3-kinase regulatory subunit 2 in porcine ovarian granulosa cells

Abstract

Objective: Numerous studies have indicated that the apoptosis and proliferation of granulosa cells (GCs) are closely related to the normal growth and development of follicles and ovaries. Previous evidence has suggested that miR-126-3p might get involved in the apoptosis and proliferation of GCs, and phosphatidylinositol 3-kinase regulatory subunit 2 (<i>PIK3R2</i>) gene has been predicted as one target of miR-126-3p. However, the molecular regulation of miR-126-3p on <i>PIK3R2</i> and the effects of <i>PIK3R2</i> on porcine GCs apoptosis and proliferation remain virtually unexplored.Methods: In this study, using porcine GCs as a cellular model, luciferase report assay, mutation and deletion were applied to verify the targeting relationship between miR-126-3p and <i>PIK3R2</i>. Annexin-V/PI staining and 5-ethynyl-2’-deoxyuridine assay were applied to explore the effect of <i>PIK3R2</i> on GCs apoptosis and proliferation, respectively. Real-time quantitative polymerase chain reaction and Western Blot were applied to explore the regulation of miR-126-3p on <i>PIK3R2</i> expression.Results: We found that miR-126-3p targeted at <i>PIK3R2</i> and inhibited its mRNA and protein expression. Knockdown of <i>PIK3R2</i> significantly inhibited the apoptosis and promoted the proliferation of porcine GCs, and significantly down-regulated the mRNA expression of several key genes of PI3K pathway such as insulin-like growth factor 1 receptor (<i>IGF1R</i>), insulin receptor (<i>INSR</i>), pyruvate dehydrogenase kinase 1 (<i>PDK1</i>), and serine/threonine kinase 1 (<i>AKT1</i>).Conclusion: MiR-126-3p might target and inhibit the mRNA and protein expressions of <i>PIK3R2</i>, thereby inhibiting GC apoptosis and promoting GC proliferation by down-regulating several key genes of the PI3K pathway, <i>IGF1R</i>, <i>INSR</i>, <i>PDK1</i>, and <i>AKT1</i>. These findings would provide great insight into further exploring the molecular regulation of miR-126-3p and <i>PIK3R2</i> on the functions of GCs during the folliculogenesis in female mammals.