LARS2 Regulates Apoptosis via ROS-Mediated Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in Ovarian Granulosa Cells

Abstract

Several studies have indicated that mutations of LARS2 are associated with premature ovarian insufficiency (POI). However, the pathogenic mechanism of LARS2 in POI has not been reported yet. In the present study, the expression levels of LARS2 and E2F1 in granulosa cells (GCs) of POI patients were examined. CCK-8 and Edu assay were performed to determine the effect of LARS2 on cell proliferation. Apoptosis rate, mitochondrial membrane potential, reactive oxygen species (ROS), and cytoplasm Ca2+ levels were analyzed by flow cytometry. Western blot was conducted to evaluate the expression level of genes affected by LARS2. Transmission electron microscopy (TEM) was used to observe mitochondrial structure in GCs. Chromatin immunoprecipitation (ChIP) was used to evaluate the regulatory effect of E2F1 on Mfn-2 expression. Our results showed that LARS2 expression was downregulated in GCs of POI patients. Silencing of LARS2 inhibited cell proliferation and promoted the apoptosis of GCs. Meanwhile, LARS2 knockdown could induce mitochondrial dysfunction and accumulation of ROS levels. Moreover, ROS was found to be involved in the antiproliferation, proapoptotic, and endoplasmic reticulum (ER) stress effects of LARS2 knockdown. Furthermore, we also found that the expression level of E2F1 was positively correlated with LARS2. In addition, E2F1 could bind at the -61/-46 region of Mfn-2 promoter and regulated MFN-2 transcription. These findings demonstrated that LARS2 could promote the expression of E2F1. E2F1 mediated the effect of LARS2 on Mfn-2 expression via targeting the promoter region of Mfn-2, in which subsequently regulated cell proliferation and apoptosis, which resulted in the etiology of POI. This study will provide useful information for further investigations on the LARS2 in the occurrence of POI.