Regulation and Mechanism of LOC112447378/miR-15a/PRLR Signaling Axis on Proliferation and Apoptosis of MAC-T Cells
GAO Meng-Jing, HAN Cheng-Rui, ZHANG Xiao-Yu, GUO Yue-Mei, LIU Si-Meng, XIE Sheng-Hao, LI Qiu-Ling, QI Ying*
Hebei Key Laboratory of Animal Diversity/Langfang Key Laboratory of Cell Engineering and Application, College of Life Sciences, Langfang Normal University, Langfang 065000, China
Abstract：Global warming has made the issue of heat stress in cows (Bos taurus) increasingly prominent, causing huge economic losses to the dairy industry. Elucidating the molecular mechanisms underlying the occurrence and response of heat stress in cows can better address the issue of heat stress. Differentially expressed long non-coding RNA (lncRNA), microRNA (miRNA), and mRNA profiles between heat and non-heat stressed conditions were screened using high-throughput sequencing in our previous study. In this study, LOC112447378/miR-15a/PRLR was identified as a key competing endogenous RNA (ceRNA) network in heat stress response through bioinformatics analysis based on the high-throughput data. By constructing an in vitro heat stress model using mammary alveolar cells-large T antigen (MAC-T), the mRNA expression levels of LOC112447378, miR-15a, and prolactin receptor (PRLR) were determined using qRT-PCR. The protein levels of PRLR were determined using Western blot. These results were consistent with high-throughput sequencing results. The mRNA expression level of LOC112447378 was significantly downregulated when miR-15a was overexpressed; The mRNA and protein levels of PRLR were significantly downregulated when miR-15a was overexpressed. After knocking down miR-15a, the mRNA expression level of LOC112447378 was significantly upregulated; The mRNA and protein levels of PRLR were significantly upregulated. The methyl thiazolyl tetrazolium (MTT) test results showed that miR-15a inhibited the proliferation of MAC-T cells. The results of Annexin V-FITC/PI dual staining showed that miR-15a promotes apoptosis of MAC-T cells. MiR-15a could target and regulate the target genes LOC112447378 and PRLR, thereby achieving regulatory effects of inhibiting MAC-T cell proliferation and promoting apoptosis. These results indicated that the LOC112447378/miR-15a/PRLR signaling axis could affect the proliferation and apoptosis of MAC-T cells, thereby playing an important regulatory role in the heat stress response process of MAC-T cells. This study provides new ideas and theoretical basis for molecular breeding of heat-resistant and high-yield cows in the future.
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