This study was aimed to characterize the distal core promoter of the porcine lncRNA NORHA gene, and explore the mechanism by which the transcription factor (TF) ZBTB6 activated its transcription and function, so as to provide a basis for analyzing the transcriptional regulatory network of NORHA.

The sequence of the distal core promoter of the NORHA gene in Yorkshire pigs was obtained by PCR amplification and sequencing. Bioinformatic method was used to predict the TF-binding sites (TFBSs) and to characterize the ZBTB6 encoding sequence. A ZBTB6 overexpression vector was constructed by restriction enzymatic digestion, and its overexpression efficiency in sow granulosa cells (sGCs) was verified by qPCR and western blot. The effects of ZBTB6 on the activity of the distal core promoter and transcription of NORHA in sGCs were determined by luciferase assay and qPCR. The binding of ZBTB6 to the ZBTB6-binding site (ZBS) in the distal core promoter in sGCs was detected by chromatin immunoprecipitation (ChIP) and DNA sequencing. The concentrations of progesterone (P4) and estradiol (E2) in follicular fluid were measured by ELISA. ZBTB6 and NORHA levels in follicles, and BCL2 and BAX levels in ZBTB6-overexpressing sGCs were detected by qPCR.

TFBSs of 255 TFs, including ZBTB6, were predicted in 526-bp of the distal core promoter of Yorkshire NORHA gene. The overexpression vector successfully achieved the overexpression of ZBTB6 levels in sGCs. The transcriptional levels of NORHA in sGCs were markedly upregulated in ZBTB6-overexpressing sGCs. Correlation analysis revealed that ZBTB6 and NORHA levels in follicles were markedly positively correlated. A ZBS motif was discovered at -2276/-2264 nt in the distal core promoter. Luciferase assay revealed that ZBTB6 activates the activity of the distal core promoter via the ZBS motif. ChIP and sequencing confirmed that ZBTB6 directly bound to the ZBS motif in the distal core promoter of the NORHA gene in sGCs. The ZBTB6 gene in pigs and other mammalian species was evolutionally conserved and all contained a conserved DNA-binding domain. ZBTB6 was markedly upregulated during sow follicular atresia and showed a marked positive correlation with the P4/E2 ratio, a marker for follicular atresia. Co-transfection experiments showed that overexpression of ZBTB6 markedly downregulated the anti-apoptotic gene BCL2 levels, upregulated the pro-apoptotic gene BAX levels, and downregulated the BCL2/BAX ratio, an apoptotic marker, whereas knockdown of NORHA reversed this.

ZBTB6 was a transcription activator of the NORHA gene in sGCs, which accelerated sGC apoptosis and follicular atresia by directly activating NORHA transcription.

miR-34a has been shown to be a pro-apoptotic factor in porcine ovarian granulosa cells (GCs) in our previous study. The aim of this study was to explore the RNA activation mechanism by which miR-34a induced apoptosis in the nucleus, so as to provide a basis for uncovering the molecular mechanism of porcine follicular atresia, and to screen small nucleic acid regulator that regulated procine reproduction.

Bioinformatics methods were used to analyze the characteristics of the porcine miR-34a, the binding sites of miR-34a to the promoter region of target lncRNA NORHA, and the binding capacity. The subcellular localization of miR-34a was analyzed in porcine GCs by using nucleocytoplasmic fractionation. The effects of miR-34a on the expression of NORHA, apoptosis marker genes BCL-2 and BAX in porcine GCs were determined by qPCR. Luciferase assay was used to verify the regulatory relationship of miR-34a on the transcriptional activity of the NORHA promoter. Chromatin immunoprecipitation (ChIP) was utilized to detect the enrichment of AGO2 and histone modifiers at the miRNA response element (MRE) in the NORHA promoter in porcine GCs by miR-34a. The regulatory effect of miR-34a on downstream factor FOXO1 expression and GC apoptosis via NORHA were analyzed by western blot and flow cytometry.

The porcine miR-34a was an intergenic miRNA, and its sequence was highly conserved among mammals. Subcellular localization analysis showed that miR-34a was distributed in both nucleus and cytoplasm in porcine GCs. Bioinformatic analysis revealed that the MRE of miR-34a was located at -194 nt - -173 nt in the NORHA promoter, and the binding free energy of the two compounds was -23.9 kcal·mol^-1. qPCR showed that miR-34a significantly upregulated NORHA expression in porcine GCs. Luciferase assay revealed that miR-34a significantly upregulated the activity of the reporter vector with NORHA promoter, whereas it had no significant effect on the activity of promoter with the mutated MRE of miR-34a. ChIP assay showed that miR-34a increased the enrichment of AGO2, a core member of RNA induced transcriptional activation (RITA) complex, and H3K4me3, a transcription activated histone modifier, while or decreased the enrichment of H3K9me3, a transcription repressed histone modifier at MRE motif of miR-34a in the NORHA promoter. Co-transfection assay showed that knockdown of NORHA significantly reversed the upregulation of FOXO1 protein level and early apoptosis rate caused by miR-34a overexpression, as well as the downregulation of the BCL-2/BAX ratio.

Nuclear miR-34a was an endogenous small activating RNA (saRNA) in porcine GCs, and might be involved in the initiation of porcine follicular atresia by inducing early apoptosis in porcine GCs through target activating the transcription of the pro-apoptotic lncRNA NORHA.