A Prelimary Functional Study On MiR-331~* Which Was Differentially Expressed In Porcine Pre-Ovulatory Follicles Between Large White And Erhualian Pigs

Mi RNAs, a class of non-coding small RNAs with an average of 21 nucleotides, reduce gene expression at the post-transcription level by binding specific m RNAs. Post-transcriptional regulatory mi RNAs have been reported to play a key role in the differentiation and development process of ovarian cells. We found mi R-331~* differentially expressed in the pre-ovulatory ovarian follilces between Erhualian and Large White pigs by Solexa sequencing. We speculated that mitochondrial gene Cytochrome B(CYTB) might be the target gene of mi R-331~* based on sequence analysis, target gene prediction, functional analysis and related literature reports. Mitochondria are not only the energy supply center, but also involved in cell metabolism, programmed cell death, cell differentiation and so on. This study researched the regulatory role and mechanism of mi R-331~* and its target genes in the process of ovulation in sows, and provided a theoretical basis for improving the reproductive performance of sows. The main results are as follows: 1. CYTB is a target of mi R-331~*Pmir GLO-CYTB-WT and pmir GLO-CYTB-mut dual luciferase reporter vectors containing the mi R-331~* binding site were constructed. Then, mi R-331~* mimics and dual luciferase reporter vectors were co-transfected into CHO cells, and the fluorescence activity was determined. The results showed that mi R-331~* contributed to up-regulation expression of CYTB gene.Real time quantitative PCR showed that mi R-331~* mimic could increase the expression level of CYTB gene in mi R-331~* mimic group compared with the control group. Western Blot showed that mi R-331~* mimic could increase the expression of CYTB gene at the protein level. Meanwhile, CYTB-si RNA could inhibit the expression of CYTB protein.2. The prelimary function study of mi R-331~* and its target gene CYTBCell proliferation of CHO cells at different time points(24h, 48 h, 96h) was detected by MTT. The results showed that mi R-331~* mimic significantly decreased cell proliferationat at different time points(24h, 48 h, 96h), while inhibition of CYTB expression did not. Compared with NC group, the percentage of early cell apoptosis presented a rising trend when mi R-331~* was overexpressed, and a decreasing trend after CYTB expression was inhibited. With the overexpression of mi R-331~*, the apoptosis protein Bax increased, and the expression level of Bcl-2 protein did not. When the expression of CYTB gene was inhibited, the apoptosis protein Bax decreased, but the expression level of Bcl-2 protein increased.CHO cell cycle was detected by flow cytometry. G1 phase of CHO cell increased and S phase obviously shortened when mi R-331~* was overexpressed. In contrast, G1 phase significantly shortened and S phase sharply increased when the expression of CYTB gene was inhibited. The content of ATP in the post-treated cells was directly detected by ELISA. The results showed that ATP content in CHO cells significantly decreased when mi R-331~* was overexpressed, and no significant difference was detected when the expression of CYTB gene was inhibited. The expression level of cell damage protein H2 AX was detected by Western blot. The results showed that the expression level of H2 AX in CHO cells significantly increased when mi R-331~* was overexpressed and decreased when the expression of CYTB gene was inhibited.Cytochrome C released into the cytoplasm, activated the caspase, thereby inducing apoptosis. Caspase can inactivate or downregulate enzymes which involved in DNA repair, block DNA replication and repair, thus affecting cell proliferation and cell cycle. Our study found mi R-331~* and target gene CYTB affected the release of Cytochrome C via regulating ATP produced by oxidative phosphorylation, and further regulated cell proliferation, apoptosis and cycle.