MiR-431 induced the apoptosis of GEECs in vitro
Previous studies have reported the first miRNA expression profile in goat endometrium during embryo implantation related to the PE and RE . Depending on the sequencing data, miR-431 was highly differential expression in goat endometrium during embryo implantation. Hence, miR-431 was invoked as a candidate miRNA to investigate its role in goat endometrial epithelial cells (GEECs). Figure 1A showed the transfection efficiency of miR-431 mimic in GEECs by stem-loop RT-qPCR. The results revealed that the mRNA expression level of miR-431 was remarkably increased compared with NC, which indicated that miR-431 could be used in the further experiments (Fig. 1A).
To further investigate the role of miR-431 in GEECs, we conducted CCK-8 and EdU assays. The CCK-8 assay revealed that miR-431 mimics significantly inhibited the viability of GEECs compared with NC. Nevertheless, there was no noteworthy difference between miR-431 inhibitor and NCH (Fig. 1B). The EdU assays also showed that miR-431 mimics notably suppressed the proliferation of GEECs while miR-431 inhibitor exhibited the opposite result (Fig. 1C).
Furthermore, Annexin V-FITC/PI apoptosis kit combined with FCM was used to estimate GEECs apoptosis. The results indicated that the number of apoptosis cells increased in miR-431 mimics group, but decreased in miR-431 inhibitor group (Fig. 1D). Similarly, we also performed the WB analysis to assess the protein of apoptosis-related, BCL2 and BAX. The results revealed that miR-431 mimics reduced the BCL2 protein level strikingly while increased the expression of BAX, whereas miR-431 inhibitor exhibited the opposite result (Fig. 1E). Altogether, these results demonstrated that miR-431 induced the apoptosis of GEECs in vitro.
MiR-431 targeted CSF1 in GEECs
As a post-transcriptional regulator of gene expression, miRNA plays a biological role in degrading mRNA of target genes or inhibiting translation . Then we wondered whether miR-431 induces cell apoptosis by negatively regulating its target gene. And we utilized the TargetScan and miRanda to screen CSF1 as the target gene of miR-431.
We constructed wild-type plasmid contained 3′UTR of CSF1(about 310bp) and mutated-type plasmid with mutated sites of CSF1 3′UTR for dual-luciferase reporter assay in HEK293T cells (Fig. 2A). As showed in the Fig. 2B, luciferase activity of miR-431 mimics co-transfection with wild-type plasmid was markedly lower compared with the NC group. However, there was no significant difference between miR-431 mimics and NC group after co-transfection with mutant plasmid (Fig. 2B). Together, the results revealed that miR-431 reduced the expression of CSF1 by binding with its complementary sequences.
To further verify the targeting effect of miR-431 for CSF1, we implemented RT-qPCR and Western blot experiment. Consistently, the results showed that overexpression miR-431 mimics dramatically depressed the expression of CSF1 in mRNA and protein level while miR-431 inhibitor had the reverse results (Fig. 2C-D). Subsequently, we tested the transfection efficiency of CSF1 in the level of mRNA and protein in GEECs. The results revealed that pc3.1-CSF1 significantly increased the CSF1 expression in mRNA and protein levels. However, miR-431 co-transfection with CSF1 reduced the expression of CSF1 protein, suggesting that the miR-431 targeted CSF1 (Fig. 2E-F). However, CSF1 with small interference RNA (si-CSF1) decreased the expression of CSF1 in mRNA and protein levels.
CSF1 promoted the proliferation of GEECs in vitro
To determine the targeted regulation effect of 431 on CSF1, we used CCK-8 and EdU assays to ascertain the function of CSF1 in GEECs. The results indicated that overexpression CSF1 elevated cell vitality while co-transfection of miR-431 abolished this stimulating effect. Similarly, we found that after interfering with the expression of CSF1, the proliferative effect was removed (Fig. 3A-B). Additionally, we sought to test whether overexpression CSF1 declined the apoptosis rate in GEECs. The results of Annexin V-FITC/PI staining presented that pc3.1-CSF1 significantly decreased the number of apoptotic cells. However, miR-431 co-transfection with CSF1 somewhat removed the effect of CSF1 on GEECs apoptosis (Fig. 3C). And the WB results also strongly support this assertion that CSF1 inhibited cell apoptosis. Conversely, silencing of CSF1 with small interference RNA induced cell apoptosis, which could weaken the trend when co-transfection with miR-431 inhibitor (Fig. 3D). Collectively, these findings suggested that CSF1 promoted cell proliferation and miR-431 induces cell apoptosis through negative regulation of CSF1.
MiR-431 inhibited MAPK signaling pathway through CSF1
Previous studies have reported that Ras/Raf/MEK/ERK signaling pathway plays a critical role in cell proliferation and cell apoptosis [6, 7, 27, 28]. It has also been reported that CSF1 can induce monocytes to produce VEGF through SP1/MAPK/ERK signaling pathway, thus promoting in vivo angiogenesis . Consequently, we speculated that miR-431 may induce GEEC apoptosis through Ras/Raf/MEK/ERK signaling pathway, on account of its negative regulation on CSF1 expression. Subsequently, we treated Western blot to evaluate the expression levels of Ras, Raf, MEK and ERK proteins and their phosphorylated proteins under the treatment of miR-431 and/or CSF1. The results indicated that miR-431 and interference CSF1(si-CSF1) both significantly decreased the expression of Ras, P-Raf, P-MEK and P-ERK proteins (Fig. 4A, 4C). However, overexpression of CSF1 observably increased the expression of Ras, p-MEK, and p-ERK proteins. When miR-431 was co-transfection with CSF1, the effect of CSF1 on MAPK signal transduction pathway was eliminated to a certain extent (Fig. 4B). Those findings suggested that CSF1 may active the MAPK cascade pathway, while miR-431 inhibit that via down-regulating CSF1.
Correlation analysis of circRNA211 and FBXO18 expression in GEECs
Increasingly evidences indicated that circRNAs, as molecular sponges of miRNAs, can further regulate the expression of downstream target genes [30, 31]. According to the expression profile of circular RNA library constructed in the laboratory and bioinformatics analysis, circRNA211 containing the binding site of miR-431 seed sequence was selected for further study. Meanwhile, sequencing results showed that FBXO18 (F-box protein, Helicase, 18), which also has targeted binding sites of miR-431 in the CDS region through bioinformatics prediction, was the host gene of circRNA211 . The transfection efficiency of FBXO18, circRNA211 and small interfering RNA of circRNA211 (si-circRNA211) shown in Fig. 5A demonstrated that they can be utilized in subsequent experiments (Fig. 5A). Furthermore, we used RT-qPCR to determine the relationship between circRNA211 and FBXO18. The results showed that high expression of FBXO18 could enhance the expression of circRNA211 (Fig. 4B), whereas circRNA211 interference had no effect on the expression of FBXO18 (Fig. 4C). In summary, all the above results suggested that the expression of circRNA211was positively correlated with FBXO18.
CircRNA211 competitively inhibited miR-431
Subsequently, full circRNA211 sequence and mutated sequence of circRNA211 with mutated sites was cloned and inserted into the psiCHECKTM-2 vector to construct wild type plasmid (WT-circRNA211) or mutated-type plasmid (MUT-circRNA211) for dual-luciferase reporter assay. The results showed that relative luciferase activity in miR-431 mimics co-transfection with WT-circRNA211 plasmid was dramatically lower than the NC group in HEK293T cells. Whereas, miR-431 mimic co-transfection with MUT-circRNA211 plasmid had no effect on the relative luciferase activity (Fig. 5D). RT-qPCR showed that the increasing expression of circRNA211 could significantly reduce the expression of miR-431 at the mRNA level. On the contrary, interfering circRNA211 markedly raised the expression of miR-miR-431 (Fig. 5E). Therefore, all the results suggested that circRNA211served as a sponge for miR-431 in GEECs, thus inhibiting the expression of miR-431.
MiR-431 targeted FBXO18 in GEECs
Furthermore, we constructed vectors including the binding site of FBXO18 and miR-431 (WT-FBXO18) or mutant fragment of FBXO18 (MUT-FBXO18) to detect luciferase activity in HEK293T cells. The results revealed that the luciferase activity of miR-431 mimics co-transfection with WT-FBXO18 plasmid was greatly declined compared with the NC group. Nonetheless, miR-431 mimics co-transfection with MUT-FBXO18 plasmid had no effect on the relative luciferase activity (Fig. 5F). In addition, miR-431 mimics also sharply declined the expression of FBXO18, while miR-431 inhibitor increased that at the mRNA level (Fig. 5G). Therefore, miR-431 targeted FBXO18 and inhibited the expression of FBXO18 in GEECs.
Effects of circRNA211and FBXO18 on CSF1 expression
To further confirm whether circRNA211 could serve as competitive endogenous RNAs (ceRNAs), we use RT-qPCR and WB to examine the expression levels of CSF1 in GEECs. The results showed that overexpression of circRNA211 significantly increased the mRNA and protein levels of CSF1 (Fig. 6A), while si-circRNA211 decreased that (Fig. 6C). Meanwhile, overexpression FBXO18 also greatly enhanced the expression of CSF1 in the mRNA and protein levels (Fig. 6B), which indicated that FBXO18 also facilitated the expression of CSF1 in GEECs. Overall, these results demonstrated that circRNA211 and FBXO18 can promote the expression of CSF1 in GEECs.
CircRNA211 and FBXO18 promoted the proliferation of GEECs
To investigate the biological role of circRNA211 and FBXO18 in GEECs, cell proliferation was evaluated using CCK-8 kit. The results showed that overexpression of circRNA211 and FBXO18 increased cell activity, and this increase was reversed while circRNA211 or FBXO18 co-transfection with miR-431 (Fig. 7A, C). On the contrary, after circRNA211 interference, the cell viability decreased dramatically (Fig. 7B). The results of EdU staining also showed that the increase in FBXO18 or circRNA211 considerably promoted cell proliferation, while interference circRNA211 inhibited cell proliferation (Fig. 7D-F). Moreover, Annexin V-FITC/PI staining results showed that FBXO18 and circRNA211 remarkably reduced the apoptosis of GEECs. Whereas, overexpression of miR-431 eliminated this decrease effect on the apoptosis of GEECs to some extent (Fig. 7G, I). Furthermore, si-circRNA211 elevated the number of apoptosis cells (Fig. 7H). On the other hand, the results of WB also showed that overexpression of FBXO18 and circRNA211 promoted cell proliferation (Fig. 7J, L), while si-circrNA211 induced cell apoptosis (Fig. 7K). Generally, those results manifested that circRNA211 and FBXO18 promote the proliferation of GEECs.
CircRNA211 and FBXO18 activated MAPK signaling pathway by inhibiting miR-431
Similarly, we also conducted WB assay to examine the effect of circRNA211 and FBXO18 on MAPK signaling pathway. The results indicated that overexpression of circRNA211 activated the activity of Ras protein, which in turn activated the phosphorylation of downstream Raf and MEK proteins (Fig. 8A). Overexpression of FBXO18 can also activate the activity of Ras protein, and then activated protein phosphorylation of downstream Raf, MEK and ERK successively (Fig. 8C). However, activity of phosphorylated proteins on the MAPK signaling pathway was inhibited under si-circRNA211 treatment (Fig. 8B). Altogether the results showed that circRNA211 and FBXO18 may promote the proliferation of GEECs by activating MAPK signaling pathway due to their ‘sponge’ effect on miR-431.
FBXO18 /circRNA211/miR-431/ CSF1 axis promoted the formation of endometrial receptivity in dairy goats
Western blot was used to investigate the effects of FBXO18/circRNA211/miR-431/CSF1 regulatory axis on CSF1R/PI3K/ AKT /mTOR signaling pathway. Firstly, we analyze the effect of miR-431 on the expression of CSF1R, PI3K, AKT and mTOR proteins and their phosphorylated proteins. The results showed that miR-431 mimics notably reduced the phosphorylation level of CSF1R protein. Interestingly enough, miR-431 further inhibited the proteins of PI3K, AKT and mTOR from phosphorylating. Conversely, miR-431 inhibitor increased the protein phosphorylation level of CSF1R, PI3K, and AKT (Fig. 9A).
In addition, the effects of CSF1 on the expression of PI3K, AKT, mTOR and their phosphorylated proteins were also detected. WB results indicated that overexpression CSF1 promoted the phosphorylation of CSF1R protein, which in turn activated the PI3K/AKT/mTOR signaling pathway. However, the activation effect in PI3K/AKT/mTOR signaling pathway was alleviated when CSF1 co-transfection with miR-431 mimics (Fig. 9B). Meanwhile, after the expression of CSF1 was interfered (si-CSF1), the ratios of CSF1R, PI3K, and AKT proteins and their phosphorylated proteins decreased (Fig. 9C).
Through the verification of targeted relationship, we found that circrNA211 promoted the expression of CSF1 via sponging miR-431. Therefore, it is speculated that circrNA211 triggered the PI3K/mTOR/AKT signaling pathway by inducing CSF1R protein phosphorylation. Western blot results revealed that circrNA211 obviously enhanced the ratio of P-CSF1R/CSF1R, P-PI3K/PI3K, P-AKT/AKT and P-mTOR/ mTOR protein levels. After co-transfection with circRNA211, miR-431 reduced the phosphorylation levels of CSF1R, PI3K and AKT proteins to a certain extent (Fig. 10A). On the contrary, interference with circRNA211 (si-circRNA211) sharply decreased the phosphorylation level of CSF1R, thereby inhibiting the activation of the PI3K/AKT/mTOR pathway (Fig. 10B). Similarly, as the host gene for circrNA211, FBXO18 also elevated the expression levels of P-CSF1R and P-AKT proteins (Fig. 10C).
Therefore, the FBXO18/circRNA211/miR-431/CSF1 regulatory axis can regulate the establishment of endometrial receptivity of dairy goats through the CSF1R/PI3K/ AKT/mTOR signaling pathway.