MFACR was overexpressed in MI and inversely correlated with miR-125b
Plasma samples from MI patients (n=61) and healthy controls (n=61) were subjected to RNA isolations and RT-qPCRs to analyze the expression of MFACR and miR-125b. Compared to Control group, significantly overexpressed MFACR (Fig.1A, p<0.001) and significantly under-expressed miR-125b (Fig.1B, p<0.001) were observed in MI group. Pearson’s correlation coefficient analysis showed that MFACR and miR-125b were inversely and significantly correlated across MI samples (Fig.1C) and control samples (Fig.1D). Therefore, a crosstalk may exist between MFACR and miR-125b.
Plasma samples from MI patients (n=61) and healthy controls (n=61) were subjected to RNA isolations and RT-qPCRs to analyze the expression of MFACR (A) and miR-125b (B). Pearson’s correlation coefficient analysis was performed to analyze the correlations between MFACR and miR-125b across MI samples (C) and control samples (D). ***,p<0.001.
Hypoxia treatment altered the expression of MFACR and miR-125b in AC16 cells
To study the effects of hypoxia on the expression of MFACR and miR-125b in AC16 cells, AC16 cells were cultivated under hypoxic conditions for 24, 48, 72 and 96h, followed by determine the expression of MFACR and miR-125b by RT-qPCR. It was observed that, in AC16 cells, hypoxia treatment increased the expression of MFACR (Fig.2A, p<0.05) and decreased the expression of miR-125b (Fig.2B, p<0.05).
To study the effects of hypoxia on the expression of MFACR and miR-125b in AC16 cells, AC16 cells were cultivated under hypoxic conditions for 24, 48, 72 and 96h, followed by determine the expression of MFACR (A) and miR-125b (B) by RT-qPCR. *,p<0.05.
MFACR overexpression decreased miR-125b expression through methylation in AC16 cells
To study the effects of crosstalk between MFACR and miR-125b, AC16 cells were transfected with either MFACR expression vector or miR-125b mimic, followed by checking the overexpression of MFACR and miR-125b every 24h until 96h. It was observed that MFACR and miR-125b were significantly overexpressed between 24h and 96h (Fig.3A, p<0.05). MFACR overexpression decreased the expression of miR-125b between 24h and 96h (Fig.3B, p<0.05), while miR-125b overexpression failed to significantly affect the expression of MFACR (Fig.3C). To study the effects of the overexpression of MFACR overexpression on the methylation of miR-125b gene, MSP was performed at 96h post-transfection. Compared to AC16 cells transfected with empty pcDNA3.1 vector, cells transfected with MFACR expression vector showed significantly increased methylation of miR-125b (Fig.3D). Therefore, MFACR may downregulate miR-125b through methylation.
To study the effects of crosstalk between MFACR and miR-125b, AC16 cells were transfected with either MFACR expression vector or miR-125b mimic, followed by checking the overexpression of MFACR and miR-125b every 24h until 96h (A). The effects of MFACR overexpression on the expression of miR-125b (B), and the effects of miR-125b overexpression on the expression of MFACR (C) were analyzed by RT-qPCR. MSP was performed at 96h post-transfection to analyze the effects of the overexpression of MFACR overexpression on the methylation of miR-125b gene (D).M, methylation; U, un-methylation; *,p<0.05.
MFACR overexpression increased AC16 cell apoptosis induced by hypoxia through miR-125b
Under hypoxic conditions, the role of MFACR and miR-125b in regulating the apoptosis of AC16 cells was analyzed by cell apoptosis assay. MFACR overexpression increased AC16 cell apoptosis, and miR-125b overexpression decreased cell apoptosis. In addition, miR-125b overexpression reversed the effects of MFACR overexpression on cell apoptosis (Fig.4, p<0.05).
Under hypoxic conditions, the role of MFACR and miR-125b in regulating the apoptosis of AC16 cells was analyzed by cell apoptosis assay.*,p<0.05.