Fig. 4: LncMDP1 regulates CHAC1 expression through the adsorption of miR-301a-5p.

a RNA expression levels of miR-301a-5p in AA broilers leg muscle at different times, n = 3 biologically independent samples. b Pearson correlation between lncMDP1 and miR-301a-5p expression at the embryonic stage of AA broilers leg muscle using qPCR (It contains the following five periods, E10, E12, E14, E16 and E18). c RNA in situ hybridization was used to detect the co-localization between lncMDP1 and CHAC1 in myoblasts. The scale is 10 μm. d The dual luciferase reporter system was used to assess the binding of CHAC1 and miR-301a-5p in DF-1 cells, n = 3 biologically independent samples. e Overexpression of miR-301a-5p decreased the RNA expression level of CHAC1, and knockdown of miR-301a-5p had the opposite effect, n = 3 biologically independent samples. The error bars are equivalent throughout the Figure. In the box line, a line in the middle of the box represents the median, and the upper and lower bottoms of the box are the upper quartile and the lower quartile, respectively. The blue circle of the left box line diagram represents mimic NC, and the orange square represents miR-301a-5p mimic. The blue circle of the right box line diagram represents inhibitor NC, and the orange square represents miR-301a-5p inhibitor. f, g Relative expression of CDK1 (n = 3 biologically independent samples), PCNA (n = 3 biologically independent samples), CCND1 (n = 3 biologically independent samples), CCNB1 (n = 4 biologically independent samples), CCNB2 (pcDNA3.1-3xFLAG is n = 4 biologically independent samples, pcDNA3.1-CHAC1-3xFLAG, si-NC and si-CHAC1 are n = 4), P21 (n = 3 biologically independent samples), CDKN1A (n = 3 biologically independent samples) and other genes after the transfection of pcDNA3.1-CHAC1-3xFLAG or si-CHAC1 in myoblasts. h Protein expression levels of CDK1 and CHAC1 after CHAC1 overexpression or interference, n = 3 biologically independent samples. The error bars are equivalent throughout the Figure. In the box line, a line in the middle of the box represents the median, and the upper and lower bottoms of the box are the upper quartile and the lower quartile, respectively. The blue circle of the left box line diagram represents pAV-3xFLAG, and the orange circle represents pAV-CHAC1-3xFLAG. The blue circle of the right box line diagram represents si-NC, and the orange circle represents si-CHAC1. Western blot was performed on different membranes for proteins with a molecular weight difference of fewer than 5 kDa (similar protein size, difficult to distinguish), and the sample size was consistent under the premise of detecting protein concentration. In this case, the sample and the loading control ran on different gels and thus transferred to different membranes. i, j Analysis of the cell cycle following transfection of myoblasts with si-CHAC1 (n = 3 biologically independent samples) or pcDNA3.1-CHAC1-3xFLAG (n = 4 biologically independent samples). k, l CCK-8 assay was used to detect the effect of CHAC1 overexpression (pcDNA3.1-3xFLAG is n = 6 biologically independent samples at 12 h,24 h and 36 h, n = 7 at 48 h, n = 5 at 36 h, pcDNA3.1-CHAC1-3xFLAG is n = 7 at 12 h and 36 h, n = 8 at 24 h and 48 h) or interference (n = 5 biologically independent samples) on myoblast proliferation. m Transfection of pcDNA3.1-CHAC1-3xFLAG (n = 5 biologically independent samples, pcDNA3.1-3xFLAG is n = 4) or si-CHAC1 (n = 3 biologically independent samples) was analyzed for proliferation of myoblasts by EdU staining at a scale of 100 μm. n MyHC staining of myoblasts after the overexpression or interference of CHAC1 n = 3 biologically independent samples. The scale is 100 μm. o–q CHAC1 promotes the RNA expression of genes related to myoblast differentiation, including MyoD (n = 3 biologically independent samples), MyoG (pcDNA3.1-3xFLAG is n = 4 biologically independent samples, pcDNA3.1-CHAC1-3xFLAG, si-NC and si-CHAC1 are n = 4), MyHC (pcDNA3.1-CHAC1-3xFLAG is n = 4 biologically independent samples, pcDNA3.1-3xFLAG, si-NC and si-CHAC1 are n = 3), and MyoMarker (pcDNA3.1-CHAC1-3xFLAG is n = 4 biologically independent samples, pcDNA3.1-3xFLAG, si-NC and si-CHAC1 are n = 3), and decreases the protein expression levels of MyHC, n = 3 biologically independent samples, the error bars are equivalent throughout the Figure. In the box line, a line in the middle of the box represents the median, and the upper and lower bottoms of the box are the upper quartile and the lower quartile, respectively. The blue circle of the left box line diagram represents pAV-3xFLAG, and the orange circle represents pAV-CHAC1-3xFLAG. The blue circle of the right box line diagram represents si-NC, and the orange circle represents si-CHAC1. Figure 4h, q was from different blots. Since the molecular weight difference of GAPDH is 2 kDa from CDK1 and CHAC1, respectively, it was not easy to distinguish between them, so the same internal control (GAPDH) was used to ensure that the protein concentration was detected. The findings are presented as the mean ± SEM of three separate experiments. (*P < 0.05; **P < 0.01, ***P < 0.001).