Fig. 3: LncMDP1 might function as a sponge for miR-301a-5p.

a RNA expression levels of miR-301a-5p in AA broilers leg muscle at different times. Among them, E10, E12, E14, E18, 1w and 3w are n = 3 biologically independent samples, E16 and 1d are n = 4 biologically independent samples. b Pearson correlation between lncMDP1 and miR-301a-5p expression profiles in the leg muscle of AA broilers was detected by qPCR (It contains the following eight periods, E10, E12, E14, E16, E18,1d, 1w and 3w). c The dual luciferase reporter system was used to evaluate the binding of lncMDP1 and miR-301a-5p in DF-1 cells, n = 3 biologically independent samples. d Overexpression of miR-301a-5p decreased the RNA expression level of lncMDP1, and interference with 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. e, f Relative expression levels of myoblast proliferation related marker genes CDK1 (mimic NC, inhibitor NC and miR-301a-5p inhibitor are n = 3 biologically independent samples, miR-301a-5p mimic is n = 5), PCNA (n = 3 biologically independent samples), CCND1 (n = 3 biologically independent samples), CCNB2 (n = 3 biologically independent samples), CCNB3 (mimic NC, inhibitor NC and miR-301a-5p inhibitor are n = 3 biologically independent samples, miR-301a-5p mimic is n = 4), CDKN1A (mimic NC is n = 5 biologically independent samples, miR-301a-5p mimic, inhibitor NC and miR-301a-5p inhibitor are n = 3), P21 (inhibitor NC and miR-301a-5p inhibitor are n = 3 biologically independent samples) and CDKN2B (n = 3 biologically independent samples) after transfection with miR-181a-5p mimics and inhibitors. g The protein expression level of CDK1 was decreased after transfection with miR-301a-5p mimic, and transfection of miR-301a-5p inhibitors increased the protein expression level of CDK1, 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 circle represents miR-301a-5p mimic. The blue circle of the right box line diagram represents inhibitor NC, and the orange circle represents miR-301a-5p inhibitor. 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. h, i CCK-8 assays were performed to assess the effect of transfection with miR-301a-5p mimics (mimic NC is n = 7 biologically independent samples at 12 h and 48 h, n = 6 at 24 h, n = 5 at 36 h, miR-301a-5p mimic is n = 6 at 12 h and 48 h, n = 7 at 24 h, n = 5 at 36 h) and inhibitors (mimic NC is n = 4 biologically independent samples at 12 h, 24 h and 36 h, n = 6 at 48 h, miR-301a-5p mimic is n = 6 at 12 h and 24 h, n = 4 at 36 h, n = 5 at 36 h) on myoblast proliferation. j Proliferation of myoblasts transfected with miR-301a-5p mimic and inhibitor was analyzed by EdU staining at a scale of 100 μm (miR-301a-5p mimic, inhibitor NC and miR-301a-5p inhibitor are n = 3 biologically independent samples, mimic NC is n = 4). k, l Cell cycle analysis after transfection of miR-301a-5p mimics and inhibitors in myoblasts, n = 3 biologically independent samples. m MyHC immunofluorescence staining of myoblasts 48 h after transfection with miR-301a-5p mimic and inhibitor, n = 3 biologically independent samples. The scale is 100 μm. n–p Transfection with miR-301a-5p mimic significantly decreased RNA expression levels of markers associated with myoblast differentiation, MyoD (n = 4 biologically independent samples), MyoG (n = 3 biologically independent samples), and MyHC (n = 4 biologically independent samples), and decreased protein expression levels of MyHC (n = 3 biologically independent samples, and 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 circle represents miR-301a-5p mimic. The blue circle of the right box line diagram represents inhibitor NC, and the orange circle represents miR-301a-5p inhibitor. The results of transfection of miR-301a-5p inhibitor were the opposite. Figure 3g, p was from different blots and since the molecular weight difference between CDK1 and GAPDH was only 2 kDa, it was not easy to distinguish between them, so the same internal control (GAPDH) was used to ensure that the sample load is consistent under the premise of detecting the protein concentration. The findings are presented as the mean ± SEM of three separate experiments. (* P < 0.05; ** P < 0.01, *** P < 0.001).