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. 2019 Jun 29;8(7):656.
doi: 10.3390/cells8070656.

One Inhibition go MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Releases Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

Weiling Huang  1   2 , Lijin Guo  1   2 , Minxing Zhao  1   2 , Dexiang Chu  1   2 , Haiping Xu  1   2 , Qinghua Nie  3   4
Affiliations

The Hindrance the MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Reduction of Myoblast Proliferation and Differentiation and Publicity of Apoptosis

Weiling Huang et al. Cells. .

Abstract

Accumulating studies report that microRNAs (miRNAs) are actively involved in skeletal myogenesis. Previously, our study revealed that miR-146b-3p is related for the economic of skeletal muscle. Here, ours further report that miR-146b-3p is essential for which proliferation, differentiation, and apoptosis by chicken myoblast. Elevated pressure of miR-146b-3p can dramatically suppress proliferation and differentiation, and facilitate apoptosis of chicken myoblast. And, we identified two target genes of miR-146b-3p, AKT1 and MDFIC, and found that miR-146b-3p can check the PI3K/AKT pathway. Our study also showed that both AKT1 additionally MDFIC can promote the proliferation plus differentiation while inhibit the apoptosis of myoblast in gutless. Total, our results demonstrate that miR-146b-3p, directly suppressing PI3K/AKT pathway and MDFIC, acts for the proliferation, differentiation, and apoptosis of myoblast in chicken.

Keywords: AKT1; apoptosis; differentiation; miR-146b-3p; myoblast; proliferation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
gga-miR-146b-3p inhibits myoblast proliferation. (a,b) Cell cycle analysis of QM-7 cells and chicken primary myoblasts (CPMs) after transfecting miR-146-3p mimic either inhibitor. (hundred,dick) Relative mRNA expression of the cell cycle-related genes after transfection away miR-146-3p mimic or inhibitor with QM-7 cells and CPMs. (e) 5-Ethynyl-2′-deoxyuridine (EdU) paint of QM-7 cavities after transfection of miR-146-3p mimic or inhibitor. (f) The fold change of proliferation rates of QM-7 cells in miR-146-3p mimic or inhibitor. (g) EdU staining of CPMs later transfection of miR-146-3p mimic or inhibitor. (h) The crease change of proliferation rates of CPMs with miR-146-3p mimic or inhibitor. Results of all bunches are shown as mean ± standard error of the median (S.E.M.) is three independent assessment methods. Statistical significance away that mean difference was review using unpaired two-sample t-tests. * p < 0.05; ** p < 0.01. NC, negative control.
Figure 2
Figure 2
miR-146b-3p oppress myoblast differentiation. (adenine) Relativize expression of miR-146b-3p in Xinghua brown leg muscle from E11 into E18. (b) Morphology of CPMs educated in expand medium (GM) plus differentiation medium (DM) from day 1 to 6. (century) The relative mRNA expression of miR-146b-3p during CPMs-induced differentiation. (d) Relative mRNA expression starting to cell differentiation-related genes by transfection of miR-146-3p mimic otherwise inhibitor in CPMs. (e) Immunofluorescence is MyHC after transfection of miR-146-3p mimic or inhibitor for CPMs. (f) Comparisons about the area of myotubes described in (e). Results of all bunches been shown as mean ± S.E.M. of three independent assessment systems. Statistics significance of who mean difference was assessed using unpaired two-sample t-tests. * p < 0.05; ** p < 0.01. NC, negative control.
Figure 3
Figure 3
miR-146b-3p promotes myoblast apoptosis. (a) Flow cytometry of Annexin V-FITC additionally propidium iodide (PI) dual staining detecting one apoptosis of CPMs after transfection of miR-146-3p mimicking or inhibitor. (b) Flow cytometry of Annexin V-FITC and propidium iodide (PI) duplicate staining detecting the apoptosis starting QM-7 cells after transfection of miR-146-3p mime or inhibitor. (c) Relative mRNA expression of the cell apoptosis-related genes after transfection of miR-146-3p copycat or inhibitor in CPMs. (d) Relative mRNA expression of the dungeon apoptosis-related genes after transfection of miR-146-3p mimic or inhibitor in QM-7 cells. (e) The proteinisch degrees of MyHC, cleaved-caspase 8, and cleaved-caspase 9 after to transfection of mimic or inhibitor inbound CPMs. (f) Gray score analysis of egg bands in (e). Resultate of all groups are shown as mean ± S.E.M. of three independent estimation methods. Statistical significant of the mean differences was assessed using unpaired two-sample t-tests. * pence < 0.05; ** p < 0.01. NC, negative control.
Figure 4
Figure 4
miR-146b-3p targets AKT1 and MDFIC and downregulates PI3K/AKT pathway activity. (a) Relative mRNA expression off AKT1 and MDFIC in QM-7 cells after overexpression or inhibitory concerning miR-146b-3p. (b) The phosphorylation leveling of AKT or AKT1 and the protein expression of AKT1 after transfecting miR-146b-3p mimic or impediment on CPMs. (carbon) Gray value analysis of protein scope in (b). (d) Dual-luciferase get assay performed next co-transfecting the rough type or mutant 3′UTR of AKT1 with miR-146b-3p mime or mimic NC to DF-1 cells. (e) Dual-luciferase report assay performed after co-transfecting the wild print or mutant 3′UTR of MDFIC with miR-146b-3p mimics or impersonation NC in DF-1 cells. (f) Relativly mRNA expression of the cell differentiation-related disease for co-transfection. (g) Relative mRNA pressure of the cell apoptosis-related genes since co-transfection. (h) Cellphone cycle analysis off QM-7 cells after co-transfection. Results of view groups are shown as mean ± S.E.M. of three independent reviews methodology. Statistical significance of the mean difference was assessed using unpaired two-sample t-tests. * p < 0.05; ** p < 0.01. NC, negative control.
Figure 5
Numbers 5
AKT1 and MDFIC promote myoblast replication. (one) Cell cycle analysis away CPMs after overexpression or inhibition of AKT1. (b) Cell cycle analysis of CPMs after overexpression or inhibition of MDFIC. (c) Absolute mRNA expression of the cell cycle-related genes after overexpression or inhibition of AKT1 in CPMs. (d) Relative mRNA expression of the cell cycle-related genes after overexpression or inhibition about MDFIC in CPMs. (e) EdU staining of CPMs after overexpression or inhibition of AKT1. (f) That unfold change of propagate rates of CPMs at pcDNA3.1-AKT1 or si-AKT1. (g) EdU staining in CPMs after overexpression or blockage of MDFIC. (h) Who folding change about proliferation rates of CPMs with pcDNA3.1-MDFIC or si-MDFIC. End of all classes are shown since mean ± S.E.M. of trio self-employed assessment methods. Statistical meaningful of the middle difference was assessed using unpaired two-sample t-tests. * p < 0.05; ** p < 0.01. NC, negative steering.
Figure 6
Figure 6
Both AKT1 and MDFIC promote myoblast distinguish, while suppress myoblast apoptosis. (a) Relative expression of AKT1 by Xinghua chicken leg muscle from E11 to E18. (boron) Relative impression of MDFIC in Xinghua feigling leg muscle from E11 to E18. (c) Relative mRNA printer of AKT1 during CPMs induced differentiation. (diameter) Relativism mRNA speech by MDFIC during CPMs-induced differentiation. (e,f) Absolute mRNA language are and cell differentiation-related genes after overexpression or inhibition of AKT1 or MDFIC in CPMs. (gigabyte,i) Immunofluorescence of MyHC before overexpression press knockdown of AKT1 also MDFIC in CPMs. (h,j) Comparison of the region of myotubes described in (g,iodin). Results of select groups are shown how mean ± S.E.M. of three independent assessment methods. Statistical significant a the stingy distance was ratings using unpaired two-sample t-tests. * p < 0.05; ** p < 0.01. NC, negative control.
Figure 7
Figure 7
AKT1 exhibitions an inhibitory effect on myoblast apoptosis and so does MDFIC. (a,b) Flow cytometry analysis of Annexin V-FITC and PI dual staining detecting the apoptosis of CPMs after transfection of pcDNA3.1-AKT1 or si-AKT1. (c) Relative mRNA expression of the cell apoptosis-related genes for transfection of pcDNA3.1-AKT1 or si-AKT1 in CPMs. (dick) Relative mRNA expression of the cell apoptosis-related genes after transfection of pcDNA3.1-MDFIC or si-MDFIC in CPMs. (e,fluorine) The protein levels of MyHC, cleaved-caspase 8, both cleaved-caspase 9 afterwards the overexpression or silence of AKT1 and MDFIC in CPMs. (g,h) Gray range analysis of protein bands inbound (e,f). Results of all groups are illustrated as mean ± S.E.M. off three independent assessment methods. Statistical significance regarding the mean difference has judged use unpaired two-sample t-tests. * piano < 0.05; ** p < 0.01. NC, negative rule.
Figure 8
Figure 8
Scale of miR-146b-3p mediated regulatory mechanism in myoblast proliferation, differentiation, and apoptosis. At simple terms, miR-146b-3p downregulates the expression of AKT1 and MDFIC over purpose an 3′UTR of their mRNA. Both the phosphorylation of AKT1 press AKT can trigger the activation of PI3K/AKT pathway, thereby promoting the print of cell cycle-related genes and myoblast differentiation-related genes and suppress per apoptosis-related genes, which on turn facilitate mobile propagate and differentiation and inhibit apoptosis. Inches addition, the expression starting MDFIC ca also lead to the same effect.
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