MiR-147 inhibits cyclic mechanical stretch-induced apoptosis in L6 myoblasts via ameliorating endoplasmic reticulum stress by targeting BRMS1

Abstract

Functional orthopedic treatment is effective for the correction of malformation. Studies demonstrated myoblasts undergo proliferation and apoptosis on certain stretch conditions. MicroRNAs (miRNAs) function in RNA silencing and post-transcriptional regulation of gene expression, and participate in various biological processes, including proliferation and apoptosis. One hypothesis suggested that miRNA was involved into the procedure via suppressing its target genes then triggered endoplasmic reticulum stress-induced apoptosis. Therefore, miRNAs play important roles in the regulation of the proliferation and apoptosis of myoblasts. In our study, the miR-147 has been explored. A cyclic mechanical stretch model was established to observe the features of rat L6 myoblasts. The detection of mRNA and protein levels was performed by qRT-PCR and western blot. L6 cell proliferation/apoptosis was checked by CCK-8 assay, DNA fragmentation assay, and caspase-3 activity assay. MiRNA transfections were performed as per the manufacturer's suggestions: (1) cyclic mechanical stretch induced apoptosis of L6 myoblasts and inhibition of miR-147; (2) miR-147 attenuated cyclic mechanical stretch-induced apoptosis of L6 myoblasts; (3) miR-147 attenuated cyclic mechanical stretch-induced L6 myoblast endoplasmic reticulum stress; (4) BRMS1 was a direct target of miR-147 in L6 myoblasts; (5) miR-147/BRMS1 axis participated in the regulation of cyclic mechanical stress on L6 myoblasts. MiR-147 attenuates endoplasmic reticulum stress by targeting BRMS1 to inhibit cyclic mechanical stretch-induced apoptosis of L6 myoblasts.

Keywords: Apoptosis; Cyclic mechanical stretch; ER-Stress; MiR-147; Myoblast.

Fig. 1

Cyclic mechanical stretch induced apoptosis and miR-147 suppression in L6 myoblasts. L6 myoblasts were continually stretched for 0, 6, 12, and 24 h. a The proliferation of L6 myoblasts was detected by CCK-8 assay. b, c The apoptosis of L6 myoblasts was detected by caspase-3 activity assay and DNA fragmentation assay. d The expression level of miR-147 in L6 myoblasts was analyzed by qRT-PCR. The data represent the mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with control group. Student’s t test

Fig. 2

MiR-147 alleviated cyclic mechanical stretch-induced apoptosis in L6 myoblasts. L6 myoblasts were not subjected to continually stretch and transfected with mimic negative control (Control + miR-NC), stretched for 24 h and transfected with mimic negative control (Stretch + miR-NC) or stretched for 24 h and transfected with miR-147 mimics (Stretch + miR-147). a qRT-PCR analyzed the expression level of miR-147 in these treated L6 myoblasts. b The proliferation of these treated L6 myoblasts was detected by CCK-8 assay. c, d The apoptosis of these treated L6 myoblasts was detected by caspase-3 activity assay and DNA fragmentation assay. The data represent the mean ± SD from three independent experiments. **P < 0.01 compared with Control + miR-NC; ^##P < 0.01 compared with Stretch + miR-NC. Student’s t test

Fig. 3

MiR-147 ameliorated cyclic mechanical stretch-induced ER-Stress in L6 myoblasts. L6 myoblasts were continually stretched for 0 h and transfected with mimic negative control (Control + miR-NC), stretched for 24 h and transfected with mimic negative control (Stretch + miR-NC) or stretched for 24 h and transfected with miR-147 mimics (Stretch + miR-147). a–c The relative mRNA levels of ER-Stress-related genes: GRP78, CHOP, and ATF4 in these treated L6 myoblasts were determined by qRT-PCR. d The protein levels of ER-Stress-related genes: GRP78, CHOP, and ATF4 in these treated L6 myoblasts were detected by western blot. The data represent the mean ± SD from three independent experiments. **P < 0.01 compared with Control + miR-NC; ^##P < 0.01 compared with Stretch + miR-NC. Student’s t test

Fig. 4

BRMS1 was a direct target of miR-147 in L6 myoblasts. a A schematic diagram showed predicted binding site of miR-147 in the 3′UTR of BRMS1, with sequences of the wild type BRMS1 3′UTR (BRMS1 3′UTR-WT) and the mutant (BRMS1 3′UTR-MT). b Luciferase reporter assays of L6 cells and HEK 293T cells co-transfected with constructed luciferase reporter vectors (psi-CHECK2, BRMS1 3′UTR-WT, BRMS1 3′UTR-MT) and miR-147 or negative control mimics (miR-NC). c Detection of BRMS1 mRNAs in biotinylated miRNA/target mRNA complex by real-time RT-PCR. The relative level of BRMS1 mRNA in the complex pulled down by using biotinylated miR-147 (Bio-miR-147) was compared to that of the complex pulled down by using the biotinylated control random RNA (Bio-miR-NC). d, e The relative expression levels of BRMS1 in L6 cells transfected with miR-147 mimics and miR-147 inhibitor or their respective negative controls were detected by qRT-PCR and western blot. The data represent the mean ± SD from three independent experiments. *P < 0.05; **P < 0.01, ns = not significant compared with the control group. Student’s t test

Fig. 5

MiR-147/BRMS1 axis was involved in the effects of cyclic mechanical stretch on L6 myoblasts. L6 myoblasts were continually stretched for 24 h and co-transfected with mimics negative control and empty vector (miR-NC + VEC), miR-147 mimics and empty vector (miR-147 + VEC), or miR-147 mimics and BRMS1 overexpressing plasmid (miR-147 + BRMS1-OE). a, b The mRNA and protein levels of BRMS1 in these treated L6 myoblasts were analyzed by qRT-PCR and western blot, respectively. c The proliferation of these treated L6 myoblasts was detected by CCK-8 assay. d, e The apoptosis of these treated L6 myoblasts was detected by caspase-3 activity assay and DNA fragmentation assay. f, g The mRNA and protein levels of ER-Stress-related genes: GRP78, CHOP, and ATF4 in these treated L6 myoblasts were detected by qRT-PCR and western blot, respectively. The data represent the mean ± SD from three independent experiments. **P < 0.01 compared with miR-NC + VEC; ^##P < 0.01 compared with miR-147 + VEC. Student’s t test