Page 119 - GPD-3-2

Gene & Protein in Disease The effect of myostatin on muscle-related miRNAs

A B A

B

Figure 2. Assessment of knockout efficiency of pX601-SaCas9-sgRNA
in C2C12 cells. (A) Agarose gel electrophoresis results demonstrating
knockout efficiency at the sgRNA1 site. Notes: M: 50 bp DNA Ladder;
1: Control cells transfected with empty pX601-SaCas9 plasmid; 2 – 4:
Gene-edited cells transfected with pX601-SaCas9-sgRNA1 plasmid. Figure 3. (A and B) Sequencing identification results for the C2C12
(B) Agarose gel electrophoresis results illustrating knockout efficiency at monoclonal cell
the sgRNA2 site. Notes: M: 50 bp DNA Ladder; 1: Control cells transfected Abbreviation: PAM: Protospacer adjacent motif.
with empty pX601-SaCas9 plasmid; 2 – 4: Gene-edited cells transfected
with pX601-SaCas9-sgRNA2 plasmid. The estimated percentage of gene sgRNA2-KO group (P < 0.01). These results indicate that
editing per sample is indicated at the bottom.
Mstn knockout upregulates the proliferative characteristics
line exhibited a 20-base deletion at the target compared of C2C12 cells, leading to an increased cell number
to the standard sequence. The 2# cell line demonstrated (Figure 4B). The cell cycle distribution of CN, sgRNA1-KO,
a minimum of 13 base mutations at the target, while the and sgRNA2-KO cell lines at the G0/G1, G2/M, and S
3# cell line had a 4-base deletion and a 6-base mutation phases was analyzed through flow cytometry. The results
at the target site (Figure 3A). Similarly, in the sgMSTN2 indicate that the proportion of cells in the G0/G1 phase
group, the 1# cell line displayed a 4-base deletion at the decreased from 81.19% in the CN group to 51.79% in the
target site compared to the standard sequence. The 2# cell sgRNA1-KO group and 73.69% in the sgRNA2-KO group.
line exhibited a 6-base deletion at the target site, and the 3# Conversely, the proportion of the G2/M phase increased
cell line had a 4-base deletion at the target site (Figure 3B). from 5.60% in the CN group to 10.83% in the sgRNA1-KO
group and 9.12% in the sgRNA2-KO group. In addition,
3.4. Identification of MSTN protein expression and the proportion of cells in the S stage increased from 13.22%
proliferative activity in the Mstn-KO C2C12 cell line in the CN group to 37.37% in the sgRNA1-KO group and
The expression levels of MSTN protein in each monoclonal 17.20% in the sgRNA2-KO group. These results indicate
cell line were determined using Western blotting. that after Mstn knockout, C2C12 cells exhibit increased
Monoclonal cell lines sgRNA1-KO and sgRNA2-KO, activity in the division phase and enhanced proliferative
exhibiting complete non-expression of MSTN protein, capacity (Figure 4C).
were selected from the sgMSTN1 and sgMSTN2 groups for 3.5. Expression of muscle-associated miRNA in the
subsequent study. The expression levels of MSTN protein Mstn-KO C2C12 cell line
in the sgRNA1-KO, sgRNA2-KO, and CN groups are
illustrated in Figure 4A. The proliferative characteristics To verify the effect of MSTN on miRNA expression, we
of C2C12 cells following Mstn knockout were assessed selected six miRNAs reported to influence or be related
using the CCK-8 method. Compared with the CN group, to muscle development for real-time PCR analysis.
the optical density (OD) value at 450 nm was significantly We observed that, compared with the CN group, the
increased in the sgRNA1-KO group (P < 0.01) and the expression levels of miR-1, miR-431, miR-206, and miR-

Volume 3 Issue 2 (2024) 6 doi: 10.36922/gpd.2991