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

targeting studies have shown that MSTN-deficient mice information as follows: C2C12 cell line from Wuhan
exhibit a dramatic increase in overall skeletal muscle mass, Punosey Life Technology Co. Ltd (China); pX601 and
with individual muscle sizes approximately twice their pKO.1 plasmid carriers from Chengdu Chuanshikewei
normal size. Subsequent studies have shown that the MSTN Biotechnology Co. Ltd (China); TRIzol reagent from
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gene is highly conserved throughout evolution, with the Invitrogen (USA); DNA glue recovery kit and cell
mature MSTN amino acid sequences remaining identical genome DNA extraction kit from Shanghai Shenggong
across different species. Targeting or naturally mutating Biotechnology Co. Ltd. (China); HiScript II 1 Strand cDNA
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st
MSTN in cattle, sheep, dogs, pigs, and humans leads Synthesis Kit from Nanjing Nuoweizan Biotechnology Co.
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to significant muscle gain. In addition to its role in muscle Ltd (China); TurboFect transfection reagent from Promega
growth regulation, MSTN also plays an important role in (USA); DH5α competent cells from Takara Bio Co. Ltd
bone development, fat browning, and metabolism. 9-11 (Japan); high glucose DMEM medium, fetal bovine serum
Therefore, understanding the molecular mechanism of (FBS), phosphate-buffered saline (PBS), and trypsin were
the MSTN gene is paramount for identifying active molecules purchased from Gibco Co. Ltd (USA); plasmid extraction
associated with MSTN regulation and for developing drugs kit from QIAGEN Corporation (Germany); Q5 DNA
that target this pathway. Previous studies have primarily polymerase, Bsa I endonuclease, BamH I endonuclease,
focused on cytokines regulated by MSTN, 12-14 with fewer EcoR I endonuclease, 10 × NEBuffer 2, T4 DNA ligase,
studies reporting on the effect of microRNA (miRNA) and T7E I endonuclease from NEB Co. Ltd (USA);
variation on muscle growth and development. MiRNAs DL2000 Marker, DL10000 Marker, 50 bp DNA Ladder,
®
are evolutionarily conserved molecules widely present in TB Green Fast qPCR Mix from Takara Co. Ltd (Japan);
various organisms and play roles in fundamental biological Murine Anti-MSTN antibody, murine anti-GAPDH
processes such as cell proliferation, differentiation, antibody, and horseradish peroxidase-coupled Goat
apoptosis, and tumorigenesis. 15,16 Notably, a subclass of anti-mouse IgG from Abcam Limited (UK); protease
miRNAs specifically exists in muscle tissues and plays an inhibitor, strong RIPA lysate, and bicinchoninic acid assay
important role in muscle development. 17,18 Rachagani et al. (BCA) protein quantitative detection kit from Biyuntian
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demonstrated that MSTN might regulate the expression of Biotechnology Co. Ltd. (China); CCK-8 kit from Dongren
miR-133a/b, miR-1, and miR-206 in skeletal muscle, with Chemical Technology (Shanghai) Co. Ltd. (China); and
these miRNAs being significantly upregulated in Mstn- fluorescent dye propidium iodide (PI) from Beijing
knockout (Mstn-KO) mice compared with wild-type and Zhuangmeng International Biological Gene Technology
heterozygous mice. In addition, the findings of Wada et al. Co. Ltd (China).
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demonstrated that miR-23a is associated with atrophy 2.2. Design of sgRNA targeting sites in the Mstn
and hypertrophy processes, inhibiting the expression gene
of ubiquitin protease (Murf-1 and Atrogin-1 mRNA)
to prevent atrophy in skeletal muscle. This inhibition is The mice Mstn gene (NM_010834.3) was downloaded
consistent with the signaling pathway effects observed after from the NCBI database (https://www.ncbi.nlm.nih.gov/).
Mstn knockout, suggesting a potential interaction. 20,21 The The Mstn exons sequence was uploaded into the sgRNA
regulatory effects of MSTN on miRNA expression related Design software (http://www.broadinstitute.org/rnai/
to muscle growth and development and the specific signal public/analysis-tools/sgrna-design). Based on the score
transduction pathways involved remain unclear. and the 5’ region of the exon, two pairs of suitable sgRNAs
were selected and named sgRNA1-MSTN and sgRNA2-
This study aims to establish a Mstn-KO C2C12 cell line
and verify whether MSTN regulates skeletal muscle growth MSTN. Fragments of ACCT and AAAC were added to the
5’ end of the sgRNA sense and antisense chain templates,
and development through associated miRNA changes. respectively, to complement the adhesive ends of the pX601
The results are intended to lay the foundation for further carrier after Bsa I endonuclease digestion. The primers for
research on the active molecules and cellular mechanisms
of MSTN at the miRNA level, ultimately shedding light detecting target site knockout were designed and identified
on the development of drugs that block MSTN signal (Table 1). The sgRNA single nucleotide chains and related
transduction. primers were synthesized by Shenggong Biotechnology
Co. Ltd. (China).
2. Materials and methods 2.3. Vector construction

2.1. Experimental materials The puromycin (Puro) resistance gene was amplified
The experimental materials used in this study were from the pLKO.1 vector using the designed hPGK-puro
purchased from various suppliers, with detailed related upstream and downstream primers. The polymerase

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