DMD is a disease characterized by severe, progressive muscle degeneration associated ultimately with cardiac and pulmonary dysfunction (Bushby et al., 2010). Our study revealed 1179 up-regulated DEGs that are mainly enriched in the immune response, ECM structure-associated activity, and viral myocarditis, as well as 324 down-regulated DEGs that are mainly enriched in muscle structure, regulation, and function. These results are consistent with the notion that the immune system plays an important role in dystrophic muscle disease pathogenesis, sustaining continuous repetitive cycles of inflammatory and fibrotic responses (Giordano et al., 2015, Mojumdar et al., 2014).
KEGG analysis of the top four modules in the PPI networks showed that ECM-receptor interaction, chemokine signaling pathways, complement and coagulation cascades, and calcium signaling pathways were dysfunctional in DMD. This is in line with previous studies that demonstrated that immune cell infiltration of the muscles in mdx mice and transforming growth factor-β (TGF-β)-mediated inflammation could cause the progressive deposition of fibrous ECM (Wehling-Henricks et al., 2008). Moreover, chronic damage and inflammation in DMD has been shown to induce elevated TGF-β activity, which allows fibroadipogenic progenitors to differentiate into fibrogenic and other ECM-secreting cells thus leading to muscle fiber calcification (Mázala et al., 2020). Encouragingly, a recent study revealed that regulating TGF-β1/Smad3 signaling by the coreceptor for TGF-β receptor type Ⅱ (TβR II) could reduce muscle-wasting (Zhang et al., 2019). Similarly, calcium homeostasis in myoblasts was altered profoundly by the mutant Dmd gene (Róg et al., 2019).
In the current study, however, CXCL12 was screened as hub nodes but not deemed as target hub gene due to the involvement of immune response, but this result was highly consistent with another study which was also based on the GSE 38417 dataset (Lai and Chen, 2021). Lai et al revealed that CXCL12 was a glucocorticoid targeted DEG and thereby a potential therapeutic target in DMD. Among the five hub nodes which were not associated with the immune response, COL1A2,FN1 and FBN1,were significantly up-regulated in older DMD patients analyzed by bioinformatics and mdx mice detected or calculated by RT-qPCR and WB. COL1A2 encodes the alpha chain of type I collagen, and whose significantly higher expression in DMD than controls has been indicated by the previous study which further determined that the alpha chain of type I collagen accumulation is responsible for the skeletal muscle fibrosis in DMD (Ieronimakis et al., 2016). As the result of our study, over-expression of FBN1 induced DMD, however, patients with Marfan syndrome (MFS), which is caused by an FBN1 mutation as well as Fbn1-deficient mice present some phenotypes similar to DMD, such as a decrease in the size and number of myofibers accompanied by an increase in fragmented fibers (Siegert et al., 2019, Percheron et al., 2007, Cohn et al., 2007, Behan et al., 2003). An additional study demonstrated that FBN1, which is a crucial component of connective tissue elastic fibers and an important extracellular regulator of TGF-β activity, could be linked to muscle atrophy and impaired muscle regeneration. Therefore, FBN1 may have a significant supporting effect on maintaining the structure and function of muscle, and both low- and over-expression of FBN1 could induce muscle dysfunction (Neptune et al., 2003, Burks et al., 2011). Moreover, over-expression of the extracellular matrix glycoprotein FN1 was also detected in our experimental data. This is highly consistent with previous studies that revealed fibronectin is a serum biomarker for Duchenne muscular dystrophy(Cynthia Martin et al., 2014).Another study also revealed that up-regulation of FN1 induced the deposition of fibronectin in the cytoplasm, which causes fibrosis (Peng et al., 2020). Finally, other studies demonstrated that activated fibroblasts proliferate and express high levels of extracellular proteins, which leads to the expansion of fibrotic tissue (Morgan and Partridge, 2020).
The lack of significantly high expression of Fyn and Prkacb in mdx mice may be attributed to the milder phenotypes that these mutations cause as compared with that seen in DMD patients. FYN is a member of the Src family of nonreceptor tyrosine kinases that plays a role in many biological processes including regulation of cell growth and survival, integrin-mediated signaling, cytoskeletal remodeling, and cell motility. One of the mechanisms of up-regulated FYN, which could account for the DMD phenotype is that the Fyn-tyrosine kinase activates the mammalian target of rapamycin 1 (mTORC1) signaling complex, which inhibits macroautophagy and induces marked muscular atrophy (Saito et al., 2010). PRKACB is another gene that plays an important role in cardiac and skeletal muscles. Several studies have asserted that upon equal stimulation, myocytes exhibit stronger contractions in the presence of β-agonists because of the induced increase in the levels of cAMP (Steinberg and Brunton, 2001, Rudolf et al., 2006). Furthermore, treatment with β-agonists up-regulated PRKACB when compared with controls (Zhao et al., 2016). Therefore, the high expression level of PRKACB in DMD, which is similar to up-regulated PRKACB upon β-agonist treatment, is probably a result of genetic compensatory response to the muscle degeneration in DMD. However, a recent study has illustrated that PRKACB has a close relationship with immune cells, especially M2 macrophages (Zhao, 2021). Therefore, the regulation mechanism of PRKACB requires further study.
siRNAs have been shown to play important roles in gene regulation that impact various diseases. In the final content of our study, rational siRNAs targeted to the coding sequence of three up-regulated hub genes were synthetized according to design principals(Reynolds et al., 2004), and transfected into C2C12 cell to regulate the expression of the targeted genes. RT-qPCR indicated that those siRNAs could significantly down-regulate the mRNA expression of the target gene. This results further suggested the therapeutic potential of these siRNAs. However, how to safely and efficiently deliver siRNA drugs to specific target cells and protect them from degradation is one of the major obstacles of current siRNA therapy. Lipid nanoparticles (LNP) are the most advanced siRNA delivery vectors in clinical practice. However, clinical studies have shown that LNP accumulates in the liver, so current LNP delivery systems are mostly liver targeted, and effective delivery of LNP in muscle needs to be addressed urgently. In a recent study, a selective organ targeting lipid nanoparticles named SORT (selective organ targeting) were developed to specifically target liver, lung, spleen and other organs by adding a new lipid SORT lipid (Cheng et al., 2020). Meanwhile, the specific mechanism of tissue-specific delivery of selective organ targeted lipid nanoparticles has also been clarified. They believe that adjusting the molecular composition of nanoparticles to bind to specific proteins in serum can be delivered to the target site. This may be an effective strategy for developing the muscle target nanocarriers, and help to deliver the siRNA-therapeutics to DMD patients to mitigate the DMD progress.
In summary, COL1A2, FBN1, and FN1 were hub genes irrespective of immune response but responsible for DMD progression. The siRNAs designed in our study were help to develop adjunctive therapy for Duchenne muscular dystrophy.