Identication of key pathways and hub genes in the myogenic process of pluripotent stem cell: A bioinformatics study

Purpose: The study aims to determine the process of myoginc differentiation in human pluripotent stem cells and to gure out that the key pathways and hub genes in the process, which do helpful for the further research of muscular regeneration. Method: Three gene expression proles, GSE131125, GSE148994, GSE149055, about the comparisons of pluripotent stem cells and myogenic stem cells from the Gene Expression Omnibus (GEO) data base. Common differentially expressed genes (DEGs) were obtained and for the further analysis as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA analysis and protein ‐ protein interaction (PPI) network. In vitro cell research to verify the hub genes and key pathways. Result:824 DEGs were co-expressed in the three GSEs. 19 hub genes were identied from the PPI network. The GO and KEGG pathway analysis were performed to determine the functions of DEGs. GSEA analysis indicated the differentiated cells were enriched in muscle cell development and myogenesis. Conclusion: Our research revealed the main hub genes and modules in the myogeinc process of stem cells which contribute to further study about the molecular mechanism of myogenesis regeneration. Paving a way for more accurate treatment for muscle dysfunction.


Introduction
As the aging of human population, muscle dysfunction has been an interrupting issue in clinical research. [1][2][3][4] A series of diseases were correlated to the atrophy of skeletal muscles and leading to dysfunction of muscular organs as Duchenne muscular dystrophy (DMD), degenerative rotator cuff tear etc [5][6][7]. Stem cells are promising cells that have the potency of multi-directional differentiation and proliferation and are widely expected to be used in the eld of tissue repair and regeneration. [8,9] In the muscle regeneration eld, stem cells also showed vigorous potency. [10] Various researches have devoted to verifying the mechanism of myoblast differentiation. Myogenic differentiation as a multistage process, there stays several regulating factors as Myf5, Myf6, myoD and myog. [11][12][13] Meanwhile, couples of pathways were veri ed to be correlated to myogenic differentiation as PI3K-MAPK, p38, p53 and actin pathway. [14][15][16][17] To up-regulate the differentiation e cacy and contribute to the repair of degenerated muscular tissues, it is especially important to clarify the differentiation mechanism at genetic level.
With the wide spread use and development of high-throughput sequencing, bioinformatics analysis showed great advantage for determining the myogenic differentiation mechanism of stem cell at genetic level. However, no study was designed to integrate the myogenic differentiation datasets in GEO. In the present study, we integrated 3 datasets in GEO comparing human pluri-potential stem cells and myogenic stem cells. Bioinformatics analysis was used to explore molecular mechanism of the pathogenesis in myogenic differentiation of stem cells.

Materials And Methods
Microarray data obtained: Three human gene expression pro les, GSE131125(GPL 20844, SurePrint G3 Human GE v3 8x60K Microarray 039494), GSE148994 and GSE149055(GPL16686, Affymetrix Human Gene 2.0 ST Array) were obtained from the GEO database. Both the GSE149055 and GSE148994 contained 6 human samples, of which 3 were undifferentiated stem cells and 3 were differentiated stem cells. GSE133125 contained 24 samples which include different timepoint of the differentiation. We choose the 3 undifferentiated stem cells and 3 differentiated for 25 days into our analysis.

Identi cation of differently expressed genes (DEGs)
The downloaded platform les were matched to the gene expression pro les by the "VLOOKUP" function of Excel 2010. Gene differential analysis was determined to summarized the differentially expressed genes (DEGs). The DEGs threshold of our study was |logFC|>1 and adj.P-value<0.01. Heatmaps of DEGs from 3 groups were generated by graphpad 8.0.2. Online tool Venn, version 2.1(bioinformatics.psb.ugent.be/webtools/Venn;version 2.1) was used to determine the common DEGs among the three pro les.

Cells sacri ced
The rabbit adipose derived stem cells were sacri ced in our laboratory from an eight-week-old Newzealand white male rabbit. The subpatellar fat pad was harvested for the further step. The remaining tissues were minced nely into the shape of chyle, mixed with collagenase type (Tianjin Haoyang Biotechnology Comparison, China) and moved to a 50 ml centrifuge tube. At the end of digestion, the tissues were transferred into a centrifuge machine (Sigma Centrifuge, Germany), and the rotator speed was set at 1700 r/min, which lasted 10 minutes. The top content and supernatant were discarded, leaving the lower white block mass content in the tube. After centrifuging the liquid for 3 minutes at 800 r/min and discarding the supernatant, the lower content containing the stem cells was obtained nally. Subsequently, we inoculated the stem cells into the ask lled with 5 ml DMEM of fetal bovine serum (FBS, Gibco Company, St Louis, MO, USA) and observed the morphological characteristics under the invert microscope (Leica, Germany). The cells were cultured with the DMEM of fetal bovine serum subjected to culture and change every three days.

Flow cytometry FCM
The cells were trypsinized, washed, and re-suspended in phosphate-buffered saline (PBS) and blocked with 3% fetal bovine serum for 15 minutes before the ow cytometry analysis. We divided the cells into 3 ADSCs tubes, labeling the three ADSCs tubes as blank, CD90 testing, and CD45 testing (Invitrogen, USA, and Gibco, USA). To every tube was added the corresponding uorescein-labeled antibody whose concentration was set at a paralleled level of 5μl/ml, and the tubes were incubated at 37℃ in a 5% CO2-saturated humidity oat tank for 30 minutes. We centrifuged the tubes and its contents at 1200 r/min for 5 minutes following the last incubation. We then discarded the supernatant, washed the remaining products twice with PBS, added appropriate amounts of 4% paraformaldehyde for 30 minutes, and assessed the mixture on the ow cytometry machine (BD FACSVerse, USA).

Tetrazolium method MTT
The passage-3 ADSCs were digested and diluted, and the mixture was transferred to a 96-well culture plate (Thermo

Quantitative real-time PCR
Total RNA was extracted from the ADSCs after induction of 9 days using Trizol lysate invitrogen . The schizolytic cells were then transferred into another tube without RNA enzymes, and 200 μl pre-cooling chloroform (Sigma Centrifuge, Germany) was added per milliliter of Trizol. The ccentrifugation yielded RNA sediments that were preserved in a -20℃ surrounding for 30 minutes. The sediments were washed with 75% ethyl alcohol and centrifuged for 5 minutes, and the supernatant was discarded after washing and centrifuging the sediments twice. The reverse transcription system was prepared using a reverse transcription kit (Thermo Scienti c, USA) according to instructions provided in the protocol of the kit.

Statistical analysis
Statistical analysis was performed on Graphpad 8.0.2. Expressed data were shown as mean±SD. Student's t test was used to evaluate the statistical signi cance of different 3 groups. P value less than 0.05 was considered as signi cant.

Identi cation of DEGs
The three datasets were standardized and the results are shown in Figure 1 Figure 1C, D and E. The DEGs in each group were mixed by the Venn plot. From the Venn polt shown in Figure 1A Figure 5B and C. The DEGs were enrich in "myogenisis" and "muscle cell development". The GSEA results were shown in Figure5 C and D, MYOD1 was "core enrichment" gene in both enriched pathways. Therefore, we set the MYOD1 as the hub gene and "actin cytoskeleton regulation" pathway as the mainly enriched functional pathway.

The expression of ADSCs and BMSCs surface markers
Speci c membrane markers con rmed the identity of ADSCs via ow cytometry. According to the results, the ADSCs results are presented in gure 6C, with a strong expression of CD90 at 82.8% positive and weak expression of CD45 at 4.58% positive and the results were shown in Figure6C.

Cell viability authenticated by MTT
The data were converted into gure6B to show cell viabilities when different concentrations of 5-Aza induced the cells. As is shown in the gure 6B,5-Aza do have does dependent and time dependent toxic effects on ADSCs. It can be calculated that the IC50 in ADSCs groups were 9.178μmol/l at 24hours, 6.469μmol/l at 48hours and 8.664μmol/l at 72hours after induction. The further induce concentration of 5-Aza was set as 0, 10 and 20μmol/l and named as group A, B and C, respectively.

Actin expression determined by immunohistochemistry
The results of the expression of actin were shown in Figure 6D. Actin was labeled and stained red by α-SAM, and the nucleus was stained blue by hochest, with the composed pictures showing that there were just parts of the cells expressing actin. The differentiated rate was calculated by graphpad 8.0.2. And the rate of each group was 0.019, 0.074 and 0.116 for groups A, B, and C, respectively.Of which the differentiate rate in group B and C was signi cantly up-regulated when compared to group A.(P<0.01) The content of myoD mRNA measured by RT-PCR We further used the RT-PCR technology to detect the content of the mRNA of myoD in each group under the induction of 5-Aza. The results were recorded at1.009, 2.391, and 4.876, respectively in each group. Of which the content in group C was signi cantly up-regulated than group B (p<0.05) whose content was also up-regulated compare to group A with signi cance(P<0.05).

Discussion
Mountainous efforts have been devoted to the research of pluripotent stem cells in our nowadays research for their regenerating and repairing damaged tissues effects. [18][19][20] In the musculoskeletal eld, the degenerated and decreased of muscle tissue have confused the clinical effects of various diseases. [21,22] The regeneration and remobilization of degenerated and damaged muscle tissues have been a hot issue in the research. [23] Stem cells, owning the myogenic differentiation, provide a possibility for current issue. However, the speci c key pathways and genes in the myogenesis of stem cells is still under mystic.
There stands various signaling pathways which were count in the myogenic differentiate process of stem cells. Fu, S reported that PI3K pathway related genes and proteins were up-regulated expressed in the myogenic differentiate courses of mouse stem cells [24]. Meanwhile, up-regulated p53 and actin signaling pathways were also proved to be responsible for the myogenesis of stem cells which were certi ed by Liu, L. [25] and Petschnik, A. E [26]. Except that, p38 signaling pathway and wnt pathway were both proved to be responsible for the process [27,28]. As for the myogenic genes, MRFs, myoD, myoG, etc [29,30] were all reported as myogenic related genes.
In the present study, a bioinformatics analysis was used to analysis the key pathways and hub genes in myogenesis of stem cells based on 3 GEO databases. According to the analysis, a total of 824 DEGs were hunted out and applied for the further GO and KEGG analysis to certify potential biological functions and pathways in myogenic differentiation. Except that, 111 genes from the top 3 clusters and 19 hub genes analyzed from the MCODE method were identi ed from the PPI network.
MyoD has been described as the decisive gene and component of diverting undifferentiated cells into myoblasts [31]. In Rudnicki [32] research, knock out of myoD and myf5 results in the prevention of formation of skeletal muscle in the embryo period. The study results revealed that myoD and myof5 were determined genes in the origination of muscle cells. In the present bioinformatic analysis, myoD showed key effects in the myogensis. In GO analysis of up-regulated genes, myoD showed signi cant in positive regulation of myoblast differentiation in BP, transcription factor complex in CC and chromatin binding, transcription factor binding, transcription factor activity in MF. Meanwhile, the GSEA analysis revealed that moyD were both core enriched elements in myogenesis and muscle cell development in the three GEO databases. The unit results from our analysis revealed that myoD can be one of the hub genes in the myogenic differentiating process. From the laboratory experiments, RT-PCR results revealed that myoD were exactly signi cantly up-regulated in myogenic induced stem cells.
Also, we performed KEGG analysis to trace out the exact relevant pathways in the myogenic differentation not only in the DEGs, but also based on the intensive module analysis from the PPI network. From the DEGs, the activation of PI3K, actin cytoskeleton regulation, p53 signaling pathway were proved to be tightly associated with myogenesis process. Meanwhile, the intensive analysis showed that actin cytoskeleton regulation pathway was also enriched.
From our laboratory experiments, the expression of actin was exactly signi cantly up-regulated in myogenic induced stem cells.
The study still has several limitations. Firstly, the included GEO pro les were still not rich enough. Secondly, the speci c genes regulations in different time point of differentiation were omitted in our study. We still need to conduct further validate experiment to proof our speculation in the future.

Conclusion
Our study identi ed a series of DEGs in the myogenic differentiation process compared to undifferentiated stem

Declarations
Ethics approval and consent to participate This study was approved by ethics of committee of Northern Jiangsu People's Hospital.

Consent for publish
Not applicable.

Availability of data and materials
The data used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing Interest
The authors declare that they have no competing interests.