3.1. Identification of primary BSMSCs
Culturing primary BSMSCs in complete medium to 80% confluence, then replacing the complete medium with 2% horse serum differentiation medium. Microscopic observation of morphological changes of myotubes differentiation at 2d, 4d, and 6d, the mRNA expression levels in differentiation at 2d, 4d, 6d, and 8d was detected by RT-qPCR. The results demonstrated that the cells were spindle-shaped during the proliferation period(Fig 1a). As cells differentiated, the number of myotubes increased gradually (Fig 1b). In addition, the mRNAs of MYOD1, MYOG, and MYH3 were differentially expressed at differentiation periods, which were closely related to myocyte differentiation. Compared to 2d, the MYOD1, MYOG, and MYH3 expression levels were higher in 6d and 8d (Fig 1c). The above results suggest that the differentiation model of primary BSMSCs has been constructed successfully in vitro.
3.2. Expression pattern analysis of bta-miR-144
To explore the influence of bta-miR-144 on skeletal muscle, this study compared the conservation and sequence features of miR-144 among different species using miRbase software (https://www.mirbase.org/). The results indicated that the mature sequence of miR-144 was not different among humans, bovines, mice, pigs, and chickens, indicating that the miR-144 mature sequence was strongly conserved (Fig 2a). In different tissues between newborn calf and adult cattle, the miR-144 expression pattern was investigated by RT-qPCR. The results found that miR-144 was expressed highly in the newborn calf ' liver, heart, and leg muscles, with the lowest expression in the rumen and subcutaneous fat (Fig 2b). In addition, compared to the newborn calf, miR-144 was higher in heart and longissimus dorsi muscle tissues in adult cattle (P<0.05), while significantly lower in subcutaneous fat (P<0.01) (Fig 2c). Total cellular RNAs were extracted when primary BSMSCs reached 30%, 50%, 70%, and 100 % confluence, and differentiation 0d (70%), 2d, 4d, 6d, and 8d, respectively, and then detecting miR-144 expression levels by RT-qPCR. It was demonstrated that miR-144 expression levels decreased significantly with increasing cell confluence(Fig 2d), but with the differentiation, which was increased and higher at 6d (Fig 2e). The above studies preliminarily revealed that bta-miR-144 expression levels in muscle tissues were higher than those in other tissues.
3.3. Effect of miR-144 on BSMSCs proliferation and differentiation
To detect the function of bta-miR-144 in the proliferation and differentiation, transfecting miR-144 mimic/inhibitor into BSMSCs. As shown in Fig 3a, the miR-144 expression levels in the mimic group were significantly increased than mimic NC, however, the results were reversed after inhibition of miR-144, indicating a better transfection efficiency. CCK-8 assay confirmed that miR-144 mimic decreased cell viability (Fig 3b), instead miR-144 inhibitor increased cell viability(Fig 3c). Moreover, compared to the mimic NC, the CyclinD1, PCNA, CDK2, and CDK4 were decreased in the miR-144 mimic (Fig 3d). In contrast, CyclinD1, PCNA, CDK2, and CDK4 were notably higher in the miR-144 inhibitor (Fig 3e). In the mimic or inhibitor group, PCNA, CDK2, and CDK4 proteins were consistent with the mRNA (Fig 3f). Furthermore, over-expression of miR-144 significantly increased MYOD1, MYOG, and MYF6 expression levels in mRNA and protein, but the opposite result was obtained after interfering with miR-144 (Fig 3g-i). These results suggest that miR-144 inhibits BSMSCs proliferation and promotes differentiation.
3.4. RNA-Seq analysis of overexpression bta-miR-144 in BSMSCs
To further clarify the miR-144 regulatory role on BSMSCs development, RNA-seq was performed after overexpression of miR-144. Table S2 lists the specific information of the RNA-seq data. Furthermore, compared to the mimic NC, 310 DEGs were increased and 166 DEGs were decreased in the miR-144 mimic(P < 0.05) (Fig 4a, Table S3). The top 20 up-regulated DEGs with the highest fold change mainly included FBP1, EGR3, and FGF18 (Table 1, Table S4), these genes were associated with muscle development and skeletal muscle cell differentiation [40, 41]. Go enrichment analysis showed that muscle tissue development (GO:0060537, P=0.00004), neuromuscular process (GO:0050905, P=0.00014), and heart development (GO:0007507, P=0.00046) were the main enriched GO terms (Fig 4b, Table S5). Meanwhile, the DEGs were mainly enriched in the adrenergic signaling pathway in myocardial cells, MAPK, and PI3K-AKT signaling pathway (Fig 4c, Table S6). To verify the RNA-seq results accuracy, 12 DEGs were validated by RT-qPCR. Based on the results, the expression levels of 12 DEGs were consistent with those in the RNA-seq results (Fig 4d).
Table 1. Top 20 differentially expressed up-regulated genes
Gene name
|
M FPKM Mean
|
NC FPKM Mean
|
FDR
|
log2FC
|
F13A1
|
7.035039
|
1.318023
|
0.00000
|
1.870328
|
ZAN
|
0.597852
|
0.091796
|
0.00000
|
1.718992
|
SCN7A
|
0.451005
|
0.098903
|
0.00000
|
1.584998
|
FBP1
|
5.458683
|
1.044844
|
0.00000
|
1.416713
|
EGR3
|
5.947703
|
1.995266
|
0.00000
|
1.314022
|
SESN1
|
15.40213
|
5.032959
|
0.00000
|
1.308761
|
TRIM29
|
1.604893
|
0.393962
|
0.00000
|
1.241698
|
GFRA3
|
10.83929
|
2.573364
|
0.00000
|
1.189316
|
EGR2
|
2.194426
|
0.657466
|
0.00000
|
1.139428
|
SUSD4
|
4.134281
|
1.345084
|
0.00000
|
1.13047
|
FGF18
|
3.708817
|
1.289289
|
0.00000
|
1.129788
|
EPHA1
|
1.09562
|
0.291105
|
0.00001
|
1.127428
|
GRHL3
|
5.50091
|
1.458888
|
0.00001
|
1.126464
|
GREB1
|
0.322969
|
0.091872
|
0.00001
|
1.11084
|
INA
|
1.502615
|
0.499747
|
0.00000
|
1.10667
|
NEXMIF
|
0.562676
|
0.183181
|
0.00000
|
1.10643
|
SERPINB10
|
2.14725
|
0.794903
|
0.00000
|
1.101352
|
CEND1
|
5.229122
|
1.590594
|
0.00002
|
1.081006
|
LY6G6C
|
3.854651
|
0.977842
|
0.00005
|
1.079324
|
ALS2CL
|
0.503287
|
0.166944
|
0.00000
|
1.075241
|
M: miR-144 mimic; NC: mimic NC
3.5. The target gene of bta-miR-144 was NACC1
To further investigate the downstream targets, bta-miR-144 potential target genes were predicted, and then 29 gene sets were screened by TargetScan, miRDB, and RNA-seq (Table 2, Fig 5a). Sequence comparison analysis showed that miR-144 was highly conserved in different species (Fig 5b). To ascertain the interaction between the bta-miR-144 and the NACC1 gene, we introduced mutations at the miR-144 binding sites within the NACC1 3′-UTR (Fig 5c). Dual-luciferase reporter assays showed that miR-144 mimic significantly inhibits the NACC1-3′-UTR-Wt luciferase activity, while there was no remarkable difference with NACC1-3′-UTR-Mut in the 293T cells (Fig 5d). Furthermore, NACC1 gene expression levels were significantly down-regulated after over-expression of miR-144(P<0.05), while transfection of miR-144 inhibitor significantly increased NACC1 gene expression levels(P<0.01) (Fig 5e). These results suggest that the target of bta-miR-144 was the NACC1 gene.
Table 2. The target gene statistics of bta-miR-144
Source
|
List of primary target genes
|
TargetScan
|
NACC1、MAFK、TFAP4、SNTB2、PHACTR2、CAV3、PIM1、ATP1B1
|
miRDB
|
NACC1、MAFK、TFAP4、SNTB2、NACC2、ATP1B1、MAFK、MDM4
|
RNA-seq
|
NACC1、MAFK、TFAP4、SNTB2、AFF4、MDM4、ADAMTS15、NACC2、ATP1B1
|
TagetScan∩miRDB∩RNA-seq
|
NACC1、UBE2D1、ADAMTS15、AFF4、ATP1B1、CALCRL、CAV3、EIF5A2、HTRA3、ITPR1、JAKMIP2、LIFR、MAFK、MDM4、MED12L、MMP15、NACC2、PHACTR2、PHTF2、PIM1、PLXNC1、RASGRF1、RGMA、RNF125、ROBO2、SLC20A2、SNTB2、TFAP4、TMED5
|
3.6 Effect of NACC1 on BSMSCs proliferation and differentiation
To explore the influence of the NACC1 gene on the BSMSCs development, the NACC1 was examined at different periods. The results revealed that the expression levels of the NACC1 gene gradually increased and were higher at 70% and 100% confluence(P<0.01)(Fig 6a). During the differentiation period, the NACC1 gene expression levels gradually increased from 0d to 4d and then decreased to 4d (P<0.01) (Fig 6b), which was the opposite of the expression pattern of miR-144. Furthermore, we designed pcDNA3.1-NACC1 and si-NACC1 vectors and then transfected them into BSMSCs. The results found that the transfection efficiency satisfied the experimental requirements(Fig 6c-d). In addition, pcDNA3.1-NACC1 significantly increased the PCNA, CDK2, and CDK4 expression levels; In contrast, knockdown NACC1 significantly decreased the PCNA, CDK2, and CDK4 expression levels(Fig 6e-g). Furthermore, overexpression of NACC1 inhibits the MYOD1, MYOG, and MYF6 expression levels, but the MYOD1, MYOG, and MYF6 expression levels were significantly increased when transfecting si-NACCA(Fig 6h-j). In conclusion, these results suggest that miR-144 regulates BSMSCs mainly by targeting the NACC1 gene.