Acta Veterinaria et Zootechnica Sinica ›› 2024, Vol. 55 ›› Issue (10): 4360-4376.doi: 10.11843/j.issn.0366-6964.2024.10.011
• Animal Genetics and Breeding • Previous Articles Next Articles
Xiaoyu MAO1(), Jiawei DU1, Jiayu TANG1, Jinhai PAN1, Lei JIANG1, Xiaolei SUN1, Linsen ZAN1,2, Hongbao WANG1,2,*(
)
Received:
2024-03-13
Online:
2024-10-23
Published:
2024-11-04
Contact:
Hongbao WANG
E-mail:maoxiaoyu@163.com;wanghongbao@nwsuaf.edu.cn
CLC Number:
Xiaoyu MAO, Jiawei DU, Jiayu TANG, Jinhai PAN, Lei JIANG, Xiaolei SUN, Linsen ZAN, Hongbao WANG. Effects of CHRNG Gene on Proliferation and Differentiation of Bovine Myoblasts and Its Mechanism[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4360-4376.
Table 1
Primers information"
基因 Gene | 转录本号 Transcript ID | 引物名称 Primer name | 引物序列(5'→3') Primer sequence | 产物长度/bp Product length |
CHRNG | NM_174273.2 | CHRNG-F | CAGTCCCAGACCTACAGCAC | 115 |
CHRNG-R | CCCACTCCCCATTTTCTGTGA | |||
GAPDH | NM_001034034.2 | GAPDH-F | AGTTCAACGGCACAGTCAAGG | 124 |
GAPDH-R | ACCACATACTCAGCACCAGCA | |||
PCNA | NM_001034494.1 | PCNA-F | CCTTGGTGCAGCTAACCCTT | 94 |
PCNA-R | TTGGACATGCTGGTGAGGTT | |||
CCNB1 | NM_001045872.1 | CCNB1-F | TACCCATTCACCATTATCAA | 103 |
CCNB1-R | ACTAACTATGCTGGACTACGA | |||
CCND2 | NM_001076372.1 | CCND2-F | CGCCAGGTTCCATTTCA | 77 |
CCND2-R | CCGACAACTCCATCAAGC | |||
CDKN1A | NM_001098958.2 | CDKN1A-F | GACCAGCATGACAGATTTCTACCA | 144 |
CDKN1A-R | TGAAGGCCCAAGGCAAAAG | |||
MYOD1 | NM_001040478.2 | MYOD1-F | AACCCCAACCCGATTTACC | 196 |
MYOD1-R | CACAACAGTTCCTTCGCCTCT | |||
MYOG | NM_001111325.1 | MYOG-F | GGCGTGTAAGGTGTGTAAG | 85 |
MYOG-R | CTTCTTGAGTCTGCGCTTCT | |||
MYH3 | NM_001101835.1 | MYH3-F | TGAACGCCCTCTCCAAATCC | 101 |
MYH3-R | AATGAAGTGCTGTCTCGGCA | |||
ANGPTL2 | NM_001109814.1 | ANGPTL2-F | GGGCACGATGAAGGTGTAGGT | 114 |
ANGPTL2-R | AAGGATGGCTTGGAGGGC | |||
HSPA1A | NM_203322.3 | HSPA1A-F | CTTCCGCAGACCCGCTAT | 120 |
HSPA1A-R | CGGTGCCCTGCCTTTT | |||
APOD | NM_001076301.3 | APOD-F | ACTCGCAAGGTAACAGAA | 127 |
APOD-R | CAAACCACCAGAGCAAC | |||
CYGB | NM_001206720.2 | CYGB-F | TTGAGCAGAAGGCCGAGTT | 81 |
CYGB-R | CCAGGAAGATGAGTCAGGGAT | |||
PI16 | NM_001024487.1 | PI16-F | CGAGCGTGAGCACTACAAC | 110 |
PI16-R | GGGAGCCACAGCCAAT | |||
TTR | NM_173967.3 | TTR-F | GCATCCAGGACCTTGACC | 115 |
TTR-R | TGGCTTCCTTCCGTCTG |
Fig. 2
CHRNG overexpression and interference adenovirus optimal MOI and expression efficiency A.Cell morphology and fluorescence brightness of bovine myoblasts infected with different MOI adenoviruses(48 h) (scale bar: 200 μm); B.The mRNA expression level of CHRNG gene when overexpressing adenovirus MOI value is 60; C.The mRNA expression levels of CHRNG gene when interference adenovirus MOI value is 80. *.P < 0.05, **.P < 0.01; the same as below"
Fig. 3
Effect of overexpressing CHRNG gene on the proliferation of bovine myoblasts A.Changes in the number of cells at different time points of proliferation after overexpression treatment (phase-contrast test); B.EdU staining analysis after overexpression(48 h); C.Analysis of myoblast proliferation changes with CCK-8 after overexpression treatment(48 h); D.The mRNA expression of PCNA, CCNB1, CCND2 and CDKN1A after overexpression; E.Protein expression level detection of PCNA, CCNB1 and CCND2 after overexpression"
Fig. 4
Effect of interfering CHRNG gene on the proliferation of bovine myoblasts A.Changes in the number of cells at different time points of proliferation after interfering treatment (phase-contrast test); B.EdU staining analysis after interfering (48 h); C.Analysis of myoblast proliferation changes with CCK-8 after interfering treatment (48 h); D.The mRNA expression of PCNA, CCNB1, CCND2 and CDKN1A after interfering; E.Protein expression level detection of PCNA, CCNB1 and CCND2 after interfering. ***. P < 0.01"
Fig. 5
Effect of overexpressing CHRNG gene on the differentiation of bovine myoblasts A.The morphologic changes of myoblasts at different differentiation times (D2, D4, D6) after overexpressing the CHRNG gene; B.Immunofluorescence staining analysis after overexpression treatment(D6); C.The mRNA expression of MYOD1, MYOG, MYH3 after overexpression of CHRNG; D-E.Protein expression level detection of MYOD1, MYOG, MYH3 after overexpression of CHRNG"
Fig. 6
Effect of interfering CHRNG gene on the differentiation of bovine myoblasts A.The morphologic changes of myoblasts at different differentiation times (D2, D4, D6) after interfering the CHRNG gene; B.Immunofluorescence staining analysis after interfering treatment(D6); C.The mRNA expression of MYOD1, MYOG, MYH3 after interfering CHRNG; D-E.Protein expression level detection of MYOD1, MYOG, MYH3 after interfering CHRNG"
Table 2
Common up-regulated genes in overexpression group and interference group"
基因 Gene | 基因ID Gene ID | 差异倍数(OE) Fold change(OE) | 差异倍数(sh) Fold change(sh) |
TFAP2A | 505849 | 3.44 | 3.44 |
IL36A | 523429 | 3.57 | 7.29 |
ESM1 | 539571 | 2.30 | 2.62 |
EREG | 100295476 | 2.20 | 2.61 |
COL13A1 | 613849 | 2.97 | 3.90 |
MYBL1 | 338034 | 2.54 | 2.18 |
NDP | 511596 | 2.09 | 2.47 |
GPR65 | 788507 | 2.87 | 2.65 |
HSPA4L | 506096 | 2.73 | 2.04 |
DEPDC1B | 540874 | 2.08 | 2.46 |
RND1 | 508869 | 2.11 | 3.13 |
CXCL8 | 280828 | 4.91 | 3.72 |
PREX2 | 520704 | 3.22 | 2.90 |
HSPA1A | 282254 | 10.78 | 2.73 |
CXCL2 | 281214 | 4.72 | 2.14 |
GRO1 | 281212 | 2.95 | 2.52 |
HSPA6 | 539835 | 695.28 | 3.42 |
H2AC6 | 506900 | 2.52 | 3.95 |
Table 3
Common down-regulated genes in overexpression group and interference group"
基因 Gene | 基因ID Gene ID | 差异倍数(OE) Fold change(OE) | 差异倍数(sh) Fold change(sh) |
PI16 | 507058 | 0.381 | 0.234 |
GFRA3 | 540009 | 0.290 | 0.497 |
CYGB | 510299 | 0.402 | 0.474 |
ABCA6 | 537351 | 0.488 | 0.463 |
ABCA10 | 504909 | 0.331 | 0.286 |
COLEC12 | 504741 | 0.333 | 0.494 |
VCAM1 | 282118 | 0.181 | 0.377 |
GALNT15 | 618078 | 0.296 | 0.471 |
HVCN1 | 616570 | 0.440 | 0.309 |
CD34 | 281051 | 0.469 | 0.189 |
DDIT4L | 510906 | 0.330 | 0.430 |
TTR | 280948 | 0.271 | 0.496 |
ANGPTL2 | 512019 | 0.461 | 0.491 |
SLC1A7 | 523673 | 0.433 | 0.238 |
PAMR1 | 513841 | 0.308 | 0.266 |
WDR17 | 783416 | 0.242 | 0.238 |
C6 | 507749 | 0.459 | 0.476 |
SH2D3C | 515820 | 0.350 | 0.433 |
C3 | 280677 | 0.352 | 0.402 |
ANGPT4 | 617915 | 0.272 | 0.220 |
APOD | 613972 | 0.344 | 0.437 |
ENTPD2 | 100126045 | 0.400 | 0.299 |
ENSBTAG-00000025258 | 515676 | 0.488 | 0.382 |
ENSBTAG-00000049395 | 100848536 | 0.220 | 0.349 |
ENSBTAG-00000052577 | 781663 | 0.438 | 0.383 |
Fig. 9
Sequencing results validated by qRT-PCR A.The genes were validated by qRT-PCR (overexpression group); B. Correlation analysis of results of RNA-Seq and qRT-PCR (overexpression group); C. The genes were validated by qRT-PCR (interference froup); D. Correlation analysis of results of RNA-Seq and qRT-PCR (interference froup)"
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