Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (12): 2407-2420.doi: 10.3864/j.issn.0578-1752.2023.12.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Effect of Valproic Acid on Reprogramming of Bactrian Camel Fibroblasts

ZHANG QiRan1(), LI ZongShuai2, MA Tian2, LI YiNa2, ZHAO XingXu1,2, ZHANG Yong1,2()   

  1. 1 College of Life Science and Technology/Gansu Key Laboratory of Animal Reproductive Physiology and Reproductive Regulation, Gansu Agricultural University, Lanzhou 730070
    2 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070
  • Received:2022-03-14 Accepted:2022-05-19 Online:2023-06-16 Published:2023-06-27

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Abstract:

【Objective】 To improve the efficiency of the reprogramming process of Bactrian camel fibroblasts and to reduce the risk of tumorigenesis caused by the introduction of proto-oncogenes. In this experiment, valproic acid (VPA) was added to the fibroblast reprogramming process to explore the effect of small molecules on the reprogramming of Bactrian camel fibroblasts. 【Method】 Given this, March-aged Bactrian camel fetal fibroblasts were used as test materials, combined with classic induction combination OSKM (Oct4, Sox2, Klf4, and c-Myc) and EGFP five retrovirus reprogramming of Bactrian camel fibroblasts (OSKM group), and the cells by adding VPA treatment for 7 days after the second viral infection (OSKM+VPA group) were collected. Endogenous and exogenous genes were examined by using PCR to confirm the modification effect of retrovirus on Bactrian camel fibroblasts. Eight genes were randomly selected from those more significantly affected by VPA. According to RNA-seq data, whether their trends before and after VPA addition were consistent with the trends of RNA-seq data was checked to verify the accuracy of RNA-seq data. The transcriptome sample genes were classified by GO analysis and significant enrichment pathways for target genes were clarified by using KEGG pathway enrichment analysis and hypergeometric validation analysis. Total RNA was extracted from the collected cells, and then, combined with RNA-seq and Real time-quantitative interpretation (RT-qPCR) techniques to detect the effect of VPA on the reprogramming of Bactrian camel fibroblasts. 【Result】 It was detected by using PCR that the expression of endogenous and exogenous genes in different groups. The results showed that nSox2, Sox2, Oct4, Klf4, and c-Myc genes were expressed in both OSKM and OSKM+VPA groups, and the expression in OSKM+VPA group was higher than that in the OSKM group, while they were not expressed in BCEFs group. Eight genes were randomly selected for testing, and the results showed that: three genes of TP53, CCNB1, and CCD20 were down-regulated in expression after the addition of VPA, which were related to the cell cycle signaling pathway. S100A4, CKS2, VIM, and MMP9 genes signaling were down-regulated in expression, which was related to the phenotypic characteristics of cancer; VEGFC gene expression was up-regulated in the PI3k-Akt signaling pathway. This expression trend was consistent with the trend of the histological data. The results showed that the expression of proliferation genes Mki67 and PCNA were down-regulated, while the expression of apoptosis gene CASP7 was up-regulated after the addition of VPA. KEGG and hypergeometric validation analyses of the transcriptome data were performed, and 959 differentially expressed genes were screened according to the analysis results, which were enriched in 276 signaling pathways, including eight signaling pathways with Q values less than 0.05: steroid biosynthesis, cell cycle, PPAR signaling pathway, progesterone-mediated oocyte maturation, fatty acid metabolism, ECM-receptor interactions, cell adhesion molecules, and cholesterol metabolism. The 26 differentially expressed genes related to cell cycle, fatty acid metabolism, cell adhesion molecule, and cholesterol metabolism were screened, and four of which were randomly selected for testing, showing that VPA upregulated the expression of L1CAM, CNTN1 and NFASC genes in the Bactrian camel fibroblast adhesion molecule signalling pathway and enhanced intercellular interactions. It was also upregulated that the expression of CD36 gene in the fatty acid signaling pathway. 【Conclusion】 The results showed that the VPA blocked cell before the split phase to reduce risk differentiation during the process of reprogramming. Meanwhile, VPA affected several signaling pathways in the reprogramming process of Bactrian camel fibroblasts, and regulated the expression trend of related genes in the signaling pathways, which effectively improved the reprogramming efficiency of the cells and played an important role in the reprogramming of Bactrian camel fibroblasts.

Key words: induced pluripotent stem cells, Bactrian camel, valproic acid, cell reprogramming

Table 1

The primer sequences and parameter"

基因 Gene 引物序列 Primer sequence (5'-3') 产物长度 Fragment lengths (bp) 退火温度 Tm (℃)
Sox2 F:GGTGCCCTGCTGCGAGTA
R: CGGCGGAAAACCAAGACG		 360 57
Oct4 F: CTCTTGTCTACCTCCCTTGCC
R: TCTGTTCCCGTCACTGCTCT		 100 57
Klf4 F: CGTTGAACTCCTCGGTCTCC
R: CGACTAACCGTTGGCGTGA		 170 57
c-Myc F: GGACTGTATGTGGAGCGGTTTC
R: TGGGCTGTGCGGAGGTTT		 487 57
TP53 F: GAGGACGCCAGTGGTAATCT
R: AGCGGCTTCTTCTTTTGTGG		 197 56
CCNB1 F: AGCGGATCCCAACCTTTGTA
R: GCATCTTCTTGGGCACACAA		 248 53
MMP9 F: GCAAACCAATCTCACCGACA
R: GGTCAGAAATTTGCCCACGT		 223 53
CDC20 F: AGAATCAGCCCGAAAACAGC
R: AGGGAAGGAATGTAACGGCA		 220 57
S100A4 F: TTCCACAAGTACTCGGGCAA
R: TTATCCGGGAAGCCTTCGAA		 242 56
VEGFC F: CTCTCTCTCAAGGCCCCAAA
R:GGGTCATCTCCAGCGTTAGA		 244 53
VIM F: GATTCAGGAACAGCACGTCC
R:TTGGATTCTTGCTTCGCCTG		 207 56
CKS2 F: GACAAGTACTTCGACGAGCAC
R:TGTGGTTCTGGCTCATGAATC		 167 53
Mki67 F: CCGTCATACCCAGCAGTGAA
R: CCTGGCGCTTTCTGGAATTT		 208 56
PCNA F: CGGACACCTTGGCACTAGTA
R: CACCCCATCTTTTGCACAGG		 215 56
CASP7 F: TCCTCTTTTGTCCCATCGCT
R: CTCGGCGTCTTTGTCTGTTC		 198 56
CD36 F: AAGTGGCAGTTAACAGCAGC
R: ACTTGGATAGAGGTGGGGTG		 230 60
NFASC F: GGTGCTGATCCTGCTCATTG
R: TCTTGCTGTTTGATGGTGCC		 195 60
L1CAM F: ATCAAGGTCCAGGCCGTTAA
R:TCTTGCTGTGCTTCCTCTGA		 235 60
CNTN1 F: TGTACCAGTGCATAGCGGAA
R: TGCGGCTTTAGGTTTGCATT		 161 60

Fig. 1

VPA-treated cell morphology A. The EGFP retrovirus packaging 72 h after transduction; B. 8 days after the second virus infection; C. Fibroblasts in culture for 8 days; D. Cells observed 8 days after the second virus infection; E. Morphology of cells treated with no VPA in the medium for seven days; F. Cell morphology in medium spiked with VPA and treated for seven days"

Fig. 2

Transcriptional and histological validation A. Semi-quantitative PCR was used to detect the mRNA expression levels of BCFFs, OSKM and OSKM+VPA; B. mRNA expression levels of TP53, CCNB1, MMP9, CDC20, S100A4, VEGFC, VIM and CKS2 (internal reference gene: β-actin). ** Showed extremely significant difference (P<0.01)"

Table 2

Comparison of reference statistics"

样本
Sample		 总计
Total		 未映射
Unmapped (%)		 单映射
Unique_mapped (%)		 多映射
Multiple_mapped (%)		 总映射
Total_mapped (%)
CK-1 61182534 5873073 (9.60%) 53914224 (88.12%) 1395237 (2.28%) 55309461 (90.40%)
CK-2 65291602 6449818 (9.88%) 57337110 (87.82%) 1504674 (2.30%) 58841784 (90.12%)
CK-3 65980288 6383674 (9.68%) 58071438 (88.01%) 1525176 (2.31%) 59596614 (90.32%)
T-1 69181666 6651458 (9.61%) 60990701 (88.16%) 1539507 (2.23%) 62530208 (90.39%)
T-2 67916304 6710952 (9.88%) 59640972 (87.82%) 1564380 (2.30%) 61205352 (90.12%)
T-3 67343960 6228926 (9.25%) 59624392 (88.54%) 1490642 (2.21%) 61115034 (90.75%)

Table 3

Statistics of genetic testing"

样本
Sample		 参考基因
Refer genes		 序列参考基因
Sequenced refer genes (%)		 新基因
Novel genes		 新基因序列
Sequenced novel genes (%)		 总基因
Total genes		 序列总基因
Sequenced total genes (%)
all 19172 15643 (81.59%) 576 576 (100.00%) 19748 16219 (82.13%)
CK-1 19172 14377 (74.99%) 576 563 (97.74%) 19748 14940 (75.65%)
CK-2 19172 14332 (74.75%) 576 562 (97.57%) 19748 14894 (75.42%)
CK-3 19172 14399 (75.10%) 576 561 (97.40%) 19748 14960 (75.75%)
T-1 19172 14367 (74.94%) 576 556 (96.53%) 19748 14923 (75.57%)
T-2 19172 14427 (75.25%) 576 564 (97.92%) 19748 14991 (75.91%)
T-3 19172 14347 (74.83%) 576 556 (96.53%) 19748 14903 (75.47%)

Fig. 3

Transcriptomic sequencing, basic analysis of correlations, and differences between groups A. Principal Component Analysis: the PC1 coordinate represents the first principal component; the PC2 coordinate represents the second principal component; while, the colored dots in the figure represent each sample; B. Correlation heat map of the samples: In the figure, the abscissa and ordinate are the respective samples and the color depth (intensity) indicates the correlation coefficient between the two samples; C. Visualization of gene abundance expression between samples or groups; D. Venn diagram showing the common and unique genes of the two groups; E. The abscissa represents the logarithm of the multiple of the difference between the two groups, the ordinate represents the negative Log10 value of the FDR of the difference between the two groups, and the red (group_2 up-regulated expression relative to group_1) and blue (down-regulated expression) indicate a difference in the gene expression levels; whereas, the black dots represent no difference; F. Genes from different samples are expressed in different colors; the redder the color, the higher the expression; whereas, the bluer the color, the lower the expression"

Fig. 4

Relative expression analysis of Mki67, PCNA and CASP7 mRNA in different groups A. Mki67 mRNA expression level (internal reference gene: β-actin); B. PCNA mRNA expression level (reference gene: β-actin); C. CASP7 mRNA expression level (reference gene: β-actin). ** showed an extremely significant difference (P<0.01)"

Fig. 5

Signaling pathway-related genes A. Heat map of 26 differentially expressed genes; B. CD36 mRNA expression level (internal reference gene: β-actin); C. NFASC mRNA expression level (internal reference gene: β-actin); D. L1CAM mRNA expression level (internal reference gene: β-actin); E. CNTN1 mRNA expression level (internal reference gene: β-actin); F. Semi-quantitative PCR was used to detect the mRNA expression levels of BCFFs, OSKM and OSKM+VPA. ** showed an extremely significant difference (P<0.01)"

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