Study on the mechanism of small molecule drug Tideglusib to promote epidermal stem cells proliferation through PI3K/PKB/KIF14 pathway

doi:10.3877/cma.j.issn.1673-9450.2021.05.004

Abstract

Abstract:

Objective

To study the effect of the small molecule drug Tideglusib on the proliferation ability and cell cycle of epidermal stem cells and its mechanism.

Methods

The human epidermal stem cells were randomly divided into the control group, and the small molecule drug Tideglusib 50 nmol/L, 100 nmol/L, and 200 nmol/L groups with the random number table method. After 48 hours of culture, changes in cell proliferation ability were assessed through the cell counting kit-8 (CCK-8) experiment (n=6). Transcriptome level changes were detected by RNA sequencing, with P. adj <0.05 as the screening criteria for differentially expressed genes. Gene ontology enrichment was used to analyze the effect of differentially expressed genes on the biology process, cell composition, molecular function of epidermal stem cells (n=3). The transcription level of the differentially expressed gene KIF14 was verified by qRT-PCR, and the protein expression level of KIF14 was verified by Western blotting and immunofluorescence staining (n=3). The expression levels of PKB and p-PKB were explored by Western blotting (n=3).

Results

After 48 hours of culture, the CCK-8 experiment showed that the difference of the effect on the proliferation of epidermal stem cells between 50 nmol/L group of small molecule drug Tideglusib and the control group was not statistically significant (P>0.05), while 100 nmol/L group and 200 nmol/L group Tideglusib could significantly promote the proliferation of epidermal stem cells (P<0.05). RNA sequencing data showed that compared with the control group, the small molecule drug Tideglusib 200 nmol/L group had a total of 107 differentially expressed genes, 106 of which were up-regulated and 1 down-regulated. GO enrichment analysis showed that differentially expressed genes were mainly related to mitosis and cell cycle. Through flow cytometry, small molecule drug Tideglusib could increase the proportion of epidermal stem cells entering mitosis in a concentration-dependent manner. There was no statistical difference in the effect of 50 nmol/L group of small molecule drug Tideglusib on the cell cycle of epidermal stem cells (P>0.05), 100 nmol/L and 200 nmol/L Tideglusib could significantly increase the proportion of epidermal stem cells in mitosis (P<0.05). Validation of the cell cycle-related differentially expressed gene KIF14 by qRT-PCR found that small molecule drug Tideglusib could promote KIF14 transcription in a concentration-dependent manner, and 50 nmol/L group of small molecule drug Tideglusib could promote KIF14 transcription (P<0.05). Western blotting proved that small molecule drug Tideglusib had a concentration-dependent effect on the expression level of KIF14 protein, and 50 nmol/L group of small molecule drug Tideglusib could increase the protein expression level of KIF14 (P<0.05). Immunofluorescence staining showed that Tideglusib had a concentration-dependent effect on the relative fluorescence intensity of KIF14. There was no statistical difference in the effect of 50 nmol/L group of small molecule drug Tideglusib on the relative fluorescence intensity of KIF14 (P>0.05). Small molecule drug Tideglusib at 100 nmol/L group and 200 nmol/L group could significantly increase the relative fluorescence intensity of KIF14 (P<0.05). The expression levels of PKB and p-PKB, the upstream protein of KIF14 were detected by Western blotting. It was found that small molecule drug Tideglusib at different concentrations did not affect the total protein level of PKB (P>0.05). The 50 nmol/L group of small molecule drug Tideglusib had little effect on the expression of p-PKB in epidermal stem cells (P>0.05). Small molecule drug Tideglusib at 100 nmol/L group and 200 nmol/L group could significantly increase the expression of p-PKB in epidermal stem cells (P<0.05).

Conclusions

The small molecule drug Tideglusib can increase the proportion of mitotic cells and promote the proliferation of epidermal stem cells. The mechanism may be related to the regulation of the cell cycle of epidermal stem cells by up-regulating the PI3K/PKB/KIF14 signaling pathway.

Key words: Cell proliferation, Cell cycle, Stem cells, Tideglusib, KIF14

Jiachen Sun, Jinglong Ma, Chuanan Shen, Wen Zhang, Xinzhu Liu. Study on the mechanism of small molecule drug Tideglusib to promote epidermal stem cells proliferation through PI3K/PKB/KIF14 pathway[J]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2021, 16(05): 389-397.

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URL: https://zhssyxfzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1673-9450.2021.05.004

https://zhssyxfzz.cma-cmc.com.cn/EN/Y2021/V16/I05/389

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References 23

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