Effect of Huntingtin-associated Protein 1 Gene on Proliferation of Mouse Fibroblasts

CAO Yuan-yuan, YANG Gui-zhi, GONG Yan-jü, ZHAO Jia, FENG Ying, ZHENG Xiang, ZHOU Xue

Abstract

To determine the effect of Huntingtin-associated protein 1 (Hap1) on fibroblast proliferation.  Methods  Hap1 knockout (Hap1-/-) primary fibroblasts were isolated and cultured in vitro. The proliferation of Hap1-/- fibroblasts was detected by EdU proliferation assay and cell flow assay. Transcriptome sequencing of the wild-type and Hap1-/- fibroblasts was screened for proliferation-related genes. Real-time quantitative PCR (qPCR) was performed to verify changes in expressions of related genes. Skin repair was examined in Hap1 knockdown mice with skin wounds. The proliferation of fibroblasts during wound repair was detected by PCNA immunohistochemical staining.  Results  Hap1-/- fibroblasts were successfully cultured. Compared with WT, EdU-positive fibroblasts decreased in Hap1-/-,with less cells entering the S phase. Transcriptome sequencing of primary fibroblasts identified genes of Cdc25C, E2f7, E2f8 and Ccl5. qPCR confirmed that Hap1 knockout increased E2f7 expression. Hap1+/- mice had larger skin lesions, slower healing and lower positive density of fibroblast proliferation than those of wild type mice.  Conclusion  Hap1 may positively regulate fibroblast proliferation by inhibiting the expression of cell cycle negative regulator E2f7.Its deletion inhibits fibroblasts entering the S phase, thereby reducing cell proliferation and affecting wound repair.

 

Keywords: Huntingtin-associated protein 1, Fibroblast, Proliferation, Wound repair

 

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References


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