Abstract
Objective We have identified a gene YOD1 encoding deubiquitinating enzyme (DUB) responsible for nonsyndromic cleft lip with or without cleft palate (NSCL/P). We aimed to determine the effects of YOD1 RNA interference (RNAi) on cell proliferation and migration, playing an important role in lip and palate formation, and to clarify whether the mechanisms involved TGF-β3 signaling associated with NSCL/P. Methods RNAi was applied to construct vectors expressing YOD1 small interference RNAs (siRNAs). The vectors were transfected into the human oral keratinocytes (HOK) cells. The cell proliferation and migration were evaluated by the cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The mRNA levels were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The protein levels were investigated by western blotting. Results The proliferation of YOD1 siRNA-transfected HOK cells was remarkably inhibited. The migration rate was significantly decreased in the YOD1 siRNA-transfected HOK cells. The TGF-β3 mRNA and protein levels were decreased significantly by siRNA-mediated knockdown of YOD1. YOD1 RNAi reduced the phosphor-Smad2/3 levels significantly. Conclusions YOD1 RNAi may inhibit cell proliferation and migration associated with the pathogenesis of NSCL/P through TGF-β3 signaling. The study indicates a novel role of YOD1 in regulating TGF-β3 signaling to affect cell proliferation and migration resulting in NSCL/P.
Highlights
As one of the most common birth defects, nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex congenital malformation with a prevalence of 1-2/1 000 births in all populations worldwide [1]
We have found that YOD1 small interference RNAs (siRNAs) decreases the mRNA levels of TGF-β3 and protein levels of phosphorSmad2/3
We provide evidence for the first time that YOD1 contributes to changing the abilities of cell proliferation and migration regulated by TGF-β3 signaling pathway, which may be involved in the pathogenesis of NSCL/P
Summary
As one of the most common birth defects, nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex congenital malformation with a prevalence of 1-2/1 000 births in all populations worldwide [1]. NSCL/P influences suckling, swallowing, and the development of language and hearing. The etiology of NSCL/P is considered to be due to both environmental and genetic factors. Many studies suggest that genetic factors play crucial roles in the pathogenesis of NSCL/P [5,6,7,8,9]. A large number of genes are suspected to be involved in NSCL/P probably to affect the embryonic development and differentiation leading to the formation of the facial structures [10,11,12,13,14]. The etiology of NSCL/P has been widely investigated [1, 6, 15], there is inconsistency across studies. Specific genes and mechanisms responsible for NSCL/P have not been fully understood
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