| Background and Objective With the rapid development of China’s manned space program,more and more astronauts will work and live in the space station.That may be accompanied by the increase of probability of astronauts’ accidental traumas and stress injuries.According to research reports,the most common trauma during spaceflight is the skin and soft tissue injuries.The skin wound healing is a complex and orderly biological process involving a variety of cellular,cytokines and extracellular matrix and other complex network.Long non-coding RNA(lnc RNA)is a kind of long-chain non-coding RNA molecule which does not encode protein and plays important roles in regulations of epigenetics,transcription and post-transcription of multiple regulatory genes.This study was designed to investigate the effects of simulated microgravity with RCCS on lnc RNA and wound healing relevant genes & proteins expression after ultraviolet C(UVC)radiation in mouse fibroblast L929,and aimed to illustrate the mechanism of wound healing under microgravity and the roles of fibroblast’s lnc RNA,in the hope of providing theoretical basis for management of astronauts accidental injuries in space missions.Methods(1)The rotary cell culture system(RCCS)was used to simulate the microgravity environment.The mouse fibroblast L929 cells were purchased and cultured in vitro.The cells were randomly divided into microgravity group(MG)and normal gravity group(NG).The gene chip(Agilent Mouse lnc RNA,4×180K,Design ID:049801)assay was applied to detect the expression of lnc RNA in fibroblast L929.The differentially expressed lnc RNAs were identified as those changed more than 2 times and had significant difference compared to the control group(P<0.05).(2)In order to investigate the differentially expressed lnc RNAs in L929 cells acquired in the first part of study,GO and Pathway analysis were applied to determine the function distribution of these target genes.The integration predictions of the mi RNA and m RNA co-expression had been proposed to refine the functional mi RNA-m RNA relationships.(3)L929 cells were randomly divided into four groups including microgravity group(MG),normal gravity group(NG),microgravity plus UVC radiation(MG+UVC),and normal gravity plus UVC radiation(NG+UVC).The cells were cultured in RCCS for 4 days,and irradiated with UVC in MG+UVC group and NG+UVC group with 30 m J/cm~2,and then cultured in RCCS for 24 hours.The genes and proteins of Caspase-3,Bcl-2,Col-I,Col-Ⅲ and TGF-β were detected by q RT-PCR and BCA protein assay.Results:(1)There were 238 differentially expressed lnc RNAs including 134 lnc RNAs up-regulated and 104 lnc RNAs down-regulated,and 237 differentially expressed m RNAs including 53 m RNAs up-regulated and 184 m RNAs down-regulated in mouse fibroblasts L929 cell line under simulated microgravity by RCCS.The RT-q PCR showed a high concordance with chip microarray results.(2)GO analysis showed that the differentially expressed lnc RNAs were related to the regulations of macrophage differentiation,wound healing,cell conjunction,and keratinocyte differentiation.Pathway analysis showed that these target genes had relationships with TGF-β signaling pathway,Vitamin digestion and absorption,and systemic lupus erythematosus,etc.The lnc RNA-m RNA co-expression networks were established and found that there was a higher enrichament of lnc RNA FR382640 and transcription factor Smad4,with 1172 genes co-expressed,and a higher enrichment of lnc RNA ENSMUST00000122952 and transcription factor MAF & MAFB,with 859 genes co-expressed.The function analysis revealed the target gene lnc RNA FR148417 which plays roles in the biological process of cytoskeleton microtubule formation,cell endocytosis,histone acetyltransferase binding,and protein kinase B signaling pathway.Another target gene is lnc RNA FR318458 which is related to the regulations of wound healing,sebaceous gland development,and protein polysaccharide binding,etc.(3)After fibroblasts L929 were under stress environment of RCCS simulated microgravity and being subjected simultaneously to UVC irradiation,Caspase-3 gene was up-regulated,while its protein was down-regulated;the expression of gene and protein of Bcl-2 were synchronously up-regulated;the expression of Col-I m RNA and its protein were higher in microgravity environment than the control,with Col-I m RNA up-regulated and Col-I protein somewhat down-regulated;both Col-Ⅲ and TGF-β,whether their m RNA or protein,were suppressed to varying degrees.Conclusion The study showed that the expression of lnc RNAs in mouse fibroblasts L929 were active and significantly affected by simulated microgravity with RCCS.Agilent Mouse lnc RNA chip assay had a good high-throughput screening efficiency for differentially expressed genes in L929 cell.Upon stress response to RCCS simulated microgravity and especially being simultaneously subjected to UVC irradiation,fibroblasts L929 exhibited various effects in the apoptosis process and extracellular matrix formation. |
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