| Backgroud and objectSkin aging are manifested as formation of wrinkles,increasing pigmentation,decreasing of elasticity,and is affected by endogenous and exogenous factors[1].The skin aging that caused by endogenous factors is also known as natural aging,it,s one manifestation of the body’s natural aging and caused by the genetic and some unavoidable reasons(such as the alteration of endocrine system,decreasing of immjuno ftmction,etc.);untill now it is still not artificial control.Exotrinsic aging is related to ultraviolet irradiation,smoking,environmental pollution,sleep,nutritional status and other factors[2].Although the mechanisms have not been studied thoroughly,but studies have shown that among them ultraviolet irradiation is the main factor of exotrinsic aging,this kind of skin aging can also be regarded as photoaging in a certain point of view[3-5].Researchers targeted in the mechanisms of photo aging and its interventions had always been hot issures in the field of dermatology.Botulinum Toxin Type A(BoNTA)is the most effective and most widely used one among the seven neurotoxins secreted by Clostridium botulinum[6].BoNTA can cause muscle relaxation,and because of this can release wrinkles(mainly dynamic wrinkles)t,it has become an important therapy of exogenous aging in dermatology field.BoNTA[7,8].In 2005 and 2008,some researchers found a face-lifting effect after intra-dermal injection of BoNTA to the mid and lower face[9,10 However,some other researchers reported that needle pricks themselves without BoNTA can make the skin become smoother as well[11]In order to understand the real utility of BoNTA in the improvement skin condition,scholars have conducted a variety of studies.In 2012,Oh et al.studied the in vitro effects of BoNTA on normal HDFs and found that BoNTA has a notable effect in increasing the level of collagen production and down regulating its degradation[12].These studies not only showed the positive effects of BoNTA on HDFs for remodeling skin,but also implied the importance of HDFs.n 2014,our research group further confirmed the direct anti-photoaging potential of BoNTA in UVB-induced premature senescence of hxuman dermal fibroblasts in vitro[13].In 2016,Jie Zhu et al.proved that topical BoNTA application could enhance the rejuvenation effect of fractional CO2 laser,further indicating that BoNTA can refine skin texture via improving the activity of HDFs[14].These studies not only demonstrate the positive effect of BoNTA on the improvement skin conditions,but also showed the importance of HDFs.Long non-coding(IncRNAs)are a group of noncoding RNA transcripts longer than 200 nucleotides which can not encode proteins[15]LncRNAs,which were previously thought to be transcriptional "noise",are now proved to have some functions by regulating gene expression at the epigenetic,transcriptional,and post-transcriptional levels and participating in ’some biologic functions,such as genomic imprinting,chromosome modification,intra-nuclear transport,transcriptional activation,interference,etc[16,17].Therefore,the understanding of cellular processes in physiological conditions will not be complete without analyzing the contributions made by hncRNAs.Therefore we study the mechanisms of photo-ageing and how BoNTA improve photo-aging by investigating the effect of UVB irradiation and BoNTA on the expression of IncRNA in normal HDFs as well as the effects of BoNTA on the expression of irx UVB-induced premature senescence(UVB-IPS)HDFsMaterials and methods1.Seperation of the experiment groupHuman dermal fibroblasts(HDFs)were isolated from the skin after circumcision.The normal HDFs were selected from 8 to 11 generations in the logarithmic growth phase,and were grouped as follows:(1)The first part:①control group;②UVB induced premature aging fibroblasts(UVB-IPS)group:UVB was continuously irradiated with 10 mJ/cm2 size of energy for 5 days and allowed to stand for 3 days,The aging of the two groups was confirmed byβ-galactosidase.(2)The second part①control group;②BoNTA treatment group(2.5U 48h):The treatment dose of BoNTA was 2.5U/106 cells and the incubation time with HDFs was 48h;③BoNTA treatment group(5U 48h):The treatment dose of BoNTA was 5U/106 cells and the incubation time with HDFs was 48h;④BoNTA treatment group(7.5U 48h):The treatment dose of BoNTA was 2.5U l 106 cells and the incubation time with HDFs was 48h;⑤BoNTA treatment group(5U 24h)The treatment dose of BoNTA was 5U/106 cells and the incubation time with HDFs was 24h;⑥BoNTA treatment group(5U 72h)The treatment dose of BoNTA was 5U/106 cells and the incubation time with HDFs was 72h;(3)The third part:①control group;②BoNTA treatment UVB-IPS group(2.5U 48h):BoNTA treatment dose of 2.5U/106 cells,and uVB-xPS HDFs co-incubation time was 48h;③BoNTA treatment UVB-IPS grosp(5U 48h):BoNTA treatment dose of 5U/106 cells,and UVB-IPS HDFs co-incubation time was 48h;④BoNTA treatment UVB-EPS group(7.5U 48h):BoNTA treatment dose of 7.5U/106 cells,and UVB-IPS HDFs co-incubation time was 48h;⑤BoNTA treatment UVB-IPS group(5U 24h):BoNTA treatment dose of 5U/106 cells,and UVB-IPS HDFs co-incubation time was 24h;⑥BoNTA treatment UVB-IPS group(5U 72h):BoNTA treatment dose of 5U/106 cells,and UVB-IPS HDFs co-incubation time was 72h;2.RNA microRNAs were screened for differential expression of RNA(lncRNA and mRNA)and bioinformatics analysisThe total RNAs of each group of HDFs were extracted using TRIzol and analyzed by RNA chip.Normalized intensity | log2(Ratio)|≥2 was set to find the differently expressed genes in each groups.And the target gene of RNA was analyzed by GO analysis and pathway enrichment analysis.In UVBvs control group,BoNTA(5U 48h)vs control group and BoNTA were used to examine the lncRNA and mRNA selected by UVB-IPSvs control group by qRT-PCR(real-time quantitative PCR)method.3.qRT-PCR method to further verify the screening of IncRNA and mRNA.FGFR3P and COL19A1 were screened by the qRT-PCR method.to further verify The expression level of FGFR3P and COL19A1 in the BoNTA vs control group and BoNTA.treated UVB-IPS vs control group.With the change of dose and time of BoNTA.Results:1.Screening and preliminary analysis of IncRNA in early HDFs induced by UVB:(1)The positive rate of β-galactosidase was significantly higher than that of HDB(10mJ/cm2)(P<0.05).(2)When the Fold change value was used to screen the Fold change value,the difference of IncRNA and mRNA detected in UVB-induced photo-aged HDFs was 3224 And 1630:bioinformatics analysis suggests that differentially expressed lncRNAs involve a variety of biological functions,and some biological functions are related to photoaging,such as cellular response to UVB,autophagy,and apoptosis.(3)The results of qRT－PCR of RNAs selected were consistent with the results of RNA chip analysis and proved the reliability of the results of chip analysis.2.Screening and preliminary analysis of long-chain non-coding RNA expression profiles of human dermal fibroblasts treated with botulinum toxin type A.(1)Compared with the control group,BoNTA was incubated at a dose of 5U/106 cells for 48 hours FC≥ 2,the expression of IncRNA and mRNA in HDFs were 2124 and 638,respectively.(2)Bioinformatics analysis suggests that differentially expressed IncRNA involves a variety of biological functions,some biological functions related to skin aging,such as collagen production,cell proliferation and so on.(3)The results of qRT-PCR showed that the expression of FGFR3P and COL19A1 was similar to that of the analysis.The results of qRT-PCR showed that the regulation of FGFR3P and COL19A1 expression had some time and dose correlation.3.The effect of botulinum toxin type A on the expression profile of IncRNA in HDFs of UVB-IPS:(1)Compared with the control group,BoNTA was incubated at 5U/106 cells for 48h,and when FC≥2 was set,UVB The expression of lncRNA and mRNA in HDFs were 1651 and 1221,respectively.(2)Bioinformatics analysis suggests that differentially expressed lncRNAs are involved in a variety of biological functions,and some biological functions are related to skin aging,such as intermolecular G1/S conversion,fibroblast proliferation,collagen production,Cell aging and so on.(3)The results of qRT-PCR of RNAs were consistent with the results of the analysis.The results of further qRT-PCR showed that the regulation of FGFR3P and COL19A1 expression was related to the time and dose.ConclusionUVB and BoNTA can significantly alter the expression of IncRNAs and mRNAs in HDFs.BoNTA can also cause the expression changes of IncRNAs in UVB-IPS HDFs.These alterations are related to various biological phenomenons including skin aging. |
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