The Effects Of CACNA1G-AS1 On Human Fibroblasts By Regulating The Expression Of MiR-205

【Background】Keloid,also known as keloid disorder or keloidal scar,is a pathologic fibro-proliferative disease caused by excessive wound healing after skin tissue injury,involving excessive growth beyond the boundary of the original wound skin,invasion of adjacent normal tissues,non-degenerative changes,and recurrent growth [1,2].Keloid patients suffer a severe impairment of quality of life,resulting from physical,psychological and social sequelae[3].As normally accepted,the abnormal proliferation of fibroblasts is the main cause for hyperplasia of pathological scars[4].Although much progress has been made,the etiology and basic mechanism underlying keloid formation are still not fully elucidated.Hence it is necessary to explore new more evidence for effective treatments of keloid.Long non-coding RNA(lnc RNA)are now identified as a class of newly discovered transcripts that contain more than 200 nucleotides in length [5,[6].More and more studies showed that lnc RNA play an important role in various cellular events including cell growth,proliferation,and death[7].It has been demonstrated that many lnc RNA are involved in the progression of fibrosis[8],such as HNF1A-AS1[9],COL1A2-AS1[10],CACNA1G-AS1,HOXA11-AS,LINC00312 and RP11-91I11.1[11].Recent study demonstrated that Calcium Voltage-Gated Channel Subunit Alpha1 G antisense RNA 1(CACNA1G-AS1)was up-regulated in keloid and had a positive effect on cell migration in keloid fibrosis[12].However,the expression and function of lnc RNA CACNA1G-AS1 remain unclear in human keloid fibroblasts up to date.Micro RNAs(mi RNAs),noncoding and single-stranded small RNAs with approximately 22 nucleotides in length,played important roles in tumorigenesis and progression[13,14].Numerous researches focused on mi RNA roles as an essential epigenetic regulator in many biological processes,including carcinogenesis and fibroblast activation[15,16].Previously,some mi RNAs have been reported to participate in proliferation,differentiation,and apoptosis of keloid fibroblasts.For example,mi R-188-5p and mi R-203 decreased the proliferation and invasion of keloid fibroblasts[17,18].It has been demonstrated that Mi R-196 a knockdown enhanced type I and type III collagen expression in keloid fibroblasts[19].Wang et al.demonstrated that mi R-21 was up-regulated in keloid tissue and affected the proliferation and apoptosis of human keloid fibroblasts[20].Pang et al. showed that mi R-152-5p was identified to play an anti-fibrotic role and suppress the progression of human keloid fibroblasts[21].However,there is no relevant study on the interaction between CACNA1G-AS1 and mi R-205 in keloid fibroblasts.In this study,we explored the relationship between CACNA1G-AS1 and mi R-205 and further studied their effects on proliferation,invasion and apoptosis of keloid fibroblasts.【Objective】1.Prepare keloid fibroblasts and normal fibroblasts,compare the biological differences between keloid fibroblasts and fibroblasts of normal skin origin,and provide the basis and basis for subsequent experiments;2.The expression level and biological function of cacna1g-as1 in keloid were further investigated to study its effect on proliferation,migration and apoptosis of fibroblasts,so as to clarify the functional role of cacna1g-as1 in fibroblasts and provide evidence for further experiments.3.Explore the biological function of mi R-205 in keloid,study its effect on proliferation,migration and apoptosis of fibroblasts,and clarify the functional role of mi R-205 in fibroblasts;4.To explore the targeting effect between cacna1g-as1 and mir-205,and the mechanism by which cacna1g-as1 regulates the expression of mir-205 and thus affects the formation of keloid,so as to provide a new target for the biological treatment of keloid.In this study,we explored the relationship between CACNA1G-AS1 and mi R-205 and further studied their effects on proliferation,invasion and apoptosis of keloid fibroblasts.【Methods】CACNA1G-AS1 and mi R-205 levels were detected using quantitative real-time polymerase chain reaction(q RT-PCR).Cell Counting Kit-8(CCK-8)assay was used to measure the proliferation and Transwell assay was performed to evaluate cell invasion.Furthermore,the apoptosis rates of cells were evaluated by flow cytometry analysis and the expression of caspase-3 in keloid fibroblasts was tested by western blot.Dual luciferase reporter assay was carried out to examine the relationship between CACNA1G-AS1 and mi R-205 and RNA immunoprecipitation(RIP)assay was conducted to further confirm the relation.【Results】CACNA1G-AS1 level was up-regulated in keloid tissues and keloid fibroblasts.CACNA1G-AS1 overexpression promoted proliferation and invasion and suppressed apoptosis of keloid fibroblasts.Moreover,mi R-205 was targeted by CACNA1G-AS1 and mi R-205 was markedly decreased in keloid tissues and keloid fibroblasts.Also,mi R-205 expression was negatively regulated by CACNA1G-AS1 and mi R-205 silencing enhanced proliferation and invasion and inhibited apoptosis.Furthermore,CACNA1G-AS1 and mi R-205 played the antagonistic role on mi R-205 expression,proliferation,invasion and apoptosis of keloid fibroblasts.【Conclusions】1.CACNA1G-AS1 is highly expressed in scar tissue and scar fibroblasts.CACNA1G-AS1 is involved in the proliferation and development of human keloid fibroblasts.2.CACNA1G-AS1 can regulate the proliferation,invasion and apoptosis of keloid fibroblasts,and CACNA1G-AS1 is involved in regulating the proliferation,invasion and apoptosis of keloid fibroblasts in vitro.3.CACNA1G-AS1 directly interacts with mi R-205 and is negatively regulated by CACNA1G-AS1.4.mi R-205 can inhibit the invasion and proliferation of keloid fibroblasts,and can promote the apoptosis of keloid fibroblasts.5.CACNA1G-AS1 regulates the proliferation,invasion and apoptosis of keloid fibroblasts by binding mi R-205 and inhibiting the expression of mi R-205.