Regulation Of LncRNA H19 On The Growth And Migration Of Keloid Fibroblasts

Background and ObjectiveKeloid is a kind of skin fibrous disease with excessive proliferation of fibroblasts and collagen hypersecretion beyond the original lesion area and grows in an "invasive" manner after skin injury,with different shapes and colors,hard and raised masses,which seriously affects the physical and mental health of patients.The keloid is a "benign skin tumor",which is not fatal but has a high recurrence rate.The treatment of keloid is very difficult,mainly including surgical excision,injection therapy and radiotherapy et al.It is easy to relapse after surgical treatment,while radiotherapy and injection therapy can only soften or contract keloid,Currently,there is no effective curative strategy.The pathogenesis of keloid has not been fully clarified,the main pathological features of keloid are excessive proliferation of fibroblasts,abnormal and excessive deposition of extracellular matrix(ECM).With the research progress in the field of molecular biology,the exploration at the molecular level is more and more in-depth,so molecular targeted therapy is very promising referring to targeted drug therapy for tumors,which provides a new treatment strategy for keloid at least.so it is of great significance to explore the pathogenesis of keloid for the treatment and follow-up prevention of keloid and improve the life quality of patients.Long non-coding RNA(LncRNA)can regulate a series of biological processes from cell growth to apoptosis via targeting microRNA(miRNA).Studies have shown that LncRNA is abnormally expressed in different types of multi-system tumors which promoting or inhibiting tumor development,and may serve as a potential biological diagnostic marker and therapeutic target for tumors.Keloid grows aggressively in a manner similar to "neoplastic" growth,previous studies have shown that LncRNA can also participate in the processes of keloid by regulating the effector cells-proliferation,migration,apoptosis and other biological behaviors.It has been reported that LncRNA H19(H19)is up-regulated in keloid tissues and fibroblasts,which can affect the activity of fibroblasts.However,there are few reports on what signal pathways can influence and regulate fibroblasts activity.In this study,bioinformatics prediction showed that there were targeted binding sites between H19 and miR-769-5p,but miR-769-5p is down-regulated in keloid tissues and fibroblasts,whether H19 can affect the development of keloid by regulating the expression of miR-769-5p is unknown.Eukaryotic translation initiation factor(eIF)is a protein involved in the translation initiation processes of eukaryotic cells.eIF is abnormally expressed in tumors and can affect cell translation,proliferation and other processes.Eukaryotic translation initiation factor 3a(eIF3a),the largest subunit of eIF3,is widely expressed in tissues,and its abnormal expression can cause a variety of diseases,it has been found that eIF3a is up-regulated in keloid tissues and fibroblasts.Meanwhile,bioinformatics prediction showed that miR-769-5p had targeted binding sites with eIF3a also,whether the expression of miR-769-5p and eIF3a is related has not been studied.Therefore,we assumed that H19 could regulate the formation and growth of keloid through miR-769-5p/eIF3a axis on the whole,and then relevant experiments were used to further verify the hypothesis,study the effect of H19 on keloid fibroblasts viability and explore its mechanism.To study the role and mechanism of H19 in the occurrence and development of keloid in this study,fibroblasts were firstly cultured,then the expression of H19 in keloid tissues and fibroblasts were detected by qRT-PCR.The effects of H19 on the keloid fibroblasts were investigated,including cell proliferation,clone formation,apoptosis,migration,invasion and protein expression of CyclinD1,Bcl2,E-cadherin.Secondly,the targeted binding relationship between H19 and miR-769-5p was verified by dual luciferase reporter gene assay,and the expression of miR-769-5p was discussed to observe whether inhibition of miR-769-5p expression could reverse the effects of H19 knockdown on the biological characteristics of keloid fibroblasts.Furthermore,similar experiments were used to verify the targeted binding relationship between miR-769-5p and eIF3a,then explored the expression of eIF3a in keloid fibroblasts.In addition,whether eIF3a overexpression could reverse the effects of miR-769-5p overexpression on the biological characteristics of keloid fibroblasts was investigated.Finally,the correlations between the expression level of H19,miR-769-5p and eIF3a were comprehensively analyzed on the basis of experiments,which confirmed that H19 could regulate the biological characteristics of keloid fibroblasts through miR-769-5p/eIF3a axis,it could provide a new potential target for clinical treatment of keloid.This study included three parts.Part one:Expression and function of lncRNA H19 in keloid tissues and fibroblasts.Part two:The regulation of LncRNA H19 on the growth and migration of keloid fibroblasts through miR-769-5p.Part three:The regulation of LncRNA H19 on the biological function of keloid fibroblasts through miR-769-5p/eIF3a axis.Part 1:Expression and Function of LncRNA H19 in Keloid Tissues and FibroblastsMethods1.Primary fibroblasts were isolated and cultured,and keloid fibroblasts were divided into two groups:si-NC group and si-H19 group.The expression level of H19 in normal skin tissues and fibroblasts,keloid tissues and fibroblasts were detected by qRT-PCR.2.The biological effects of H19 knockdown on keloid fibroblasts were detected respectively:MTT assay and EDU assay were used to detect cell proliferation,cell clone formation ability was detected by plate clone formation assay,the apoptosis rate was detected by flow cytometry,cell migration and invasion ability were detected by scratch assay and transwell chamber assay,the protein expression level of CyclinDl,Bcl2,E-cadherin,α-SMA,Collagen I,Collagen III and Fibronectin were detected by Western blot.Results1.H19 was highly expressed in keloid tissues and fibroblasts.2.Knocking down H19 could inhibit the proliferation,clone formation,cell migration and invasion of keloid fibroblasts while promote cell apoptosis,also could inhibit the protein expression of CyclinD1 and Bcl2,promote the expression of E-cadherin,and inhibit the protein expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ and Fibronectin,further inhibit the accumulation of ECM.Part 2:The Regulation of LncRNA H19 on the Growth and Migration of Keloid Fibroblasts through miR-769-5pMethods1.The targeting relationship between H19 and miR-769-5p was detected by dual luciferase reporter gene assay.2.The study was divided into si-NC group,si-H19 group,si-H19+anti-miR-NC group and si-H19+anti-miR-769-5p group.The expression level of miR-769-5p in normal skin tissues and fibroblasts,keloid tissues and fibroblasts were detected by qRT-PCR.The correlation between the expression level of H19 and miR-769-5p in keloid tissues was analyzed by Pearson method.3.The effects of H19 knockdown on the biological characteristics of keloid fibroblasts by increasing the expression of miR-769-5p were respectively detected by the same methods as Part I.Results1.The expression of miR-769-5p was down-regulated in keloid tissues and fibroblasts,H19 could target and bind to miR-769-5p,correlate negatively with miR-769-5p and negatively regulate the expression of miR-769-5p.2.Inhibition of miR-769-5p expression could reverse the inhibitory effects of H19 knockdown on proliferation,clone formation,migration and invasion of keloid fibroblasts,the promoting effect of cell apoptosis,also could reverse the inhibiting effects of protein expression of CyclinDl and Bcl2,and the promoting effect of protein expression of E-cadherin.3.Inhibition of miR-769-5p expression could reverse the inhibitory effects of H19 knockdown on α-SMA,Collagen Ⅰ,Collagen Ⅲ and Fibronectin protein expression in.keloid fibroblasts.Part 3:The Regulation of LncRNA H19 on the Biological Function of Keloid Fibroblasts through miR-769-5p/eIF3a AxisMethods1.MiR-NC,miR-769-5p mimics,miR-769-5p and pcDNA,miR-769-5p and pcDNA-eIF3a were transfected into keloid fibroblasts,which were classified as miR-NC group,miR-769-5p group,miR-769-5p+pcDNA group,and miR-769-5p+eIF3a group respectively.2.The expression level of eIF3a in normal skin tissues and fibroblasts,keloid tissues and fibroblasts were detected by qRT-PCR and Western blot respectively.3.Dual luciferase reporter gene assay was used to detect the targeting relationship between miR-769-5p and eIF3a;The correlation between miR-769-5p and eIF3a expression level,the correlation between H19 and eIF3a expression level in keloid tissues were analyzed by Pearson method.4.The effects of over-expression of miR-769-5p on the biological characteristics of keloid fibroblasts by inhibiting eIF3a were detected respectively by the same methods as Part I.Results1.eIF3a was highly expressed in keloid tissues and fibroblasts,miR-769-5p could target and bind to eIF3a,correlate negatively with eIF3a and regulate negatively the expression of eIF3a.2.The over-expression of miR-769-5p could inhibit the proliferation,clone formation,migration and invasion of keloid fibroblasts while promote apoptosis,also could inhibit the expression of CyclinDl and Bcl2,promote the expression of E-cadherin,and inhibit the expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ and Fibronectin,then could inhibit the deposition of ECM.The overexpression of eIF3a could reverse the biological characteristics of keloid fibroblasts induced by miR-769-5p overexpression.3.H19 was correlated positively with eIF3a which could negatively regulate the expression of miR-769-5p and positively regulate the expression of eIF3a.Conclusions1.Compared with normal skin tissues and fibroblasts,H19 and eIF3a were highly expressed,miR-769-5p was low-expressed in keloid tissues and fibroblasts.2.Knocking down H19 could inhibit the proliferation,clone formation,migration,invasion and ECM deposition of keloid fibroblasts,also could promote cell apoptosis,and further inhibit the growth and migration of keloid.3.H19 could target and bind to miR-769-5p,negatively correlate with miR-769-5p and negatively regulate the expression of miR-769-5p.miR-769-5p could target and bind to eIF3a which could negatively correlate with eIF3a and regulate negatively the expression of eIF3a.H19 could positively regulate the expression of eIF3a via negatively regulating the expression of miR-769-5p.4.H19 could participate in the processes of cell growth and migration by regulating miR-769-5p/eIF3a axis,which providing a potential target for clinical treatment of keloid.