The Role Of Endothelin-1in The Uterine Fibroids Fibroblast Transdifferentiation And Its Potential Molecular Mechanism

Uterine fibroids are the most common benign of the femalereproductive system, its incidence is increasing year by year, and whichpathogenesis is not yet entirely clear. The development of uterine fibroidsto the emerges of clinical symptoms with its volume increases graduallyclosely related, and play a key role in this process is not fibroids rapid cellproliferation, but the excessive accumulation of ECM in the tumormicroenvironment. Recent studies have raised the role of media in thepro-inflammatory response and pro-fibrotic, uterine fibroids is mainlycharacterized by a fibrosis progression of the disease. Recent studies haveraised the perspectives is that uterine fibroids is mainly characterized by afibrosis progression in the role of the pro-inflammatory response andpro-fibrotic. Fibrotic diseases characterized by ECM abnormalprecipitation. ECM mainly secretes by fibroblasts, the performance of thesynthesis of extracellular matrix ability of fibroblasts significantlyenhanced at the same time can produce a variety of cytokines, growth andinflammatory factors when the fibroblasts differentiate into myofibroblasts. Endothelin-1(ET-1), a potent vasoconstrictor, has been implicated in thepathogenesis of inflammation, collagen accumulation, scar formation,extracellular matrix remodeling, and renal and cardiac fibrosis. The presentstudies show that ET-1influences the cell proliferation and apoptosis ofuterine fibroids. However, there are no related investigations about ET-1whether play an important role and thus affecting the accumulation of ECMon fibroblast transdifferentiation to myofibroblasts, and also no studiesabout the correlation between ET-1and the important fiber media TGF-β1.Section one Isolation, culture and identification of the primary cellsof uterine fibroids myofibroblastA total of30human uterine fibroids samples and another30normalmyometrium of cervical intraepithelial neoplasia hysterectomy werecollected from the Department of Gynecology at the First AffiliatedHospital of Chongqing Medical University, China. Inclusion criteria fornormal myometrium were consistent with follows:(1) Patients aged32-49years old;(2) Premenopausal women;(3) Not suffering from uterinefibroids and uterine malignancies; and (4) The past six months did notundergo hormone therapy.Specimens histological features Observed by HE staining, thedifference of myofibroblasts between normal myometrium and leiomyomawere distinguished by immunofluorescence; myofibroblast in human uterine fibroid was established by primary culture, and was tested byimmunocytochemical method. Specimen histological features consistentwith uterine fibroids and normal myometrium. The myofibroblasts inuterine fibroid tissue more than in normal myometrium（P<0.05）. Theprimary uterine fibroids myofibroblasts was isolated and culturedsuccessfully, and proved by the immunocytochemistry results showed thatin primary cells the expression of Vimentin and α-SMA positive,meanwhile the expression of H-Caldesmon negative. The excessiveaccumulation of ECM is the one of the main reasons in the process ofgradually increasing uterine fibroids, and myofibroblast is the main sourceof the secretion of ECM synthesis. The results of the study confirmed thatthe uterine fibroids fibroblasts conversion into myofibroblasts than innormal myometrium, which may be one of the key factors in thedevelopment process of the uterine fibroids.Section two ET-1in human myometrium into fibroblast trans-differentiationA total of10human normal myometrium samples were collected fromthe Department of Gynecology at the First Affiliated Hospital ofChongqing Medical University, China. Inclusion criteria for normalmyometrium were consistent with follows:(1) Hysterectomy by Cervicalintraepithelial neoplasia (CIN);(2) Patients aged32-49years old;(3) Premenopausal women;(4) Not suffering from uterine fibroids and uterinemalignancies; and (5) The past six months did not undergo hormonetherapy. The human myometrial fibroblasts were established by primaryculture, and were tested by immunocytochemical method. The cells wasdivided into experimental group and control group, the differentconcentration of ET-1(0.1、1、10、100nmol/L) was added and reacted in thecells for24h. In addition, we added the10nmol/L level of ET-1to reactionfor6h,12h,24h,48h, and10nmol/L ET-1,10ug/L TGF-β1,10nmol/LET-1+10microg/L TGF-β1experimental group, the normal cells wasconsidered to be the control. The α-SMA mRNA and protein expressionwere tested by Quantitative PCR and Western blot.Experimental results showed that the myometrium fibroblastmorphology fusiform, more protruding and the nucleus flat oval.Immunocytoc y showed that α-SMA staining was negative and Vimentin(Vimentin) stained positive, and consistent with protein markerscharacteristic of fibroblasts, which means primary culture of success.RT-qPCR and Western blot showed that the myometrium fiber cellsα-SMA mRNA and protein expression positively after treated with0.1,1,10,100nmol/L ET-1Respectively， and the expression level werestatistically significant compared to control group (P <0.05). The mRNAand protein expression levels of10nmol/L treatment group are highest,and the difference was statistically significant compared to0.1,1nmol/L group (P <0.05), On the other hand, the mRNA and protein expressionlevels were no statistically significant (P>0.05) compared to100nmol/Lgroup.RT-qPCR and Western blot showed that the myometrium fiber cellsα-SMAmRNAand protein expression positively after treated with6，12，24，48h ET-1（10nmol/L） respectively，and the expression level werestatistically significant compared to control group (P <0.05). The mRNAand protein expression levels of48h group are highest, and the differencewas statistically significant compared to6、12、24h groups (P <0.05).RT-qPCR and Western blot showed that the myometrium fiber cellsα-SMA mRNA and protein expression positively after treated with10nmol/L ET-1,10μg/L TGF-β1,10nmol/L ET-1with10μg/L TGF-β1respectively， and the expression level were statistically significantcompared to control group (P <0.05). The mRNA and protein expressionlevels of10nmol/L ET-1with10μg/L TGF-β1group are highest, and thedifference was statistically significant compared to10nmol/L ET-1and10μg/LTGF-β1groups (P <0.05).ET-1-induced myometrial fibroblasts transformed into myofibroblasts,and the optimal concentration may be10nmol/L; ET-1andTGF-β1-induced fibroblast to myofibroblast transdifferentiation may besynergistic effect. Section three The possible mechanism of ET-1on TGF-β1-inducedfibroblast transdifferentiationThe shRNA lentiviral packaging plasmid targeting primary humanuterine fibroids myofibroblast ET-1gene was established. Interference theET-1gene after cells transfection efficiency were observed, screening theinterference effect plasmid and optimal multiplicity of infection (MOI),andthe expression of ET-1、α-SMA、TGF-β1、Smad2、Smad3、p-Smad2、p-Smad3mRNA and protein were tested by Quantitative PCR and Westernblot.Western blot showed that in the three siRNA interference sequences,and the LV-shRNA1lowered ET-1protein expression is the most obvious,with the other two siRNA sequences and blank control group and negativecontrol group difference was statistically significant（P <0.05）.After interference by shRNA1, The mRNA expression of uterinefibroids myofibroblast cells α-SMA, TGF-β1, Smad2, Smad3tested byquantitative PCR. The results showed that compared with the control groupand negative control group, the experimental group α-SMA, TGF-β1,Smad2, Smad3mRNA expression reduced was statistically significant（P <0.01）.After interference by shRNA1, the protein expression of uterinefibroids myofibroblast cells α-SMA, TGF-β1, Smad2, Smad3tested by Western blot. The results showed that compared with the control group andnegative control group, the experimental group α-SMA, TGF-β1, Smad2,Smad3protein expression reduced was statistically significant（P <0.05）.Targeting with the ET-1gene can inhibit the mRNA and protein levelsof α-SMA and TGF-β1, thus may be reversed the transdifferentiation offiber cells to myofibroblast. ET-1promotes the TGF-β1-induceddifferentiation of fibroblasts into myofibroblasts, which may be achievedby adjusting the level of expression of Smad2, Smad3, p-Smad2, p-Smad3.Conclusion: Normal myometrium fibroblasts transformed intomyofibroblasts may be one of the main reasons of the increase in size ofuterine fibroids. ET-1can induce primary myometrial fibroblast tomyofibroblast transdifferentiation; which may be consistent with TGF-β1.Primary isolation and culture of uterine fibroids myofibroblast for the firsttime as the research object. Confirmed that ET-1may promote the role ofTGF-β1-induced fibroblast to myofibroblast transdifferentiation by upregulating the expression levels of Smad2, Smad3, p-Smad2, p-Smad3.ET-1may be Play an important role in the development andprogression of uterine fibroids by induction of fibroblast differentiation andaffect the important fibrosis media TGF-β1-induced differentiation.