Relationship Between TGF-β1/Smad2,3Signal Pathway, Mir-29a And Intrauterine Adhesions

BackgroundIntrauterine adhesions (IUA), also named Asherman syndrome, first detailedly described and reported by Asherman in1984, was originally defined as a consequence of trauma to the endometrium, producing partial or complete obliteration in the uterine cavity and/or the cervical canal, resulting in conditions such as menstrual abnormalities, infertility, and recurrent pregnancy loss. The clinical manifestations include hypomenorrhea, secondary amenorrhea, and periodic pelvic pain. It can lead to many obstetric complications, such as placenta previa, placenta adhesion or implantation, and bring about recurrent miscarriages or female infertility. All of these damages from IUA seriously affect the physical and psychological health and fertility of childbearing age women. With the frequency of intrauterine operation, the popularity of hysteroscopic surgery and the raising awareness of clinicians, the incidence and diagnosis rates of IUA are increasing gradually during the recent years, but the recovery and pregnancy rates still keep low. The treatment of IUA requires a comprehensive and individualized method. The precondition is to separate adhesions. The key point is to prevent the recurrence of adhesions. And the emphasis is to promote restoration of endometrium. The admitted systematic treatment of IUA is transcervical resection of adhesions, then using intrauterine devices to build up a separative enclosure, and taking large doses of estrogen after operation. But the recurrence rate of IUA remains high, especially severe IUA, which is up to62%. Therefore, to explore the pathogenesis of IUA and seek effective targets to prevent the occurrence of endometrial fibrosis and the formation of IUA, has become the common goal of clinical doctors and scientific researchers.IUA is the result of endometrial abnormal restoration and endometrial fibrosis after damage. Just like other organs, the common pathological process of fibrosis also exists in the process of IUA forming——extracellular matrix (ECM) accumulating, organizational reconstructing and scar formation. Excessive proliferation of collagen fibers occur after damage of endometrial basal layer, and endometrial hyperplasia is suppressed at the same time. As a result, endometrial tissue is gradually replaced by fibrous connective tissue, leading to endometrial fibrosis and cicatrization. Therefore, to block the endometrial fibrosis process after injury and the accumulation of ECM might be able to prevent the formation of IUA.Transforming growth factor-β1(TGF-β1) has been found the paramount cytokine to promot fibrosis up to now. Its signal is transmitted by the downstream intermediary molecules called Smad2and Smad3. After trauma, TGF-β1is first released to injury site by the platelets. Then fibroblast cells, monocytes and neutrophils are chemotactic by TGF-β1dose dependently. When TGF-β1combines with specific membrane receptors in fibroblasts and other cells, Smad2and Smad3transmit the TGF-β1signal from cytoplasm into nucleus and regulate the transcription of target ECM genes. In normal physiological conditions, TGF-β1can promote injured tissue repair and wound healing. At that time, the synthesis and degradation of ECM keep in dynamic balance. While in pathological conditions, TGF-β1can stimulate genetic transcription of fibronectin and collagen, accelerating ECM synthesis, and inhibiting ECM degradation. Consequently, ECM accumulates excessively and adhesion forms.MicroRNA-29(miR-29) is a newly discovered family of small RNA closely related to fibrosis diseases, and has become the research hotspot in biology. Expression of miR-29decreases in many fibrotic tissues, such as myocardium of myocardial infarcion, hepatic stellate cells, pulmonary fibrosis and so on. Its decreasing level is positively associated with the degree of fibrosis. MiR-29can not only inhibit the synthesis of ECM directly, but also participate in many signal pathways related to fibrosis such as the TGF-β1/Smad pathway. TGF-β1can decrease miR-29, while overexpression of miR-29can decrease TGF-β1and inhibit Smad3in reverse. All these indicate miR-29may have a mighty potency of anti-fibrosis, and provide a new direction for diagnosis and treatment of fibrosis diseases. How miR-29expresses in endometrium is still without reported. It is given that miR-29in human includes miR-29a, miR-29b1, miR-29b2and miR-29c, and among them it is the miR-29a that has most target genes related to fibrotic proteins. So miR-29a is chosen to be one of our research goals. Make it clear that how miR-29a expresses in endometrium, and how it works during endometrial fibrosis, may be able to provide a new direction of research for prevention and treatment of IUA.In the present study, we detected the expression of TGF-β1, Smad2,3and miR-29a in endometrium of women with IUA using fluorescent quantitative PCR (FQ-PCR) and immunohistochemistry (IHC), and discussed their relations with IUA, in order to provide some theoretical basis for further research and seek for some effective targets to prevent or cure IUA. Research data1. Grouping:80patients hospitalized in the Zhujiang Hospital Affiliated to Southern Medical University because of IUA from March2012to January2014were chosen to be the experimental groups, including30cases in minimal IUA,26cases in moderate IUA, and24cases in severe IUA.27patients hospitalized at the same time because of other diseases were chosen as the control group. The general conditions, such as age, times of pregnance, times of intrauterine operation and so on, have no statistical significance among the four groups(.P>0.05). All the patients have signed the information consent form.2. Classification of IUA:①Minimal IUA:Less than one-fourth of uterine cavity; thin or filmy adhesions; ostial areas, and upper fundus minimally involved or clear.③Moderate IUA:One-fourth or three-fourth of uterine cavity; no agglutination of walls; ostial areas and upper fundus only partially occluded.③Servere IUA:More than three-fourth of uterine cavity; agglutination of walls or thick bands; ostial area and upper cavity occluded.3. Exclusion criteria:①having endocrine disorders, such as diabetes, hyperthyroidism and so on.②demonstrated to have any organ fibrosis, such as liver cirrhosis, pulmonary fibrosis, systemic sclerosis and so on.③) amenorrhea caused by dysfunction of ovary, pituitary, hypothalamus.④other intrauterine diseases, such as polyps, myoma.⑤HE stain shows secretory phase endometrium.Chapter1Expression and significance of TGF-β and Smad2,3in endometrium of women with intrauterine adhesionsObjective:To detect the mRNA level and protein level of TGF-β1and Smad2,3in endometrium of women with intrauterine adhesions, explore their potential significance and effect on the formation of intrauterine adhesions, and provide some theoretical basis for further research.Method:1. Detecting the mRNA level of TGF-β1and Smad2,3by FQ-PCR.Fresh endometrium was obtained during hysteroscope operations. Total RNA was extracted from endometrium. First-strand cDNA was synthesized from total RNA by reverse transcription. The reverse transcription reaction condition was37℃for15minutes,85℃for5seconds and4℃. Then FQ-PCR reactions were performed. FQ-CR incubation and cycling parameters were95℃for30seconds, and a two-step temperature cycle consisted of a denaturing step at95℃for5seconds and an annealing cycle of60℃for34seconds, repeated40times. The melt curve was performed using the following parameters:95℃for15seconds,60℃for1minute and95℃for15seconds. Here β-actin was used as an internal standard of mRNA expression. Each sample was repeated fot three times. The expression levels of target genes were calculated by using the relative quantitative method2-△△Ct.2. Detecting the protein level of TGF-β1and Smad2,3by IHC.Endometrium was infused by4%paraformaldehyde solution and made into paraffin specimens and sections with4μm in thickness. Endometrial type was observed by HE stain after dehydrated and hydration, and those in proliferative stage were chosen for further research. For TGF-β1and Smad2/3antigen retrieval, slides were boiled in a microwave oven in citrate buffer solution (PH6.0). Sections were incubated in3%H2O2, goat serum, primary antibody (1:100in concentration), secondary antibody according to the manufacturer’s instructions. At last, immunoreactivity was detected using DAB. Here PBS was used to replace primary antibody as negative control. Images were captured with the use of Leica Application Suite. The IOD value was obtained by image analysis system.Results:1. The mRNA level of TGF-β1, Smad2and Smad3in endometrium(1) The genetic expression of TGF-β1had significant difference among groups and it showed a trend of increasing with the aggravating of adhesion degree (0.61±0.58in control group,0.84±0.53in minimal IUA,1.11±0.54in moderate IUA,1.66±1.62in severe IUA). There was no significant difference between minimal IUA and control(P=0.343), neither minimal IUA and moderate IUA(P=0.261). While it showed significant differences between control group and moderate IUA(P=0.046). And the TGF-β1genetic expression in severe IUA had significant difference compared with control group(P<0.001), minimal IUA (P=0.001) and moderate IUA (P=0.035).(2) An increasing thend of the genetic expression of Smad2was observed with the aggravating of adhesion degree and the difference was significant among groups (0.63±0.45in control group,0.84±0.43in minimal IUA,1.05±0.52in moderate IUA,1.41±0.76in severe IUA). There was no significant difference between control group and minimal IUA(P=0.144), neither between minimal IUA and moderate IUA(P=0.155). There was significant difference between control group and moderate IUA(P=0.006). And the Smad2genetic expression in severe IUA had significant difference compared with control group(P<0.001), minimal IUA (P<0.001) and moderate IUA (P=0.024).(3) The genetic expression of Smad3had significant difference among groups and it showed a trend of increasing with the aggravating of adhesion degree (0.57±0.49in control group,0.95±0.51in minimal IUA,1.19±0.47in moderate IUA, 1.34±0.59in severe IUA). There was significant difference between experimental groups and control group(PminimalIUA=0.006、Pmoderate IUA<0.001、PsevereIUA<0.001), the same as minimal IUA and severe IUA(P=0.008). But moderate IUA had no significant difference respectively compared with minimal IUA(P=0.092) and severe IUA(P=0.316).2. The correlation analysis of Smad2,3and TGF-β1mRNAA strong positive correlation between the genetic expression of Smad2, Smad3and TGF-β1was observed(rs Smad2=0.647, PSmad2<0.001; rs Smad3=0.663, Psmad3<0.001).3. The protein level of TGF-β1and Smad2,3in endometrium(1) The TGF-β1protein was observed in both glandular epithelial cells and mesenchymal cells in endometrium. The protein expression of TGF-β1in experimental groups was significantly higher than of control group and the ascending order of TGF-β1protein was control group(134635.90), minimal IUA(266384.27), moderate IUA(464274.10) and severe IUA(719535.75). There was significant difference between experimental groups and control group (PmiidiuA=0.001Pmoderate IUA<0.001, Psevere IUA<0.001). The TGF-β1protein expression in severe IUA had significant difference compared with mild IUA (P<0.001) and moderate IUA (P=0.004), the same as minial IUA compared with moderate IUA(P=0.002).(2) The Smad2,3protein was observed in both glandular epithelial cells and mesenchymal cells in endometrium. The protein expression of Smad2,3had significant difference among groups and the ascending order of Smad2,3protein was control group(78607.57), minimal IUA(113200.78), moderate IUA(188062.00) and severe IUA(472224.86). The protein level in control group had no significant difference compared with minimal IUA(P=0.160) but had significant difference compared with moderate IUA(P<0.001) and severe IUA(P<0.001). The protein level in severe IUA also had significant difference compared with minimal IUA(P<0.001) and moderate IUA(P=0.005), the same as minial IUA compared with moderate IUA(P=0.040).4. The correlation analysis of Smad2,3and TGF-β1proteinA strong positive correlation between the protein expression of Smad,3and TGF-β1was observed(rs=0.467, P<0.001).Conclusion:1. The protein of TGF-β1and Smad2,3can be observed in both glandular epithelial cells and mesenchymal cells in endometrium.2. The overexpression of mRNA and protein of TGF-β1in endometrium may play an important role in process of IUA.3. The overexpression of mRNA and protein of Smad2,3in endometrium may participate in development of IUA.4. The excessive regulation of the TGF-β1/Smad2,3signal pathway may take part in the formation of IUA.Chapter2Expression and significance of miR-29a in endometrium of women with intrauterine adhesionsObjective:To detect the expression of miR-29a in endometrium of women with intrauterine adhesions, explore its potential significance and effect on the formation of intrauterine adhesions, and provide some theoretical basis for further research. Method:Fresh endometrium was obtained during hysteroscope operation. Total RNA was extracted from endometrium. First-strand cDNA was synthesized from total RNA by reverse transcription. The reverse transcription reaction condition was42℃for15minutes,85℃for5seconds and4℃. Then FQ-PCR reactions were performed. FQ-CR incubation and cycling parameters were95℃for30seconds, and a two-step temperature cycle consisting a denaturing step at95℃for5seconds and an annealing cycle of60℃for34seconds, repeated40times. The melt curve was performed using the following parameters:95℃for15seconds,60℃for1minutes and95℃for15second. Here U6was used as an internal standard of miR-29a expression. Each sample was repeated for three times. The expression levels of target genes were calculated by using the relative quantitative method2-△△CT.Results:1. The expression of miR-29a in endometriumThe expression of miR-29a in experimental groups was significantly higher than that of control group. The descending order of miR-29a expression was control group(1.62±0.55), minimal IUA(1.33±0.40), moderate IUA(1.04±0.41) and severe IUA(0.85±0.54). The expression of miR-29a in control group had significant difference compared with minimal IUA(P=0.024), moderate IUA(P<0.001), and severe IUA(P<0.001) respectively. The expression of miR-29a in minimal IUA also had significant difference compared with moderate IUA(P=0.024) and severe IUA(P<0.001). But there was no significant difference between moderate IUA and severe IUA(P=0.183).2. The correlation analysis of miR-29a and TGF-β1mRNA: A strong negative correlation between the genetic expression of miR-29a and TGF-β1was observed(rs miR.29a=-0.335, PmiR-29a<0.001).Conclusion:1. MiR-29a exits in endometrium and was closely related to the degree of adhesion of uterine cavity.2. The low expression of miR-29a in endometrium may take part in occurence of IUA.3. There may be interaction between miR-29a and the TGF-β1mRNA in endometrium.