| Uterine fibroids are the most common benign tumor in women of reproductive age,with a prevalence of 30%to 70%,of which approximately one third of patients have clinical symptoms,including abnormal uterine bleeding(AUB),anemia,pelvic pressure and pain,dysmenorrhea and reproductive dysfunction.Traditionally,it was believed that the presence and severity of symptoms depended on the size and location of the fibroids(subserosal,intramural,or submucosal),but now most scholars believe that the growth rate of fibroids and the presence or absence of malignant transformation are the causes of symptoms of uterine fibroids.Serious reasons,and relatively small relationship with the size and number of fibroids.The main factors that lead to the occurrence of uterine fibroids in patients are genetic factors,long-term oral contraceptives and repeated abortions.At present,surgical treatment and drug treatment are mainly used in the clinical treatment of patients with uterine fibroids.So far,no therapeutic drug with ideal treatment effect and low recurrence rate has been found.Therefore,in the actual clinical treatment process,the Surgery is the main treatment.However,surgical treatment will seriously affect the quality of life of patients.Therefore,finding and developing more effective therapeutic targets is the way to completely cure uterine fibroids.Epithelial-mesenchymal transition(EMT)plays a crucial role in the invasion and metastasis of uterine fibroids.E-cadherin,a member of the cadherin family,whose expression is absent or reduced promotes cell migration and invasion.E-cadherin is a marker molecule of epithelial phenotype cells and has important value in maintaining cell polarity;N-cadherin and Vimentin are marker molecules of mesenchymal phenotype cells that promote cell motility and invasion;Snail,a transcription factor that regulates the EMT process,binds to the E-cadherin promoter region,impeding gene transcription,attenuating the epithelial phenotype and promoting its transformation to a mesenchymal phenotype.Therefore,inhibition of EMT plays an important role in the invasion of uterine fibroids.Signal transducers and activators of transcription(STAT)are a unique family of DNA-binding proteins.Contains SH2 and SH3 domains that can bind to specific phosphorylated tyrosine-containing peptides.When STAT is phosphorylated,it aggregates into an activated transcription activator in the form of homologous or heterodimers,enters the nucleus and binds to a specific site of the target gene promoter sequence to promote its transcription.STAT3 is required for mediating oncogene cell transformation and is directly associated with human cancers.Activated STAT3 can further stimulate c-Myc,VEGF,MMPs and other molecules to promote tumor cell proliferation,invasion,metastasis and angiogenesis.Our study showed that STAT3 can increase the expression of cyclins Cyclin D1 and c-Myc,and promote the proliferation of uterine fibroids.In addition,the STAT3 signaling pathway enhanced the invasion of uterine fibroids by increasing the expression of MMP2 and MMP9.Tip:STAT3 signaling pathway plays an important role in the occurrence and development of uterine fibroids.miRNAs can degrade or inhibit mRNA translation,thereby participating in the regulation of cell proliferation,apoptosis and invasion.The miR-29family consists of miR-29a,miR-29b and miR-29c.The miR-29 family can act as tumor suppressors,inhibiting the proliferation of gastric,lung,liver and colon cancers.It has been reported that miR-29a can regulate the expression of BMI1 gene through the NF-κB and Wnt/β-catenin signaling pathways,thereby inhibiting the growth,migration and invasion functions of tumor cells.miR-29a can target CDK6 and downregulate JNK and p38/MAPK/ERK signaling pathways,thereby inhibiting the proliferation of schwannomas.miR-29b is inhibited in cervical cancer,thereby promoting extracellular matrix accumulation and cell proliferation rate.miR-29b is downregulated in head and neck squamous cell carcinoma and exerts tumor suppressor effect by blocking integrinβ1-mediated oncogene signaling pathway.According to Liu’s research,miR-29b regulates STAT3 in lung adenocarcinoma cells,and STAT3 can promote lung adenocarcinoma cell proliferation while inhibiting apoptosis.There are relatively few studies on miR-29c.It has been reported that the expression of miR-29c is transcriptionally regulated by NF-k B and SP1,and that miR-29c can promote colon cancer metastasis.Some researchers believe that the differential expression of miR-29c in different tumors may provide the possibility for tumor targeted therapy.Based on this,we speculate that miR-29c may play an important role in the growth and development of uterine fibroids.We demonstrate using high-throughput sequencing of miRNAs and appropriate basic experiments that when miR-29c is overexpressed,it binds to STAT3,leading to STAT3 degradation.Subsequently,the expression of Cyclin D1 and c-Myc decreased,and the proliferation of uterine fibroids was inhibited.Furthermore,STAT3 was identified as an essential mediator of MMP2 and MMP9 expression,and STAT3 directly activates the transcription of MMP2 and MMP9 in cancer cells.When STAT3 was degraded by miR-29c,the expression of MMP2 and MMP9 was reduced,and the metastasis and invasion of uterine fibroids were attenuated.miR-29c is down-regulated in tumor cells,reduces the expression of stem cell-related transcription factors and their ability to form spheroids,activates the STAT3signaling pathway,increases STAT3 expression,and promotes tumor pathogenesis.Therefore,the interaction of miR-29c with STAT3 plays a key role in extracellular matrix degradation,enhanced tumor invasion,proliferation and metastasis.Despite the rigorous miRNA sequencing analysis performed in this paper,there are still some deficiencies,and in this study,we did not perform gene overexpression or knockout animal experiments to further verify its function.Therefore,in future research,we should investigate this aspect in depth.In conclusion,our findings suggest that high expression of miR-29c can inhibit the proliferation,invasion and metastasis of uterine fibroids,which may be related to the inhibition of STAT3 signaling pathway,providing a new target for the diagnosis and treatment of uterine fibroids.Most of the clinical diagnosis and observation of uterine fibroids are achieved by ultrasound.We collected and statistically analyzed various ultrasound indicators of the uterine artery.The results showed that the uterine artery blood supply in patients with uterine fibroids was rich,and the ultrasound index S/D was higher than that of the control group.The observation and treatment of uterine fibroids provides a new choice of observation indicators.Part one Screening of differential proteins and miRNAs in uterine fibroidsObjective:By detecting the differentially expressed miRNAs in uterine fibroids and myometrium samples,the core miRNAs regulating uterine fibroids were screened out and further clarified through basic experiments.Methods:1.Using the R language limma software package,theRNA-seq data were subjected to quantile normalization and differential gene analysis(|log FC|<1,p-value<0.05),and cluster analysis was performed at the same time.2.Cut the tissue pieces obtained during the operation into small pieces(about 1mm~3 in volume)and homogenize them,then wash them 2-3 times with phosphate buffer containing penicillin and streptomycin.Tissues were digested in 1 mg/ml IV collagenase for 3-5 h.Cells were successfully filtered through a 100um cell strainer to remove impurities.Then,the cells were centrifuged(1500 rpm/5 min),and the pellet was collected and suspended in DMEM medium.During the culture of uterine fibroids,the state of uterine fibroids was observed under a microscope.After the cells were fully grown,the cells were passaged,and the cells used in the experiment were the third-generation cells.Uterine fibroids and myometrium cells were collected separately,and whole-cellRNA was extracted.The mRNA levels of miR-29c were compared by RT-PCR detection.3.The uterus-related tumor cell lines(Hela,HT-3,Ect1/E6E7,HTP6136)were cultured and passaged until the third passage of cells was collected,and whole-cellRNA was extracted.The mRNA levels of miR-29c were compared by RT-PCR detection.4.The fibroids and myometrium cells are extracted and cultured until the third passage of cells is collected,lifting the whole cell protein.Western blot analysis was used to analyze the expression changes of tumor migration-related proteins(E-cadherin,N-cadherin,Vimentin,snail),invasion-related proteins(MMP2,MMP9),and proliferation-related proteins(Cyclin D1,c-Myc).5.The fibroids and myometrium cells are extracted and cultured until the third passage is collected,and whole-cellRNA is extracted.The mRNA expression changes of tumor migration-related proteins(E-cadherin,N-cadherin,Vimentin,snail),invasion-related proteins(MMP2,MMP9),and proliferation-related proteins(Cyclin D1,c-Myc)were analyzed by RT-PCR technology.Results:1.Screening of differentially expressed genes:Screening of uterine fibroids miRNA sequencing results according to the criteria of p-value<0.05and|log FC|<1.The results showed that there were 204 differentially expressed genes in miRNAs of uterine fibroids,of which 139 were up-regulated and 65were down-regulated.Among them,miR-29c was significantly reduced in uterine fibroids,and the results suggested that miR-29c may be at the core of gene regulation in uterine fibroids.2.Compared with myometrial tissue,miR-29c was significantly lower expressed in uterine fibroids.3.Compared with other tumor cell lines in the uterus,the uterine fibroids cell line HTP6136 had the lowest expression of miR-29c and was significantly reduced.4.In uterine fibroids,the expression of E-cadherin was decreased at both protein and gene levels,while other migration-related proteins N-cadherin,Vimentin and snail were significantly increased.5.In uterine fibroids,invasion-related proteins(MMP2,MMP9)were significantly increased at both protein and gene levels.6.In uterine fibroids,proliferation-related proteins(Cyclin D1,c-Myc)were significantly increased at both protein and gene levels.Summary:1.Based on miRNA sequencing analysis,miR-29c plays a central role in the gene regulation of uterine fibroids.2.miR-29c was significantly down-expressed in uterine fibroids tissues and cells.3.miR-29c may regulate the migration,invasion and proliferation of uterine fibroids.Part Two The effect of miR-29c on migration,invasion and proliferation of uterine fibroidsObjective:To clarify the regulatory effect of miR-29c on uterine fibroids by means of molecular biologyMethods:1.Uterine fibroids cells were seeded into 6-well plates for culture.The experiment was divided into(1)control group(NC group):transfected with miR-NC mimic;(2)miR-29c group:transfected with miR-29c mimic;(3)inhibitor NC group:transfected with miR-NC inhibitor;(4)inhibitor group:miR-29c inhibitor was transfected;4 parallel wells were set in each group.When the uterine fibroids were cultured to about 80%confluence,transfection was carried out according to the instructions of lipofectamine 3000.After 48 h,cells in each group were collected,totalRNA was extracted,and the transfection effect was detected by RT-PCR.2.Repeat method 1 above to collect cells and extract total protein.The expression changes of tumor migration-related proteins(E-cadherin,N-cadherin,Vimentin,snail),invasion-related proteins(MMP2,MMP9),and proliferation-related proteins(Cyclin D1,c-Myc)were detected by Western blot.3.Cell invasion experiment:Matrigel diluted with serum-free medium was coated on a polycarbonate microporous membrane with a pore size of 8μm in a Transwell chamber,and fully fused for 2 hours.Cells were resuspended in serum-free medium and placed in Transwell chambers in24-well plates.500μl of serum-containing medium was added below the Transwell chamber,and 200μl of cell suspension was added to the upper chamber and incubated for 24 hours.To detect the invasive ability of uterine fibroids cells.4.Cell proliferation experiment:after the uterine fibroids in logarithmic growth phase were digested with trypsin,resuspended in complete medium to form a cell suspension and counted,and the cells were seeded in a six-well plate at a density of 400 cells/well Cultivate until the number of cells in most single clones is more than 50,change the medium every 3 days and observe the cell status.After cloning is completed,wash once with PBS,fix with 4%paraformaldehyde for 30 min,and wash each well with PBS.Add 0.1%crystal violet staining solution,stain the cells for 10-20 min,wash with PBS for 2-3times,and take pictures with a digital camera after drying.To detect the proliferative capacity of uterine fibroids cells.Results:1.In uterine fibroids,compared with the control group transfected with miR-NC,the mRNA levels of miR-29c were significantly increased in uterine fibroids transfected with miR-29c mimics;transfected with miR-29c inhibitors In uterine fibroids,the mRNA level of miR-29c was significantly reduced.2.In uterine fibroids,compared with the control group transfected with miR-NC,the expression of E-cadherin protein increased after uterine fibroids were transfected with miR-29c mimics,while other migration-related proteins N-cadherin,Vimentin and snail were significantly decreased;after transfection of miR-29c inhibitor,E-cadherin protein expression was decreased,while other migration-related proteins N-cadherin,Vimentin and snail were significantly increased.3.In uterine fibroids,compared with the control group transfected with miR-NC,the expression of invasion-related proteins(MMP2,MMP9)decreased after uterine fibroids were transfected with miR-29c mimics;transfected with miR-29c inhibitors After treatment,the expression of invasion-related proteins(MMP2,MMP9)was significantly increased.4.In uterine fibroids,the expression of proliferation-related proteins(Cyclin D1,c-Myc)decreased after uterine fibroids were transfected with miR-29c mimics,compared with the control group transfected with miR-NC;After miR-29c inhibitor,the expression of proliferation-related proteins(Cyclin D1,c-Myc)was significantly increased.5.The results of cell invasion assay showed that compared with the control group transfected with miR-NC,the invasion ability of uterine fibroids cells was significantly reduced after transfection with miR-29c mimic;after transfection with miR-29c inhibitor,the invasion ability was significantly increased.6.The results of cell proliferation experiments showed that compared with the control group transfected with miR-NC,the proliferation ability of uterine fibroids was significantly reduced after transfection with miR-29c mimic;after transfection with miR-29c inhibitor,the proliferation ability was significantly increased.Summary:1.miR-29c affects the migration of uterine fibroids by changing the expression of E-cadherin,N-cadherin,Vimentin and Snail.2.miR-29c affects the invasion of uterine fibroids by changing the expression of MMP2 and MMP9.3.miR-29c affects the proliferation of uterine fibroids by changing the expression of Cyclin D1 and c-Myc.Part three miR-29c targets STAT3 to regulate the occurrence and development of uterine fibroidsObjective:Using molecular biology methods,it is clear that miR-29c targets STAT3 and regulates the occurrence and development of uterine fibroidsMethods:1.Luciferase reporter gene detection:Construction of STAT3 mutant:STAT3-MUT.miR-29c mimic was transfected into STAT3-WT uterine fibroids and STAT3-MUT uterine fibroids,respectively.After that,follow the instructions of Dual-Lumi?Firefly Luciferase Detection Kit,and finally use a multi-functional microplate reader with chemiluminescence detection function to detect the fluorescence value of firefly luciferase.2.Isolate and culture uterine fibroids cells and myometrium cells,carry out cell passage,until the cells are the third generation cells.Whole cellRNA and protein were extracted separately.The mRNA levels of STAT3 were detected and compared by RT-PCR,and the protein expression levels of STAT3 were detected by Western blot.3.The uterus-related tumor cell lines(Hela,HT-3,Ect1/E6E7,HTP6136)were cultured and passaged until the third passage of cells was collected,and whole-cellRNA was extracted.The mRNA levels of STAT3 were compared by RT-PCR detection.4.In uterine fibroids cells,miR-29c mimic was transfected,the cells were collected,the whole protein was extracted,and the protein expression level of STAT3 was detected by Western blot.5.In uterine fibroids cells,si-STAT3 was transfected,the cells were collected,the whole protein was extracted,and Western blot was used to detect tumor migration-related proteins(E-cadherin,N-cadherin,Vimentin,snail),invasion-related proteins(MMP2,MMP9),the expression changes of proliferation-related proteins(Cyclin D1,c-Myc).Results:1.The results of luciferase reporter gene detection showed that:in STAT3-WT uterine fibroids,transfection of miR-29c will reduce the fluorescence intensity,but in STAT3-MUT uterine fibroids,transfection of miR-29c fluorescence intensity constant.2.Western blot and RT-PCR results showed that the expression of STAT3was increased in uterine fibroids.3.Among the uterine-related cancer cell lines,the uterine fibroids cell line HTP6136,the mRNA expression of STAT3 was the highest and significantly increased.4.In uterine fibroids,after transfection of si-STAT3,the tumor migration-related protein E-cadherin was significantly increased by Western blot,while N-cadherin,Vimentin and snail were significantly decreased.5.In uterine fibroids,after transfection of si-STAT3,the invasion-related proteins MMP2 and MMP9 detected by Western blot were significantly decreased.6.In uterine fibroids,after transfection of si-STAT3,the proliferation-related proteins(Cyclin D1,c-Myc)detected by Western blot were significantly decreased.Summary:1.The target of miR-29c is STAT3.2.miR-29c inhibits the proliferation,invasion and migration of uterine fibroids by binding to STAT3,leading to the degradation of STAT3.Part four Analysis of clinical characteristics of uterine artery on Hysteromyoma by ultrasonic monitoringObjective:Doppler ultrasound was used to monitor the uterine artery and analyze the clinical features of hysteromyoma.Methods:The uterine artery ultrasound indexes of clinical 310 cases(women between the ages of 20 and 60)were analyzed,including peak arterial systolic blood velocity(PS),end-diastolic flow velocity(ED),peak arterial systolic flow velocity/end-diastolic flow velocity(S/D),and resistance index(RI).To evaluate the diagnostic and therapeutic value of these variables in the diagnosis and treatment of uterine Leiomyoma,the differences of these variables in different age stages and uterine Leiomyoma were observed.Results:1.The incidence of uterine fibroids is related to age(women between the ages of 20 and 60),with extremely low incidence under 30 years,and gradually increasing with age.2.There was no significant difference between the two uterine arteries detected by Dopler ultrasound.3.The right uterine artery was studied.Compared with the normal group,the S/D of myoma group was significantly lower.However,there was no significant difference in each index for different size of Hysteromyoma,less than 50 mm group and more than 50 mm group.Summary:1.Within a certain age range,the incidence of uterine fibroids was associated with age.2.There was no difference between the left and right uterine arteries.3.The sonographic parameters of uterine artery(S/D)were changed in patients with hysteromyoma and normal controls.The size of Hysteromyoma had no correlation with the parameters of uterine artery.Conclusions:1.miR-29c plays a central role in the regulation of uterine fibroids.2.miR-29c was significantly down-expressed in uterine fibroids tissues and cells.3.The target of miR-29c is STAT3.4.miR-29c inhibits the proliferation,invasion and migration of uterine fibroids by binding to STAT3,leading to the degradation of STAT3.5.Uterine artery ultrasound index is correlated with the incidence of uterine fibroids,and uterine artery ultrasound index can also be used as a reference for selecting treatment methods. |
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