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OBJECTIVES@#Cervical squamous cell carcinoma is the most common cancer in female reproductive system. This study aims to explore the effect of microRNA-9-5p (miR-9-5p) on the migration, invasion, and epithelial-mesenchymal transition (EMT) process of cervical squamous cells.@*METHODS@#Bioinformatics were used to predict the miRNAs that could bind to E-cadherin (E-cad). The Cancer Genome Atlas (TCGA) database was used to analyze and extract significantly differentially expressed miRNAs from part of cervical squamous cell carcinoma tissues and normal cervical tissues, and miR-9-5p was selected as the main research target. The translated regions (UTR) of wild-type E-cad (E-cad-WT 3'-UTR) or the 3'-UTR of mutant E-cad (E-Cad-MUT 3'-UTR) was transfected with miR-9-5p mimic normal control (NC), and miR-9-5p mimic was co-transfected human embryonic kidney cells (293T). The relationship between miR-9-5p and E-cad was detected by double luciferase assay. The expression of miR-9-5p in normal cervical epithelial cell lines (H8) and cervical squamous cell lines (C33A, siha, caski and Me180) were detected by quantitative real-time PCR. Then, the experiments were divided into groups as follows: a block control group, an overexpression control group (mimic-NC group), a miR-95p overexpression group (mimic group), an inhibitory expression control group (inhibitor-NC group), and a miR-9-5p inhibitory expression group (inhibitor group). The changes of migration ability were detected by scratch assay. Transwell invasion assay was used to analyze the changes of invasion ability, and the mRNA and protein changes of E-cad and vimentin were detected by quantitative real-time PCR and Western blotting.@*RESULTS@#MiR-9-5p had a targeting binding relationship with E-cad. Compared with the normal cervical tissue H8 cell line, the miR-9-5p was highly expressed in cervical cancer cell lines (C33A, siha, caski and Me180) (all P<0.05). The luciferase activity of E-cad-MUT was increased compared with that of E-cad-WT in miR-9-5p mimic cells (P<0.05). Compared with the blank control group, the protein and mRNA expressions of E-cad were decreased in the miR-9-5p mimic group (both P<0.05), which were increased in the miR-9-5p inhibitor group (both P<0.05). Compared with H8 cell line, the miR-9-5p was highly expressed in the cervical squamous cell lines (all P<0.05). Compared with the mimic-NC group, the distance of wound healing, the number of caski and Me180 cells invaded below the membrane, and the mRNA and protein expressions of vimentin were all increased in the miR-9-5p mimic group (all P<0.05), while the mRNA and protein of E-cad were decreased (both P<0.05). Compared with the inhibitor-NC group, the distance of wound healing, the number of caski and Me180 cells invading the membrane, and the mRNA and protein expressions of vimentin were decreased in the miR-9-5p inhibitor group (all P<0.05), but the mRNA and protein expressions of E-cad were increased (both P<0.05).@*CONCLUSIONS@#The miR-9-5p is highly expressed in cervical squamous cell carcinoma, which can increase the migration and invasion ability, and promote the EMT process of cancer cells.
Subject(s)
Humans , Female , Cell Line, Tumor , Vimentin/metabolism , Uterine Cervical Neoplasms/genetics , Epithelial-Mesenchymal Transition/genetics , MicroRNAs/metabolism , Carcinoma, Squamous Cell/genetics , Cell Movement/genetics , RNA, Messenger , Cell Proliferation/genetics , Gene Expression Regulation, NeoplasticABSTRACT
Objective To investigate the effects of specific methyltransferase inhibitor of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on the promoter methylation of E-cadherin (E-cad) gene, protein expression in human cervical cancer SiHa cells, and the cell biological behavior. Methods SiHa cells were treated with 5-Aza-CdR at different concentrations. Quantitative methylation-specific polymerase chain reaction (QMSP) was used to examine CpG island promoter methylation level of E-cad gene before and after treatment. The experimental group of the optimum concentration was selected. The expression levels of E-cad mRNA and its protein in SiHa cells line were detected by quantitative real-time polymerase chain reaction (RT-PCR) and western blot respectively. Cell adhesion test was used to measure cell adhesion ability and Transwell test was used to detect cell invasion and migration ability. Results E-cad gene promoter methylation index (PMR) of 5-Aza-CdR at 0, 1, 5, 10, 15 μmol/L level was (53.0 ±1.6) %, (50.0 ±1.2) %, (44.0 ±1.4) %, (27.0 ±1.7) %, (15.0±8.2) %respectively, and PMR value decreased gradually with the increase of 5-Aza-CdR concentration. Furthermore, PMR value was the lowest at 15μmol/L, and the difference was statistically significant compared with other 4 groups (P< 0.01). Then 5-Aza-CdR at 15 μmol/L was selected as the following experimental concentration. The expression of E-cad mRNA and its protein in the 5-Aza-CdR group were significantly higher than those in the blank control group (P<0.05). The rates of cell adhesion , cell invasion inhibition and migration inhibition were all increased with significant differences (P<0.05). Conclusions 5-Aza-CdR can upregulate E-cad mRNA and protein expression level in cervical cancer SiHa cells, reduce cell invasion and migration ability, and promote the adhesion of SiHa cells, which has reversed hypermethylation in the promoter region of E-cad gene partly.
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Objective To investigate the effects of specific methyltransferase inhibitor of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on the promoter methylation of E-cadherin (E-cad) gene, protein expression in human cervical cancer SiHa cells, and the cell biological behavior. Methods SiHa cells were treated with 5-Aza-CdR at different concentrations. Quantitative methylation-specific polymerase chain reaction (QMSP) was used to examine CpG island promoter methylation level of E-cad gene before and after treatment. The experimental group of the optimum concentration was selected. The expression levels of E-cad mRNA and its protein in SiHa cells line were detected by quantitative real-time polymerase chain reaction (RT-PCR) and western blot respectively. Cell adhesion test was used to measure cell adhesion ability and Transwell test was used to detect cell invasion and migration ability. Results E-cad gene promoter methylation index (PMR) of 5-Aza-CdR at 0, 1, 5, 10, 15 μmol/L level was (53.0 ±1.6) %, (50.0 ±1.2) %, (44.0 ±1.4) %, (27.0 ±1.7) %, (15.0±8.2) %respectively, and PMR value decreased gradually with the increase of 5-Aza-CdR concentration. Furthermore, PMR value was the lowest at 15μmol/L, and the difference was statistically significant compared with other 4 groups (P< 0.01). Then 5-Aza-CdR at 15 μmol/L was selected as the following experimental concentration. The expression of E-cad mRNA and its protein in the 5-Aza-CdR group were significantly higher than those in the blank control group (P<0.05). The rates of cell adhesion , cell invasion inhibition and migration inhibition were all increased with significant differences (P<0.05). Conclusions 5-Aza-CdR can upregulate E-cad mRNA and protein expression level in cervical cancer SiHa cells, reduce cell invasion and migration ability, and promote the adhesion of SiHa cells, which has reversed hypermethylation in the promoter region of E-cad gene partly.
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Cervical cancer is women's common malignant tumor.With the extensive application of screening and early surgery,cervical cancer incidence declines year by year,but no decrease mortality trend.The patient died of cancer metastasis and recurrence.E-cadherin is the central link of intercellular adhesion,and junction complex has been confirmed by a large number of literature and closely associates cervical cancer development.This review summarizes the relevant research between E-cadherin and the occurrence and development of cervical cancers.
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Objective To investigate the influence of human papillomavirus (HPV) 16 E6 gene silencing by small interfering RNA (siRNA) on the expression and the promoter hypermethylation status of E-cadherin (E-cad) in cervical cancer SiHa cell line. Methods siRNA which used lentivirus as the vector was used to knock down the HPV16E6 gene in cervical cancer SiHa cell line. The expression levels of HPV16E6 mRNA, E-cad mRNA and protein in siRNA-HPV16E6 SiHa cell line were detected by RT-qPCR and Western blot, respectively. Methylation specific PCR (MSP) method was used to detect the methylation status of E-cad gene (CDH1) promoter in siRNA-HPV16E6 SiHa cell line. Results The E-cad mRNA expression levels in siRNA E6 group, empty vector group and blank control group were 4.755±1.085, 1.224± 0.840, 1.327±1.221, respectively. The protein expression levels were 0.616±0.019, 0.325±0.016, 0.299±0.015, respectively. The expressions of E-cad mRNA and protein in siRNA E6 group were significantly higher than those in the empty vector group and blank control group (F = 21.346, P 0.05). After knocking down HPV16E6 gene, the methylation status of E-cad gene was weakly positive, and the intensity of the amplified products was significantly weaker than that in the empty vector group and blank control group, while the unmethylation amplification was positive. Conclusions Knocking down the HPV16E6 gene increases the expression of E-cad in cervical cancer SiHa cell line, and decreases the level of CDH1 promoter methylation. To a certain extent, it partly reverses the hypermethylation status of CDH1 promoter, and causes E-cad to be re-expressed.
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Objective To investigate the expressions of human papillomavirus (HPV) 16E6 and E-cadherin in different cervical tissues.Methods Immunohistochemistry expressions of HPV16E6 and E-cadherin were analyzed in 30 normal cervical tissues,34 cervical intraepithelial neoplasias (CIN),and 60 squamous cervical cell carcinomas.Results The differences in positive expression rates of HPV16E6 and E-cadherin were statistically significant among normal cervical tissues,CIN Ⅰ,CIN Ⅱ-Ⅲ,cervical squamous cell carcinomas (x2 =40.166,P =0.000;x2 =31.295,P =0.000),respectively.The abnormal expressions of HPV16E6 and E-cadherin expression were significantly related to clinical pathological stage,tumor size,histological grade,invasion depth of squamous cervical cancers (P ＜ 0.05),but not correlated with age (P ＞0.05).The expressions between HPV16E6 and E-cadherin protein were negatively correlated (rs =-0.647,P =0.000).Conclusions There may be relationship between HPV16E6 and E-cadherin,and the abnormal expressions of two proteins may be associated with poor prognosis of cervical carcinomas.
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The emergence of fertility-sparing surgery for early-stage cervical cancer is one of the important progresses in the treatment of cervical cancer.How to optimize the selection and assessment of patients,how to improve the efficacy and reduce complications,how to improve tumor outcome and increase the likelihood of pregnancy,are the current hot issues.The new progress and controversial issues of the treatment in recent years were reviewed in order to promote the future treatment in the direction of safe,efficient and minimally invasive surgery,and hope the surgery can better improve the quality of life of patients in clinical cure.
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Objective To explore the security,pregnancy outcomes,and the tumor recurrence related factors of young patients with cervical cancer treated with different radical trachelectomy (RT).Methods Thirty-two young patients ＜ 40 years of age with early cervical cancer from May 2004 to July 2012 admitted in Tumor Hospital Xiangya School of Medicine of Central South University were divided into two groups based on different operation methods:vaginal radical trachelectomy (RVT) group and abdominal radical trachelectomy (RAT) group.The clinical data were analyzed by One-way Anova and multivariate Cox stepwise regression analysis.Results The operation duration,number of lymph node dissection,the height of the cervical resection,postoperative hospitalization time,incidence of vascular injury and incidence of postoperative lymphocele were respectively (250 ± 82) min,15 ± 6,(2.31 ± 0.21) cm,(9.2 ± 2.9) d,1/18 and 1/18 in RVT group,while (263 ±60) min,16 ±8,(2.32 ±0.26) cm,(10.3 ±3.5) d,0 and 1/14 in RAT group.There was no statistically significant difference between the two groups (all P ＞ 0.05).The blood loss (281 ±201) ml in RVT group was significantly lower than that in the RAT group (492 ±320) ml (P ＜0.05).The length of Vaginal hysterectomy[(2.61 ±0.50) cm] and the width of parametrial resection[(2.38 ±0.36) cm] in RVT group were significantly less than those[(2.95 ±0.10),(2.81 ±0.22) cm] in the RAT group (all P ＜ 0.05).The pregnancy rate between RVT group (3/18) and RAT group (2/14) were no significant difference (P ＞ 0.05).One-way Anova analysis showed that the recurrence of early cervical cancer was related to tumor size in diameter (F =4.911,P =0.047),while there were no correlation with age,clinical stage,histological type and surgical approach (all P ＞ 0.05).Multivariate analysis showed that tumor diameter size was an independent risk factor for tumor recurrence (3 =0.259,P =0.031).Conclusions RT for young patients with early cervical cancer is feasible.Pregnancy outcomes after RT need to be study in the future.Tumor size in diameter is the major risk factor for tumor recurrence.