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Objective Design and synthesize short hairpin RNA (shRNA) expression vector of RNA for specific silencing of heparanase (HPA) gene,screened plasmid which silence effects is the best.Observe the function of ceil invasion after inhibiting the expression of HPA in cervical carcinoma cell lines (HeLa).Methods The genomic sequence of HPA gene was retrieved from GenBank database.Designed four pairs of specific oligonucleotide sequences and a negative control according to the shRNA design principles.They were inserted into the vector pYr-1.1,vectors,and transfected into HeLa cells via lipofectamine.Reverse transcription (RT)-PCR and immunofluorescence were employed to detect the expression of HPA gene in the transfected cells at the mRNA and protein levels,respectively.The plasmid were screened and transfected into HeLa cells,then transwell small room stromal invasion experiment were employed to observe the cervical carcinoma cell invasion.Results RT-PCR results of transfected HeLa cells shown that the mRNA amplification multiples were 0.54 ±0.05 in the HPA-592 group,0.89 ±0.18 in HPA-995 group,0.82 ±0.22 in the HPA-1351 group,0.91 ±0.47 in HPA-1658 group.While,they were 1.31 ±0.72 and 1.09 ±0.16 in negative control and blank control group,respectively.Green fluorescence was visible in the cytoplasm,which indicated that the HPA protein was expressed in the cytoplasm,of them the weakest green fluorescence in the HPA-592 group.The relative numbers of invasive cells among the HeLa cells were as follows:182 ±6 in the blank control group,258 ± 17 in the negative control group,and 44 ± 4 in the HPA-592-specific interference group(P ＜ 0.01).Conclusion Successfully screened shRNA vector targeting human HPA,efficiently inhibit expression of HPA gene when transfected into HeLa cells,and significantly reduced the invasion capacity of cervical carcinoma cells.
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Objective To detect the expression of heparanase (HPA) in cervical cancer cells and investigate the impact of quercetin on the expression of HPA,and the molecular mechanism that quercetin inhibits the growth of cervical cancer cells.Methods The experimental groups included cervical cancer cell lines (HeLa and Caski) exposed to different concentrations of quercetin (20,40 and 80 μmol/L) in the culture medium.The control groups included a negative control group,which was cultured with RPMI 1640 only,and a positive control group,in which cervical cancer cells were transfected with HPA small interference RNA (siRNA) to silence HPA expression.The cellular expression levels of HPA were detected with fluorescence quantitative real-time PCR and western blot analysis at 24,48 and 72 hours after treatment.Results (1) HPA was significantly expressed in both cervical cancer cell lines (HeLa and Caski),and it exists both nucleus and cytoplasm.(2)The real-time PCR shows as follows:as the quercetin concentration increased (20,40 and 80 μmol/L),the mRNA expression level of HPA decreased (P ＜0.01),in which the inhibition of HPA expression was concentration dependent.In addition,the inhibition of HPA expression was also time dependent.As time growth,the expression level of HPA mRNA (24,48 and 72 hours) in HeLa and Caski cells decreased (P ＜ 0.01).Compared with negative control group,the expression level of HPA mRNA decreased in different concentrations of quercetin (40 and 80 μmol/L) in both HeLa and Caski cells (all P ＜ 0.05) ; Compared with positive control group,the expression level of HPA mRNA expressed no obvious difference in quercetin (80 μmol/L) group (P ＞ 0.05) in HeLa cells,while it was opposite in Caski cells(P ＜0.01).(3)The result of western blot shown that,as the quercetin concentration increased(20,40 and 80 μmol/L)and time growth (24,48 and 72 hours),the expression level of HPA protein decreased (P ＜ 0.01),and the inhibition of HPA protein expression was concentration and time dependent.Compared with negative control group,the expression level of HPA protein decreased in different concentrations of quercetin (40 and 80 μmol/L) in both HeLa and Caski cells (all P ＜ 0.05) ;Conpared with positive control group,the expression level of HPA protein expressed no obvious difference in quercetin (80 μmol/L) group (all P ＞ 0.05) in both HeLa cells and Caski cells (all P＞0.05).Conclusion Quercetin could inhibits the expression of HPA in cervical carcinoma cell lines,which inhibition is concentration and time dependent.
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Objective To study the efficacy and safety of Gonadotropin-releasing hormone agonists (GnRH-a) combined with laparoscope conservative surgery in treatment of moderate or severe endometriosis.Methods From Jan.2007 to Jan.2010,68 patients with moderate or severe undergoing treatment in Renmin Hospital of Wuhan University were enrolled in this retrospective study.Three groups were classified,which were 25 patients in GnRH-a group,subcutaneous injection Leuprorelin on the second day of menstruation,every 4 weeks for 3 months.Twenty-three patients in Marvelon group,orally one marvelon tablet on the second day of menstruation,continuous 21 days for one period of treatment for 3 courses.Twenty patients in surgery group,without any medicine used preoperatively.All patients were followed by 12 months and compare their surgery time,blood loss,recovery,visual analog scale (VAS),and recurrence and so on.Results The operating time were (68 ± 18) min in GnRH-a group,(80 ± 21) min in Marvelon group and (90± 24) min in surgery group.The amount of bleeding were (118 ± 15) ml in GnRh-a group,(161 ± 18) ml in Marvelon group and (193 ± 13) ml in surgery group.There was significant lower in the operating time and amount of bleeding in GnRH-a group than those in other two groups (P ＜ 0.05).The activity time and the anus exhaust time were shorter in patients in GnRh-a group than those in the other two groups significantly(P ＜ 0.05).When followed up in 12 months after treatment,visual analogue scale had dropped from 3.8 (1.9-6.8) to 1.9 (1.1-2.8) in GnRh-a group,from 2.7 (1.3-5.5) to 1.8 (1.2-3.2) in Marvelon group and from 1.9(1.0-4.9) to 1.6(1.0-3.6) in surgery group.It was showed the most remarkable decreased VAS in GnRHa group when compared with the other two groups(P ＜ 0.05).The recurrence rates were 12％ (3/25) in GnRH-a group,22％ (5/23) in Marvelon group and 25 ％ (5/25) in surgery group.It was found that the most significant lower recurrence was in GnRH-a group when compared with the other two groups (P ＜ 0.05).Conclusions It was safe and efficacy that GnRH-a combined with laparoscopic conservative surgery were used in treatment of endometriosis.It could bring shorter operation time,less intraoperative blood loss,quick postoperative recover,the lower recurrence rate.
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ObjectiveTo study the expression of EZH2 gene in cervical squamous carcinoma and its clinicopathologic significance. MethodsThe expression of EZH2 mRNA was detected in 21 samples of normal cervical tissue, 27 samples of CIN tissue and 48 samples of cervical squamous carcinoma tissue by RT-PCR. And the relationship between EZH2 expression and the clinical pathological characteristic was analyzed. ResultsThe level of EZH2 mRNA in cervical squamous carcinoma tissues(1.67±0.01)were significantly higher than that in the normal cervical tissues (0) and CIN tissues (0.36±0.02) (P ＜ 0.01).There was no correlation between EZH2 and ages (P ＞ 0.05), while the expression of EZH2 was highly correlated with histologic stage,clinical stage,muscular invasion depth and lymph node metastasis of cervical squamous carcinoma (P ＜ 0.01).ConclusionThe over-expression of the EZH2mRNA may play an important role in the pathogenesis and progression of cervical squamous carcinoma,suggesting that EZH2 might be a new biomarker for diagnosing cervical squamous carcinoma.