Cancer cellular immunotherapy has made inspiring therapeutic effects in clinical practices, which brings new hope for the cure of cervical cancer. CD8+T cells are the effective cytotoxic effector cells against cancer in antitumor immunity, and T cells-based immunotherapy plays a crucial role in cellular immunotherapy. Tumor infiltrated Lymphocytes (TIL), the natural T cells, is approved for cervical cancer immunotherapy, and Engineered T cells therapy also has impressive progress. T cells with natural or engineered tumor antigen binding sites (CAR-T, TCR-T) are expanded in vitro, and re-infused back into the patients to eradicate tumor cells. This review summarizes the preclinical research and clinical applications of T cell-based immunotherapy for cervical cancer, and the challenges for cervical cancer immunotherapy.

Targeting integrin α7 (ITGA7) suppresses malignant progression of several types of cancer, including tongue squamous cell carcinoma, hepatocellular carcinoma and non-small cell lung cancer, while the effect of its knockdown on cell function and its association with clinicopathological features in endometrial cancer (EC) is unclear. The present study aimed to investigate this issue. ITGA7 was knocked down by short-interfering (si)RNA in Ishikawa and RL95-2 cells followed by western blotting and reverse transcription-quantitative PCR assays. Subsequently, cell proliferation, apoptosis, invasion and expression levels of PI3K, phosphorylated (p-) PI3K, AKT and p-AKT were determined using Cell Counting Kit-8, TUNEL, Transwell assays and western blotting. Moreover, ITGA7 in tumor and adjacent tissues from 50 patients with endometrial cancer was detected using immunohistochemical assay. ITGA7 expression was increased in EC cell lines (HEC-1A, RL95-2, Ishikawa and KLE) compared with telomerase-immortalized human endometrial stromal cells (THESCs). In both Ishikawa and RL95-2 cells, three ITGA7 siRNAs all demonstrated good efficiency on ITGA7 knockdown, amongst which the one with the highest efficiency was selected for the following experiments. ITGA7 knockdown reduced cell proliferation and invasion, while inducing apoptosis; moreover, it suppressed p-PI3K/PI3K and p-AKT/AKT ratios. In patients with EC, ITGA7 expression was increased in tumor tissues compared with adjacent tissues, and its lower tumor expression was associated with myometrial invasion (<1/2), non-lymphovascular invasion and decreased FIGO stage. In conclusion, ITGA7 knockdown repressed proliferation, invasion and the PI3K/AKT pathway while inducing apoptosis in EC cell lines, and its insufficiency was associated with less advanced tumor features in EC patients. These results indicated that ITGA7 may be a potential target for the treatment of EC.

Background: Bevacizumab-combined chemotherapy is a new regimen for advanced/recurrent endometrial cancer.Aims: To evaluate the efficacy and safety of bevacizumab-combined chemotherapy in advanced/recurrent endometrial cancer.Study design: Systematic review and meta-analysis.Methods: Eligible studies were retrieved from Embase, PubMed, and Cochrane Library.The data of primary outcomes including progression-free survival and overall survival and secondary outcomes including overall survival, response rate, and adverse events (grade ≥2) were extracted, pooled, and used for the meta-analysis to compare the efficacy and safety of bevacizumab-combined chemotherapy with other treatments in patients with advanced/recurrent endometrial cancer.Results: Notably, 2 randomized-controlled and 5 single-arm trials of bevacizumab-combined chemotherapy or bevacizumab single-agent therapy for endometrial cancer were included.Meta-analysis indicated that bevacizumab-combined chemotherapy significantly increased the progression-free survival rate (hazard ratio=0.82,95% confidence interval=0.70,0.97) and overall survival rate (hazard ratio=0.83,95% confidence interval=0.70,0.98) compared with chemotherapy alone.The rates of overall, complete, and partial response to bevacizumab-combined chemotherapy were 76%, 22%, and 21%, respectively.The 6 and 12-month disease-free progression rates after bevacizumab-combined chemotherapy were 79% and 62%, respectively.Anemia (23%), leukopenia (46%), neutropenia (51%), hypertension (16%), and fatigue (24%) were the general adverse events after bevacizumab-combined chemotherapy.Conclusion: Bevacizumab-combined chemotherapy may have a higher efficacy in improving the overall and progression-free survival in patients with advanced/recurrent endometrial cancers compared with chemotherapy alone.

Background Endometrial carcinoma (EC) is one common gynecological tumor, threatening physical and psychological health of females. Huge amount of essays indicated that long non-coding RNAs (lncRNAs) were widely reported to serve as a crucial regulator in the biological movements among multiple carcinomas, including EC. Methods RT-qPCR was implemented to detect the expression of target genes. Loss/gain-of-function experiments certified the impacts of OIP5-AS1 and miR-152-3p on EC cell progression. Results Data of this research suggested that powerful expression of OIP5-AS1 was discovered in EC cell lines. Loss/gain-of-function assays inferred that OIP5-AS1 promoted proliferative, migratory and invasive abilities, and Epithelial-Mesenchymal Transition (EMT). In addition, we identified miR-152-3p expression was negatively modulated by OIP5-AS1. OIP5-AS1 accelerated the development of EC cells via downregulating miR-152-3p expression. SLC7A5 was selected out as a downstream target of miR-152-3p. The competing relationship between OIP5-AS1 and SLC7A5 was corroborated by luciferase reporter assay. Eventually, the results of rescue assays indicated that SLC7A5 overexpression could restore the impacts of OIP5-AS1 ablation on the progression of EC cells. Conclusion Our research confirmed that OIP5-AS1 propeled the development of EC cells through targeting miR-152-3p/SLC7A5. OIP5-AS1 could be utilized as a target for EC treatment.

Endometrial cancer is a common gynecological malignancy, and the incidence of this disease has increased in recent years. Recently, some studies suggested that the expression of miR-379-5p suppressed the metastasis of breast cancer cells. However, whether the expression of miR-379-5p could affect the proliferation, migration and invasion of endometrial cancer is unclear. In this study, we established miR-379-5p overexpression and miR-379-5p inhibition in endometrial cancer cells. Next, EdU and colony formation assays were performed to measure proliferation of endometrial cancer cells. Wound healing and transwell assays were carried out to examine the migration and invasion of these cells. Then, luciferase reporter assay was performed to test the relationship between miR-379-5p and ROR1. Finally, we overexpressed ROR1 in miR-379-5p overexpressing endometrial cancer cells. Colony formation, wound healing and transwell assays were used to measure proliferation, migration and invasion of these cells. The results showed that overexpression of miR-379-5p repressed proliferation, migration and invasion of endometrial cancer cells. Higher levels of miR-379-5p repressed expression of N-cadherin, Vimentin and ZEB1. Overexpression of miR-379-5p also promoted expression of E-cadherin and ZO-1. In addition, miR-379-5p targeted and suppressed expression of ROR1. Overexpression of ROR1 abolished the inhibitory effect of miR-379-5p on proliferation, migration, invasion and EMT of endometrial cancer cells. All of these results indicated that miR-379-5p suppressed proliferation, migration and invasion of endometrial cancer cells by inhibiting the expression of ROR1 and the EMT process.

Department of Obstetrics and Gynecology, Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology, Wuhan City, Hubei Province 430022, People’s Republic of China Background: RUNX1-IT1 suppresses colorectal cancer and liver cancer, while its role in other cancers is unknown. This study was performed to investigate the role of RUNX1-IT1 in endometrial cancer (EC). Methods: EC and paired non-tumor tissues were collected from 62 EC patients, and the expression of RUNX1-IT1, mature miR-21 and miR-21 precursor in these tissue samples were determined by RT-qPCR. Correlations were analyzed by linear regression. Overexpression of RUNX1-IT1 was achieved in EC cells and the expression of mature miR-21 and miR-21 precursor were analyzed by RT-qPCR. CCK-8 assay was used for cell proliferation analysis. Results: We found that RUNX1-IT1 was downregulated in EC and inversely correlated with mature miR-21 but not miR-21 precursor. RUNX1-IT1 was predicted to bind with miR-21 precursor. The interaction between them was verified by dual-luciferase activity assay and RNA pull-down assay. In EC cells, overexpression of RUNX1-IT1 downregulated mature miR-21, but not miR-21 precursor. Overexpression of RUNX1-IT1 suppressed the role of miR-21 in increasing cell proliferation. Conclusion: RUNX1-IT1 is downregulated in EC and inhibits cancer cell proliferation by suppressing the maturation of miR-21.

Background: RUNX1-IT1 suppresses colorectal cancer and liver cancer, while its role in other cancers is unknown. This study was performed to investigate the role of RUNX1-IT1 in endometrial cancer (EC). Methods: EC and paired non-tumor tissues were collected from 62 EC patients, and the expression of RUNX1-IT1, mature miR-21 and miR-21 precursor in these tissue samples were determined by RT-qPCR. Correlations were analyzed by linear regression. Overexpression of RUNX1-IT1 was achieved in EC cells and the expression of mature miR-21 and miR-21 precursor were analyzed by RT-qPCR. CCK-8 assay was used for cell proliferation analysis. Results: We found that RUNX1-IT1 was downregulated in EC and inversely correlated with mature miR-21 but not miR-21 precursor. RUNX1-IT1 was predicted to bind with miR-21 precursor. The interaction between them was verified by dual-luciferase activity assay and RNA pull-down assay. In EC cells, overexpression of RUNX1-IT1 downregulated mature miR-21, but not miR-21 precursor. Overexpression of RUNX1-IT1 suppressed the role of miR-21 in increasing cell proliferation. Conclusion: RUNX1-IT1 is downregulated in EC and inhibits cancer cell proliferation by suppressing the maturation of miR-21.

Enhancer of zeste homolog 2 (EZH2) is overexpressed in various malignancies and associated with poor prognosis and drug-resistance. A recent study suggested that there is a link between EZH2 expression and the mediation of gene silencing in association with aberrant DNA methylation. In the present study, we showed an inverse correlation between EZH2 and human mutL homolog 1 gene (hMLH1) expression in 30 epithelial ovarian cancer (EOC) tissues. Moreover, we found that EZH2 downregulation could induce the re-expression of the unmethylated, basally expressed hMLH1 gene without affecting DNA methylation in the hMLH1 promoter. These results suggest that EZH2 can modulate the transcription of basally expressed hMLH1 via a non-DNA-methylation-dependent pathway, but it has no effect on hMLH1 silencing that is mediated by DNA hypermethylation.

Objective To study the effect of silencing Aurora B on paclitaxel chemosensitivity in ovary cancer cell line A2780 using RNAi.Methods A2780 cells were transfected with various concentrations of AURKB siRNA as experimental group,meanwhile,negative control and blank control groups were set up,and then the cells in each group were treated with different doses of paclitaxel.MTT assay was used to test the cell viability.The apoptosis rate was evaluated by using Annexin Ⅴ-FITC/PI;Caspase-3 protein expression was detected by using Western blot.The apoptosis was observed by Hoechst-stained method.Results With the increase of siRNA concentration,the cell viability was decreased.There was significant difference in the cell viability among AURKB siRNA(50 pmol/L)-transfected group,negative control group and blank control group(P<0.05).With the increase of paclitaxel dose,the cell viability was decreased in AURKB siRNA-transfected group,and there was significant difference among AURKB siRNA-transfected group,negative control group and blank control group in the presence of 10 μmol/L of paclitaxel(P<0.05).In the cells treated with 10 μmol/L paclitaxel 48 h after transfection with 50 pmol/L AURKB siRNA,the apoptosis rate of cells was significantly higher in AURKB siRNA-transfected group than in negative control group and blank control group(P<0.01).In the AURKB siRNA-transfected group,the Caspase-3 protein expression was significantly increased after addition of paclitaxel,and the apoptosis increased obviously.Conclusion Silencing of Aurora B induces cell apoptosis and enhances the paclitaxel chemosensitivity in human ovary cancer cell line A2780.

Objective To study the effect of silencing Aurora B on proliferation and cell cycle of ovary cancer cell line A2780 using RNAi.Methods AURKB RNAi was transfected into A2780 cells.Reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to examine the expression of Aurora B.Cell proliferation was evaluated by MTT and Trypan blue staining.Cell cycle changes and apoptosis were examined by flow cytometry.Results ①The expression levels of Aurora B mRNA and protein were obviously detected in five cell lines of ovary cancer by RT-PCR and Western blot;②The mRNA and protein expression level of Aurora B was completely silenced in 200 pmol/L AURKB RNAi-transfected A2780 cells 48 h after transfection;③Cell proliferation was significantly inhibited 4 days after transfection,and the absorbance value was significantly lower than that in control cells and negative control cells(P<0.05);④After A2780 cell were transfected with the AURKB RNAi for 24 h,entry of mitosis was detected with polyploidysation,and after 72 h,20.54% of AURKB RNAi-transfected A2780 cells had DNA content>4 N,indicating polyploidisation.After 96 h,the AURKB RNAi-transfected A2780 cells showed a significantly higher apoptosis rate(11.67%)than in control cells(0.57%);⑤As compared with controls and negative controls,the apoptosis rate was significantly increased in A2780 cells after transfected with 200 pmol/L AURKB RNAi for 96 h.Conclusion Silencing of Aurora B inhibits cell proliferation,and induces cell polyploidisation and apoptosis of A2780 cells,suggesting Aurora B may be an effective target for ovary cancer.

目的:探讨Aurora B在正常子宫内膜和子宫内膜癌组织中的表达及其与子宫内膜癌临床病理参数之间的关系.方法:采用免疫组织化学链霉素抗生物素蛋白-过氧化物酶(streptavidin-biotin peroxidase,SP)法对10例正常增殖期子宫内膜组织、10例正常分泌期子宫内膜组织和72例子宫内膜癌组织Aurora B的表达情况进行了检测,分析其在正常子宫内膜组织中的表达规律及其与子宫内膜癌临床病理特征的关系.结果:Aurora B表达的阳性指数在正常增殖期子宫内膜组织中明显高于分泌期子宫内膜组织,差异有极显著性意义(P＜0.01);增殖期的表达也高于子宫内膜癌组织的表达,差异有极显著性意义(P＜0.01);Aurora B表达阳性指数在高分化、低手术分期和肌层浸润深度≤1/2的子宫内膜癌组织中低于中、低分化、高手术分期和子宫肌层浸润深度＞1/2的子宫内膜癌组织,差异有显著性意义(P＜0.01、P＜0.01、P＜0.05,respectively).结论:Aurora B参与正常增殖期子宫内膜腺体卜皮细胞的增生和复制;Aurora B在子宫内膜癌组织中的表达与其生物学行为有关,Aurora B的表达升高提示子宫内膜癌的恶性生物学行为增加.

Objective:To explore the role of chemokine receptor 4(CXCR4) and its ligand stromal derived factor-1α(SDF-1α) in the processes of adhesion and invasion of ovarian cancer cells through activating the mitogen-activated protein kinases(MAPK) signaling pathway and its possible mechanism.Methods:Intracellular calcium mobilization in SKOV3 cells was detected with a laser scanning confocal fluorescence microscopy before and after SDF-1α treatment.Western blotting was used to detect the phosphorylation of extracellular signal-regulated kinase(ERK1/2) in SKOV3 cells after exposure to SDF-1α.Adhesion capability and matrix metalloproteinase(MMP) activity of ovarian cancer cells after exposure to SDF-1α were mesured by adhesion assay and gelatin zymography,respectively.Results:SDF-1α induced rapid intracellular calcium mobilization in a metastatic ovarian cancer cell line SKOV3,as well as rapid phosphorylation of ERK-1/2.Adhesion capability of ovarian cancer cells to fibronectin(FN) and collagen Ⅳ(COL) was increased by SDF-1α treatment,while the addition of PD98059,an inhibitor of ERK-1/2 signaling,reduced the effects of SDF-1α.SDF-1α also increased the activities of MMP-2 and MMP-9 secreted by SKOV3 cells.Conclusion:SDF-1α/CXCR4 regulates adhesion ability of ovarian cancer cells by activating MAPK signaling pathway and stimulates secretion of MMP-2 and MMP-9,thereby participates the processes of invasion and metastasis of ovarian cancer.

This study was aimed to explore the role of stromal-derived factor 1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis in mediating the metastasis of ovarian cancer cells through activation of extracellular signal-regulated kinase-1/2 (ERK-1/2) signaling pathway. A highly metastatic ovarian cancer cell line, SKOV3, was used in the study. Intracellular calcium mobilization was detected by using laser scanning confocal fluorescence microscopy. Western blotting was used to detect the phosphorylation of ERK1/2 in SDF-1α-treated SKOV3 cells. Adhesion capability and matrix metalloproteinase (MMP) activity of ovarian cancer cells after exposure to SDF-1α were measured by adhesion assay and gelatin zymography. The results showed that SDF-1α induced rapid intracellular calcium mobilization in SKOV3 cells, as well as the phosphorylation of ERK-1/2. The adhesion of ovarian cancer cells to fibronectin and collagen IV was increased after SDF-1α treatment. An inhibitor of ERK-1/2 signaling, PD98059, could antagonize such effects of SDF-1α. SDF-1α could also increase the secretion of active MMP-2 and MMP-9. It was concluded that the SDF-1/CXCR4 axis played a critical role in the metastasis of human ovarian cancer by increasing the adhesion capability of cancer cells and the activity of MMP-2 and MMP-9 via ERK1/2 signaling pathway.

BACKGROUND & OBJECTIVE:The chemokine receptor CXCR4 and its sole ligand stromal cell-derived factor-1 (SDF-1) not only actively participate in inflammation, hematopoiesis, infection of HIV, but also play a pivotal role in migration, invasion and metastasis of some malignant tumors. This study was to investigate the role of CXCR4/SDF-1 axis in mediating metastasis in cervical cancer cells through activating the mitogen-activated protein kinase (MAPK) pathway and its possible mechanism.METHODS:Intracellular calcium mobilization was observed under laser scanning confocal fluorescence microscopy. The phospharylation of extracellular signal-regulated kinase (ERK) 1/2 in HeLa cells after binding of SDF-1alpha to CXCR4 was measured by Western blot. Adhesion of CXCR4/SDF-1 to cervical cancer cells and secretion of matrix metalloproteinase (MMP) were detected by adhesion assay and gelatin zymography, respectively.RESULTS:After SDF-1alpha was bound to CXCR4, a rapid and robust mobilization of intracellular calcium in Hela cells was initiated. The difference between the average baseline fluorescence intensity (FI) and the peak FI was significant (P < 0.01). ERK-1/2 was rapidly phosphorylated in Hela cells after its exposure to SDF-1alpha, and the strongest phosphorylation occurred at 30 min. The adhesion ability of Hela cells to fibronectin (FN) and laminin (LN) was increased after SDF-1alpha treatment (P < 0.05 and P < 0.01, respectively), while pretreatment of Hela cells with an ERK-1/2 inhibitor, PD98059, decreased adhesion of Hela cells to extracellular matrix (ECM) with the presence of SDF-1alpha (P < 0.05). Increased amounts of active MMP-2 were secreted in response to increased SDF-1alpha concentrations. When the concentration of SDF-1alpha was 800ng/mL, the secretion of active MMP-2 reached the peak and started to decrease afterwards.CONCLUSION:CXCR4/SDF-1 participates in tumor invasiveness and metastasis in cervical cancer through regulating the adhesion ability by activating the MAPK signaling transduction pathway and promoting secretion of MMP-2.

Objective To investigate the expression of proliferating cell nucleus antigen(PCNA)and Langerhans cell(LC) infiltration in endometrial carcinoma,in an attempt to evaluate the clinical significance of these markers.Methods Streptavidin-biotin peroxidase(S-P) immunohistochemical technique was used to detect the expression of PCNA and LC infiltration in 10 cases of normal proliferative endometrium and 72 cases of endometrial carcinoma respectively.Results PCNA was expressed in both normal proliferative endometrium and endometrial carcinoma,but positive rate and PCNA(PI) score in the latter were significantly higher than that in the former(P0.01);S-100 protein positive LC had not been observed(0%) in normal proliferative endometrium,but it was observed(81.9%) in endometrial carcinoma,a statistically significant difference was found between two groups(P0.01);PCNA(PI) score were lower in tumors with well differentiation(G1),low surgical staging(Ⅰ~Ⅱ),≤1/2 myometrial invasion than that in tumors with moderate and low differentiation(G2~G3),higher surgical staging(Ⅲ~Ⅳ),1/2 myometrial invasion(P0.01);S-100 protein positive LC infiltration were higher in fomer conditions than that in latter ones(P0.01);PCNA(PI) score was negatively related to the S-100 protein positive LC infiltration(r=-0.471,P0.01).Conclusion PCNA(PI)score and the LC infiltration are significantly correlated with biologic behavior of endometrial carcinoma,our results indicate that PCNA(PI)score and Langerhans cell infiltration may be favorable prognostic factors in endometrial carcinoma.

Objective:Using genetic engineering technology,fuse the latency-associated protein(LAP)of transforming growth factor-β1(TGF-β1)to human soluble tumor necrosis factor receptor Ⅰ(hsTNFRⅠ)via a matrix metalloproteinase(MMP)cleavage site,in order to construct eukaryotic expression vector of pcDNA3.1 /LAP-MMP-hsTNFR Ⅰ fusion protein and obtain LAP-MMP-hsTNFR Ⅰ fusion protein.Methods:A double-stranded deoxyoligonucleotide coding for MMP cleavage site was cloned into plasmid pcDNA3.1(+),obtained pcDNA3.1/MMP recombinant ;The TGF-β1-LAP and hsTNFRⅠcDNA were inserted into the two sides of MMP cleavage site respectively,then constructed pcDNA3.1/LAP-MMP-hsTNFRⅠrecombinant vector.After nucleotide sequencing,the transfection of recombinant plasmid into COS-7 cells was mediated by lipid,and the expression of fusion gene in COS-7 cells was detected by RT-PCR.Results:The results of restriction endonuclease digestion and sequencing demonstrated that pcDNA3.1/LAP-MMP-hsTNFRⅠ recombinant vector was constructed successfully.Efficiently expression of fusion gene LAP-MMP-hsTNFRⅠ was confirmed by RT-PCR.Conclusion:The recombinant eukaryotic expression vector pcDNA3.1/LAP-MMP-hsTNFRⅠhas been constructed and expressed successfully,which will contribute to further studies about the role of fusion protein in targeting treatment of endometriosis.

Objective To investigate the relationship between P38 MAPK activity and apoptosis in drug-resistant cell line of ovarian carcinoma.Methods The effect on the cell apoptosis of A2780/Taxol cells which have been treated with P38 MAPK inhibitor SB203580 was analyzed by PI staining and flow cytometry(FCM).The 50% inhibition concentration(IC50) of paclitaxel on A2780/Taxol was determined by MTT method in vitro.The effect of SB203580 on the P38 MAPK expression was detected with cytoimmunochemistry.P-gp expression was detected by Western blot.Results The apoptosis rate of A2780/Taxol cells treated with SB203580 for 24 hours was 23.7%,higher than that of the control group and untreated group(P0.01).When adding 10μmol/L SB203580 to A2780/Taxol cell for 24h,the P38 MAPK expression decreased,the density and count of the P38 MAPK declined too.The IC50 of Taxol on A2780/Taxol cell declined greatly(P0.01).The P-gp expression was higher than the control group and untreated group.Conclusion P38 MAPK signal pathway plays an important role in drug resistance of ovarian carcinoma cell and has a close relationship with the drug resistance.P38 MAPK can protect A2780/Taxol cells from inhibiting apoptosis.Blocking the P38 MAPK signal way can promote the apoptosis of ovarian carcinoma drug-resistant cells.

Objective To investigate the effect of gene silence of MDR1 gene by small interference RNA( siRNA) in drug resistant A2780/Taxol cell line. Methods From July 2005 to December 2005,two siRNAs which were specifically targeted MDR1(Multidrug resistance 1) gene were synthesized by Union Hospital of Tongji Medical Schod, then the plasmid was transformed into E.coli. From positive clone, the plasmid was obtained and purified.The purified plasmid was co-transfected with pEGFPN-1 plasmid into A2780/Taxol cells. The positive expression of transfection was determined by the flow cytometry (FCM).50% inhibition concentration (IC_ 50 ) of paclitaxel on A2780/Taxol was determined by MTT method in vitro. P-gp protein was detected by Western blot. MDR1 mRNA was assessed by RT-PCR. Results The transfection efficiency had no difference between the two constitutive plasmids and the EGFP plasmid. The relative reversal efficiency of A2780/Taxol cells to paclixtel for two siRNA was 63.8 % and 51.2% respectively. The expression of p-gp decreased greatly. Treatment of A2780/Taxol cell with the 2 kinds of siRNAs resulted in a silence of MDR1 gene to different extent. The MDR-A siRNA was more effective in the suppression of MDR1 with a significant reduction of (67.3±0.8)% of the MDR1 mRNA expression. Conclusion The siRNA can effectively reverse the MDR1/P-gp-mediated multidrug resistance of A2780/Taxol cells.

Objective To explore methylation status of Ras-association Domain Family 1A Gene (RASSF1A) in promoter region and its expression at mRNA level in gynecological malignancy cell lines.Methods RT-PCR and Methylation-specific PCR were performed to detect RASSF1A gene expression and DNA methylation status of its promoter region in 9 cell lines as followes: A2780, Anglne, CaOV3, COC1, SKOV3, Hela, HEC-1B, Ishikawa, and Siha. Results RASSF1A gene promoter region showed DNA hypermethylation and low levels or loss of mRNA expression was found in A2780, Anglne, CaOV3, SKOV3, HEC-1B and Ishikawa cells, whereas partial DNA methylation was identified and its mRNA detected in COC1, Hela and Siha cells.Conclusion DNA mehtylation in RASSF1A gene promoter region is ubiquitous in gynecological malignancy cell lines, which could play some role in pathophysiology of these cancers.

OBJECTIVE:To investigate the reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxel.METHODS:shRNA plasmid vector specifically targeting MDR1 and MDR3 genes was transfected into A2780/taxol cells. The early stage cell apoptosis and the effect of intracellular rhodamine 123 (Rh123) accumulation were detected by flow cytometry (FCM). The late stage cell apoptosis rate was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). The 50% inhibition concentration (IC(50)) of paclitaxel on A2780/taxol cells was determined by methyl thiazolyl tetrazolium (MTT) assay. MDR1 and MDR3 mRNA were assessed by RT-PCR, and caspase-3 protein was detected by western blot.RESULTS:After treatment with MDR1 and MDR3 shRNA plasmid vector, early apoptosis rate of A2780/taxol cells was (20.21 +/- 0.56)% and (10.87 +/- 1.24)%, respectively. MDR1 and MDR3 shRNA could increase cellular Rh123 accumulation (116.6 +/- 8.1 and 98.4 +/- 3.8, respectively). The late stage apoptosis rates detected by TUNEL displayed the same tendency as FCM results did. The IC(50) for paclitaxel of A2780/taxol cells was decreased significantly. The mRNA levels of MDR1 and MDR3 in A2780/taxol cells were decreased by (73.3 +/- 0.8)% and (51.6 +/- 0.4)% of control, and the reduction of MDR1 and MDR3 mRNA was in a time-dependent manner. The expression of caspase-3 protein of MDR1 and MDR3 shRNA vector transfected group in A2780/taxol cells was significantly increased [(80.8 +/- 2.6)% and (72.0 +/- 4.7)%, respectively].CONCLUSION:MDR1 and MDR3 gene silencing could recover sensitivity of A2780/taxol cells to paclitaxel and induce cell apoptosis, thus reversing cell resistance to paclitaxel.