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@#[Abstract] Objective: To explore the regulatory effect of lncRNA maternal imprinting gene 3 (MEG3) on proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of cervical cancer cells via miR-9-5p/SOCS5 axis. Methods: A total of 20 pairs of cancer and para-cancerous tissue specimens resected from cervical cancer patients in Chongqing Hospital of Traditional Chinese Medicine from January 2017 to June 2019 were collected for this study. Using liposome transfection technology, pcDNA3.1-MEG3,si-MEG3, miR-9-5p mimics, miR-9-5p inhibitor and their control plasmids were transfected into cervical cancer HeLa and SiHa cells respectively to construct overexpression and silence cell model. qPCR was used to detect the expression levels of MEG3, miR-9-5p and SOCS5 in cervical cancer tissues and cell lines. CCK-8 method and Transwell chamber method were used to detect cell proliferation, migration and invasion ability. The expression levels of E-cadherin and vimentin in cells were detected by cellular immunofluorescence experiments. Target genes were predicted through the Online Bioinformatics TargetScan database. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-9-5p and MEG3, SOCS5, respectively. Results: Compared with para-cancerous tissues and cervical epithelial HcerEpic cells, the expressions of MEG3 and SOCS5 were significantly down-regulated and the expression of miR-9-5p was significantly up-regulated in cervical cancer tissues and cell lines (all P<0.01). TargetScan database analysis and Dual luciferase reporter gene assay confirmed the targeting relationship between miR-9-5p and MEG3 or SOCS5. MEG3 and SOCS5 significantly inhibited while miR-9-5p significantly promoted cell proliferation, migration and invasion ability (all P<0.01). MEG3 and SOCS5 promoted E-cadherin expression and inhibited vimentin expression, while miR-9-5p inhibited E-cadherin expression and promoted vimentin expression (P<0.05 or P<0.01). Conclusion: lncRNA MEG3 regulates proliferation, migration, invasion and EMT of cervical cancer cells via miR-9-5p/SOCS5 axis.
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@# Objective: : To investigate the effects of GBX2 gene on the proliferation, migration and invasion of human cervical carcinoma SiHa cells and to explore the mechanism. Methods: Recombinant plasmid over-expressing GBX2 gene (pCMV6-entry-GBX2, experimental group) and empty vector plasmid (pCMV6-entry, negative control group) were transfected into cervical cancer SiHa cells by plasmid transfection technique. The proliferation, colony formation and cell cycle of transfected cells were detected by WST-1 method, Colony formation assay and flow cytometry, respectively. The cell migration and invasion were detected by wound healing assay and Transwell assay. The expression level of IL-6 in cell culture supernatant was detected by ELISA. WB was used to detect the expression changes of EMT-related proteins and to explore its possible mechanism. Results: Compared with the SiHa/pCMV6 negative control group, after up-regulation of GBX2, (1) the proliferation, colony formation, migration and invasion of SiHa/GBX2 cells in the experimental group were significantly enhanced (all P<0.01); The proportion of cells in G0/G1 phase decreased while the proportion of cells in S phase and G2/M phase increased (all P<0.01); (2) the expression of E-cadherin decreased, and the expressions of N-cadherin, vimentin and snail increased (all P<0.01); (3) the expression of IL-6 in the culture supernatant of SiHa/GBX2 cells was significantly up-regulated (P<0.01); (4) STAT3 phosphorylation in SiHa/GBX2 cells was enhanced, and could be inhibited by STAT3 inhibitor S31-201 (P<0.01). Conclusion: GBX2 may induce EMT of cervical cancer SiHa cells through IL-6/STAT3 pathway, thereby promoting the proliferation, migration and invasion of cervical cancer cells.
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@#Objective: To investigate the expression of microRNA(miR) -760 in human cervical squamous cell carcinoma (CSCC) tissues and cells, and it’s effects on the proliferation, apoptosis, invasion, migration and epithelial-mesenchymal transition (EMT) of SiHa cells, as well as its molecular mechanism. Methods: Eighty pairs of CSCC cancerous and corresponding para-cancerous tissue specimens which were pathologically confirmed and 40 cases of normal cervical tissue specimens obtained by myomectomy in Liaocheng Maternal and Child Health Hospital of Shandong Province from April 2015 to August 2018 were selected. The expression of miR-760 in CSCC tissues, para-cancerous tissues and normal cervical tissues, human CSCC cell lines (SiHa, HCC94) and human cervical squamous epithelial immortalized H8 cells were detected by qPCR, and the relationship between miR-760 and clinicopathological characteristics of CSCC patients was analyzed. miR-760 mimics and NC-mimics plasmids were transfected into SiHa cells by liposome transfection. The expression of miR-760 in SiHa cells was detected by qPCR, the proliferation activity and apoptosis rate were detected by CCK-8 test and flow cytometry, respectively. The invasion and migration of SiHa cells were detected by Transwell assay. The expressions of EMT-related proteins, such as E-cadherin, vimentin and N-cadherin, in SiHa cells were detected by WB. Bioinformatics was used to predict the targeting relationship between FOXA1 and miR-760, and double luciferase reporter gene assay was used to verify the direct regulation of miR-760 on FOXA1. Results: The expression of miR-760 in CSCC tissues was significantly lower than that in para-cancerous tissues and normal cervical tissues (all P<0.01), and the expression of miR-760 was closely related to lymphnode metastasis and clinical stage (all P<0.01). The expression of miR-760 in SiHa and HCC94 cells was significantly lower than that in H8 cells (all P<0.01). Up-regulation of the expression of miR-760 could significantly inhibit the proliferation, invasion and migration of SiHa cells (all P<0.01), promote apoptosis (P<0.01), up-regulate the expression of E-cadherin protein (P<0.01), and down-regulate the protein expressions of vimentin and N-cadherin (all P<0.01).FOXA1 was a direct target gene of miR-760 (P<0.01). Up-regulation of miR760 significantly inhibited the mRNA and protein expressions of FOXA1 in SiHa cells (all P<0.05). Conclusion: The expression of miR-760 is down-regulated in CSCC tissues and cells, and it can regulate the proliferation, invasion, migration and apoptosis of CSCC cells by targeting FOXA1. ··
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Objective To study the effect of human homologue of polycomb 2 (HPC2) on the growth of cervical cancer cells siha and the regulation of E7 gene.Methods HPC2 and E7 genes of siha cells were silenced by siRNAs respectively.Detected the expression of HPC2 gene and protein in siha cell lines after E7 gene silencing,cell proliferation activity and the rate of cell apoptosis.Results The expressions of HPC2 mRNA and protein were decreased in siha cells with E7 gene silencing,cell proliferation was inhibited,and apoptosis was increased,which was similar to HPC2 gene silencing.Conclusion HPC2 may be involved in the regulation of cell proliferation and apoptosis,and its expression may be closely related to E7 gene in SiHa cells.
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Objective After the detection of FAT10 protein in human cervical cancer tissue behind clinical pathological examination,investigate the effect of up regulation and down regulating the expression of FAT10 gene on the radiation sensitivity of cervical cancer cell line SiHa cells in nude mice.Methods The expression levels of FAT10 mRNA in 30 cases cervical cancer and adjacent tissues were detected by RT-PCR,FAT10 siRNA and recombinant expression vector pcDNA3.1-FAT10 were respectively transfected into cervical cancer SiHa cells by electroporation,and stable transfection cell lines was obtained by G418 screening.After transfection 48h,the expression levels of FAT10 mRNA and protein were respectively detected by fluorescence quantitative RT-PCR and Western blot.Cell proliferation was detected with CCK-8.The effect of FAT10 expression on the radiation sensitivity of cervical cancer cell SiHa line was observed by the clonogenic experiment.The cell apoptosis was detected by TUNEL.The growth of cervical cancer cells was detected by Xenograft tumor assay combined with X-ray irradiation.Results The relative expression level of FAT10 protein in human cervical cancer tissues was significantly higher than that in adjacent tissues,and the difference was statistically significant(P ＜ 0.05).The expression of FAT10 gene in cervical cancer SiHa cells was significantly lower in FAT10 down group,and the FAT10 up regulation group was significantly higher.The radiation sensitivity of cervical cancer SiHa cells was increased in the FAT10 down group,and the radiation sensitivity of SiHa cells was decreased in the FAT10 up regulation group (P ＜ 0.05).FAT10 down group of cervical cancer SiHa cell proliferation was inhibited,the cell apoptosis rate was increased (P ＜ 0.05).The proliferation ability of FAT10 cells was significantly enhanced,and the cell apoptosis rate was decreased in the FAT10 up regulation group (P ＜ 0.05).The average volume of transplanted tumor in the FAT10 down group was significantly smaller than that of the control group (P ＜ 0.05);the average volume of the tumor in the FAT10 up regulation group was larger than that of the control group (P ＜ 0.05).The average volume of the tumor was significantly decreased in the FAT10 down group after irradiation of 20Gy gamma ray(P ＜ 0.05),and the average volume of subcutaneous implanted tumor in nude mice was not significantly different (P ＞ 0.05).Conclusion Downregulation of FAT10 expression can inhibit the growth of human cervical cancer SiHa cells in nude mice,enhance the radiation sensitivity of tumor cells,up regulation of FAT10 expression will make tumor radiation resistance.
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Objective: To explore the inductive effect of galangin on HPV-positive human cervical cancer cells and the possible mechanism. Methods: Two HPV-positive human cervical cancer cell lines (SiHa cell and HeLa cell) and one HPV-negative human cervical cancer cell line (C-33-A cell) were given different concentration of galangin (20, 40, and 80 μmol/L) for 24, 48, and 72 h. Three human cervical cancer cell lines and relative cell viabilities were determined by the MTT method. Apoptosis and cell cycle were analyzed by flow cytometry. Western blotting analysis was used to determine the protein expression levels of Bcl-2 family proteins. Results: Cell proliferation of two HPV-positive human cervical cancer cells was significantly inhibited by galangin in a dose- and time-dependent manner, and galangin had no effect on cell proliferation of HPV-negative human cervical cancer cells. Cell cycle detection results showed that galangin could reversibly arrest the two HPV-positive cell lines, either in G1 or in G2/M phases. Flow cytometry results showed that beyond certain galangin concentration or/and over 24 h exposure, the cells underwent apoptosis. The data of Western blotting showed that 40 μmol/L galangin up-regulated the expression levels of Bad, Bid, and Bax, but down-regulated Bcl-2 and Bcl-w. Conclusion: Galangin can inhibit the proliferation of HPV-positive cervical cancer cells and promote apoptosis, which may be associated with the regulation of Bcl-2 family proteins expression.
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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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ObjectiveTo study the effects of tanshinoneⅡA on proliferation of cervical squamous cancer Siha cells; To discuss its possible molecular mechanism.Methods Cervical squamous cancer Siha cells were treated with different doses of tanshinoneⅡA. The effects of tanshinoneⅡA on proliferation of Siha cells were measured by MTT assay and flow cytometry analysis. The effects of tanshinoneⅡA on expression levels of phospho-extracellular regulate kinase (p-ERK) and Cyclin D in Siha cells were measured by Western blot.Results 1×10-5, 5×10-6, 1×10-6, 5×10-7 mol/L tanshinoneⅡA significantly inhibited Siha cell proliferation and such effect could be enhanced by ERα antagonist MPP and attenuated by ERβ antagonist PHTPP. 1×10-5, 5×10-6, 1×10-6 tanshinoneⅡA could significantly decrease the proliferation index of Siha cells. 1×10-5, 5×10-6, 1×10-6, 5×10-7 mol/L tanshinoneⅡA could significantly reduce the protein expression levels of p-ERK and Cyclin D of Siha cells.ConclusionTanshinoneⅡA can inhibit cervical squamous cancer Siha cell proliferation and such effect is realized via estrogen receptor pathway. TanshinoneⅡA plays anti-proliferation roles by reducing the expression levels of p-ERK and Cyclin D.
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ABSTRACT:Objective To explore the effects of miRNA-1246 (miR-1246)on cell proliferation,invasion and migration in human cervical squamous cell carcinoma (CSCC)cell line SiHa.Methods SiHa cells were assigned into 3 groups:miR-1246 analog group,miR-1246 antagonist group and control group.Transfection efficiency was determined.The MTT assay,transwell assay and wound healing assay were performed respectively to evaluate the proliferation,invasion and migration abilities of SiHa cells.Western blot was carried out to detect the expression of thrombospondin-2 (THBS2)before and after transfection.A THBS2 3’-UTR-containing dual luciferase plasmid was synthesized and co-transfected with miR-1246 into SiHa cells to observe the luciferase enzyme activity.Results MTT assay,transwell assay and wound healing assay revealed that the abilities of proliferation,migration and invasion were significantly enhanced (P<0.01)in SiHa cells transfected with miR-1246 analog,but suppressed in SiHa cells transfected with miR-1246 antagonist.Western blot showed that SiHa cells transfected with miR-1246 analog had significantly decreased THBS2 expression (gray value = 6 .2 8 ± 1 0 .2 2 , P=0 .0 1 3 ) while those transfected with miR-1246 antagonist had significantly increased THBS2 expression (gray value = 12.90±19.81, P=0.037).After co-transfected with miR-1246 and THBS2 3’-UTR-containing plasmid,SiHa cells exhibited a decreased level of luciferase enzyme expression.Conclusion miR-1246 promoted the proliferation,invasion and migration of CSCC SiHa cell, and it might promote CSCC tumorigenesis and progression by suppressing the expression of its target gene THBS2 .
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Trichomonas vaginalis induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-alpha production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-alpha production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-alpha production was significantly decreased compared to the control; however, TNF-alpha reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-alpha production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-alpha production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.
Subject(s)
Female , Humans , Cell Line , Cervix Uteri/enzymology , Epithelial Cells/enzymology , MAP Kinase Signaling System , Mucous Membrane/enzymology , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , Trichomonas Vaginitis/enzymology , Trichomonas vaginalis/physiology , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Objective:This paper aimed to investigate the effects of isocorydinone on cell proliferation in SiHa human cervical carcinoma cell lines. Methods:Different concentrations of isocorydione (100, 200, 400, 800, and 1200 μmol/L) were used to treat SiHa human cervical carcinoma cells in vitro for 24, 48, and 72 h. Methyl thiazolyl tetrazolium (MTT) assays were conducted to determine the inhibitory action of isocorydione. Flow cytometry was performed to detect the cell cycle in SiHa human cervical carcinoma cells af-ter treatment with 400 μmol/L isocorydione. Hoechst 33342 staining was used to observe the micro-morphological changes of SiHa cell nucleus after the treatment. The expression of Bcl-2, Bax, and caspase-3 proteins in cervical carcinoma SiHa cell lines was determined using western blot analysis. Results: MTT assays showed that isocorydione inhibits the proliferation of SiHa cells in a dose- and time-dependent manner (P<0.05). The flow cytometry results showed that SiHa cervical carcinoma cells treated with different concen-trations of isocorydione exhibited increased cell cycle. Compared with the control group, Hoechst 33342 staining showed that SiHa cells became narrow, with nuclear pyknosis and fragmentation, and formed an apoptotic body after treatment with 400 μmol/L isocoryd-ione for 48 h. Furthermore, western blot analysis proved that isocorydione significantly inhibited the proliferation of SiHa cell lines, and the expression of Bax protein was increased. By contrast, the expression of Bcl-2 protein decreased gradually. Consequently, the ra-tio of Bax/Bcl-2 increased, as well as the expression of caspase-3 protein. Conclusion:Isocorydione exhibited an overt inhibitory ac-tion on SiHa cells. Isocorydione promoted the occurrence of cell apoptosis, which may be associated with related proteins of mitochon-drial apoptotic pathway.
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Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite.
Subject(s)
Humans , Blotting, Western , Cell Line, Tumor , Epithelial Cells/metabolism , Metalloproteases/genetics , Proteolysis , Sequence Analysis, DNA , TOR Serine-Threonine Kinases/metabolism , Trichomonas vaginalis/enzymologyABSTRACT
Objective: To explore the influence of silencing Survivin expression by RNA interference on the radiosensitivity of cervical carcinoma SiHa cells. Methods: The recombinant eukaryotic expression plasmid pSurvivin-shRNA was constructed and transfected into SiHa cells. The mRNA and protein expressions of Survivin were determined by RT-PCR and Western blotting, respectively. The activity of caspase-3 was measured by kinase activity determination method. The apoptotic rate was determined by flow cytometry. The changes of radiosensitivity were explored by colony formation test. Results: Compared with SiHa cells transfected with pNeg-shRNA (SiHa/ pNeg-shRNA cells) and untransfected SiHa cells, the expressions of survivin in the cells transfected with pSurvivin-shRNA (Si-Ha/ pSurvivin-shRNA cells) were inhibited significantly at both mRNA and protein levels. The activity of caspase-3 in SiHa/pSurvivin-shRNA cells was significantly enhanced, and the D405 was 1.34 ± 0.05 (P < 0.05). The apoptotic rate of SiHa/pSurvivin-shRNA cells was (5.13 ± 0.81)% and (11.27 ± 1.89)% after 24 hand 48 h X-ray irradiation at 6 MV and 8 Gy, respectively, which was significantly higher than SiHa/pNeg-shRNA cells and untransfected cells (P < 0.05). The colony formation ability of SiHa/pSurvivin-shRNA cells was markedly decreased after irradiation (P < 0.05), which indicated that the sensitivity of SiHa/pSurvivin-shRNA cells was enhanced. Conclusion: The inhibition of Survivin by RNAi could enhance the radiosensitivity of SiHa cells by increasing caspase-3 activity and inducing apoptosis.
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OBJECTIVE: Green tea polyphenol (GTP) has been shown to have anti-tumor properties in a wide variety of experimental systems. In this study, we evaluated the effects of GTP on the cytotoxic effects of cisplatin in cultured HeLa and SiHa cells. METHODS: The cell lines from Korean Cell Culture Bank were cultured in a RPMI-1640 medium supplemented with a 10% fetal bovine serum, antibiotics streptomycin and penicillin. GTP was extracted from tea leaves (Camellia scinensis) by water extraction and organic solvent fractionation. Cells were seeded at 1 x 10(4) cells/well in RPMI1640 media in triplicate wells on a Nunc Labware 96 well flat bottom microculture plate, with and without GTP (100 microgram/mL) and at different concentrations of cisplatin (0-1000 microgram/mL). After incubating the plates at 37 degrees C in 5% CO2 for 2 days, cell viability was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; thiazolyl blue] assay. RESULTS: The viability of the HeLa cells was decreased to 14% at a 600 microgram/mL concentration of cisplatin, and to 16% at 600 microgram/mL in the SiHa cells as measured by the MTT assay. However, in the HeLa cell, co-cultured with GTP (100 microgram/mL), the cell viability decreased to 68% at 200 microgram/mL of cisplatin and to 17% at 400 microgram/mL of cisplatin. And in the SiHa cell, co-cultured with GTP (100 microgram/mL), the cell viability decreased to 48% at 200 microgram/mL of cisplatin and to 17% at 400 microgram/mL of cisplatin. CONCLUSION: This study showed that cisplatin with GTP seems to have a potentiating effect on Cisplatin cytotoxicity than cisplatin alone.