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Objective:To explore the effect of miR-21 on cell proliferation, apoptosis, invasion and radiosensitivity of cervical cancer HeLa cells and unravel the underlying mechanism.Methods:RT-qPCR assay was used to detect the expression levels of miR-21 in cervical cancer tissues and adjacent non-tumor tissues, normal cervical epithelial cells (H8) and cervical cancer cell lines (HeLa, SiHa, ME180). HeLa cell line with inhibition of miR-21 or knockdown of RECK were constructed. CCK-8, Caspase3/7 live cell apoptosis detection, wound healing test, Transwell invasion, clone formation assay, Western blot and immunofluorescence were performed to detect cell viability, apoptosis, migration, invasion, radiosensitivity and related proteins. The dual luciferase assay verified whether miR-21 targeted RECK.Results:MiR-21 level in the cervical cancer tissues was significantly higher than that in its corresponding adjacent non-tumor tissues ( P<0.05). The expression levels of miR-21 in cervical cancer cell lines HeLa, SiHa and ME180 were significantly up-regulated compared with those in normal cervical epithelial cells H8(all P<0.05). MiR-21 knockdown significantly inhibited HeLa cell viability, promoted cell apoptosis, reduced radiation tolerance, down-regulated the expression of Cyclin D 1,Bcl-2, MMP-2 and MMP-9, and up-regulated the expression P21 and Bax proteins (all P<0.05). miR-21 targeted the 3’-UTR of RECK mRNA and negatively regulated the expression of RECK. Silencing RECK reversed the effects of miR-21 knockdown on HeLa cell apoptosis, migration, invasion and radiosensitivity. Conclusions:Inhibiting the expression of miR-21 significantly decreases cell viability, induces cell apoptosis, weakens cell migration and invasion capabilities, and enhances the radiosensitivity of HeLa cells. The potential mechanism is closely related to the targeted up-regulation of RECK.
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Objective:To evaluate the effect of resveratrol combined with γ-ray irradiation on the biological behavior of cervical cancer cells, and to explore its possible mechanism.Methods:The proliferation of cell populations after different concentrations of resveratrol solution±γ-ray irradiation was detected by CCK-8 assay. Scratch test and Transwell chamber test were used to detect cell migration and invasion. Flow cytometry and Annexin V-FITC/PI double staining were employed to assess cell apoptosis. Western blot was performed to measure the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR proteins.Results:Compared with the normal control (NC) group, the resveratrol group±γ-ray irradiation could inhibit the proliferation, migration, and invasion and promote cell apoptosis of human cervical cancer Hela cells, and the combined effect was more obvious. Compared with the NC group, resveratrol and γ-ray irradiation could significantly down-regulate the expression levels of Bcl-2, PI3K, p-Akt and p-mTOR proteins, up-regulate the expression level of Bax protein, but did not significantly alter the expression levels of Akt and mTOR proteins in human cervic1 255al cancer Hela cells.Conclusions:Resveratrol combined with γ-ray irradiation can dramatically inhibit the proliferation, migration, invasion, and promote the apoptosis of cervical cancer Hela cells. The mechanism may be related to the inhibition of the PI3K/Akt/mTOR signaling pathway and down-regulating the expression levels of downstream related proteins.
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The study aimed to achieve enhanced targeted cytotoxicity and cell-internalization of cisplatin-loaded deoxyribonucleic acid-nanothread (CPT-DNA-NT),mediated by scavenger receptors into HeLa cells.DNA-NT was developed with stiff-topology utilizing circular-scaffold to encapsulate CPT.Atomic force microscopy (AFM) characterization of the DNA-NT showed uniformity in the structure with a diameter of 50-150 nm and length of 300-600 nm.The successful fabrication of the DNA-NT was confirmed through native-polyacrylamide gel electrophoresis analysis,as large the molecular-weight (polymeric) DNA-NT did not split into constituting strands under applied current and voltage.The results of cell viability confirmed that blank DNA-NT had the least cytotoxicity at the highest concentration (512 nM) with a viability of 92％ as evidence of its biocompatibility for drug delivery.MTT assay showed superior cyto-toxicity of CPT-DNA-NT than that of the free CPT due to the depot release of CPT after DNA-NT inter-nalization.The DNA-NT exhibited targeted cell internalizations with the controlled intracellular release of CPT (from DNA-NT),as illustrated in confocal images.Therefore,in vitro cytotoxicity assessment through flow cytometry showed enhanced apoptosis (72.7％) with CPT-DNA-NT (compared to free CPT;64.4％).CPT-DNA-NT,being poly-anionic,showed enhanced endocytosis via scavenger receptors.
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Objective:To establish a paclitaxel (PTX)-resistance cell line of human cervical carcinoma Hela/PTX and investigate its biological features. Methods:Using Hela as the negative cells,drug-resistant cervical cancer Hela/PTX cells were applied as the target cells. The morphology changes were observed under an optical microscope;the drug resistance of Hela/PTX to different chemotherapeutic drugs was detected by CCK-8 method. The accumulation of drugs in the cells was determined by HPLC. The expression of P-glycoprotein (P-gp), Survivin protein and mRNA was detected by Western blot and RT-PCR. Results:Hela/PTX multidrug resistant cell line was successfully established. The resistance index to PTX was 58.40 and the resistance index of cross-resistance to docetaxel and 5-fluorouracil was 8.11 and 4.62,respectively. The mRNA expression level of P-gp and Survivin protein in Hela/PTX cells respectively was(9.93 +0.90) times(P < 0.05) and(3.02 +0.57) times(P <0.05) of that in Hela cells. The expression of P-gp and Survivin protein in drug-resistant cells was significantly higher than that in parental cells.Conclusion:Human cervical cancer resistant cell line Hela/PTX with the basic biological characteristics of multidrug resistance is successfully established, which can be used as an in vitro model for the studies on paclitaxel resistance mechanism and provide experimental basis for the studies of multidrug resistance mechanism of cervical cancer.
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Objective To explore the effects of Rab11 on biological functions of human cervical cancer cell line HeLa through regulating the expression levels of Rab11. Methods The Rab11 siRNA was transfected into HeLa cells and the expression of Rab11 was detected by Western blot. CCK8 assay, colony formation experiments, EdU assay and Transwell assay were adopted to observe the effect of Rab11 on HeLa cells proliferation and invasion. Results The expression of Rab11 was decreased significantly in HeLa cells transfected with Rab11 siRNAs than that in control siRNA (1.096 ±0.091 vs 1.735 ±0.084, P< 0.01). The proliferation was markedly inhibited in Rab11 siRNA group compared with that in control siRNA group (48 h:0.721±0.092 vs 1.090±0.099; 72 h: 0.956±0.105 vs 1.482±0.096; 96 h: 1.231±0.099 vs 1.720±0.174, P< 0.01), the number of colonies was lower than that in control siRNA group (36±1 vs 75±8, P< 0.01) and so was proliferation rate [(33.880±1.902) % vs (45.570±2.025) %, P< 0.05]. The cell invasion rate of Rab11 siRNA group was lower than that of control siRNA group [(38.6 ±0.8) % vs (100.0 ±0.2) %, P< 0.01]. Conclusion Down-regulation of Rab11 expression can inhibit the growth of HeLa cells.
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Objective To study the effect of lentinan on cisplatin’s inhibition on cervical cancer Hela cell line. Method Cervical cancer Hela cell line were divided into LEN group (only lentinan treatment), CIS group (only Cisplatin treatment), LEC group (Both Lentinan and Cisplatin treatment) and CON group (no Lentinan and Cisplatin treatment) according to their treatment methods. The differences of cell proliferation, cell cycle, PI and apoptosis were compared. Results The differences of absorbance between LEN group and CON group were not significant d1-d8 after treatment, but significant between CIS and LEC group (P<0.05). The absorbance in LEC group was significantly lower than in CIS group (P<0.05). The differences of PI and apoptosis rate in LEC and CIS group were significant (P<0.05), but not between LEN and group. The PI in LEC group was lower than in CIS group (P<0.05), while apoptosis rate was higher (P<0.05). Conclusion Lentinan could significantly promote Cisplatin’s inhibition effects on cervical cancer Hela cell line.
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Objective To construct recombinant plasmids containing HPV18E7 gene ,and explore the optimization condition of its expression in Escherichia coli .Methods The genomic DNA extracted from HeLa cell line which served as a template to the HPV18 E7 gene was amplified using PCR method ;and the amplified product of HPV18E7 gene was connected to the pET-32a(+ ) vector ,which composed the pET-32a(+ )-HPV18E7 recombinant plasmid ;the positive recombinant plasmids were transformed into BL21-DE3-pLysS competent cells and the optimized expression condition was explored in order to obtain a large amount of HPV18E7 oncogenic protein .Results The fragment length of PCR products of HeLa cell genomic DNA was consistent with that of HPV18 E7 gene .In LB medium ,the expression level of the target protein was not high under such conditions as different concentra-tion of IPTG and lactose ,different temperatures and different induction starting amount .Therefore the ZYM-5052 auto-induction medium was tried in this experiment ,and the expression amount of the fusion protein was much higher than that induced with IPTG and lactose .Conclusion The amount of HPV18E7 fusion protein in ZYM-5052 automatic induction medium is much higher than that induced with IPTG and lactose .
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A cell line is a permanently established cell culture which proliferates indefinitely under given appropriate conditions. The oldest and commonly used human cell line is HeLa.DNA fingerprinting using multi-locus probes, STR profiling, L1 retro transposon marker and Temperature-sensitive SV40 large T antigen are technologies for the identification and characterization of cell lines. Cell lines cost less to maintain, are easier to genetically manipulate, handle and store, moreover their immortal nature enables them to be continuously cultured, distributed and studied in many labs. Cell lines are in vitro models to study the pathway of malignant progression, induction of cellular apoptosis, DNA methylation, histone modifications & tumor suppressor gene expressions, cell culture cytotoxicity assay and so on. The collection of human material for culture must be passed by the relevant hospital’s ethics committee. Cell cultures should be handled in Class II biosafety cabinets and all discarded materials should be autoclaved, incinerated or chemically disinfected. The challenge of improving protein yield and quality is done by generating cell lines that can produce high yields of recombinant proteins, methods of stable gene integration and quality of culture media. Finally, the efficacy of recombinant glycoproteins as a human therapeutic is strongly dependent on their glycosylation. However, cell lines show slow population doubling time, have a finite lifespan, are prone to contamination, genotypic and phenotypic drift and behave differently in culture as compared to when they are a part of a tissue/organ.
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Objective To investigate the inhibition and radiosensitization effects of Topotecan(TPT)on cervical cancer cell line HeLa,and make comparison with Cisplatin(DDP)and Taxol(TAX).Methods The effects of TPT,DDP and TAX on proliferation of cervical cancer cell line HeLa were evaluated by MTT assay.The radiosensitization effects of TPT,DDP and TAX on HeLa cells were detected by clone formation assay,and the sensitization enhancement ratios of TPT,DDP and TAX were calculated.Results The IC_(50) of TPT at different reaction time(24 h,48 h and 72 h)were 8.0μg/mL,2.6 μg/mL and 0.8 μg/mL,respectively,those of DDP were 2.4 μg/mL,0.7 μg/mL and 0.1μg/mL,respectively,and those of TAX were 0.3 μg/mL,0.1μg/mL and 0.0 μg/mL, respectively.The apoptosis rates of tumor cells treated by radiosensitization were significantly higher than those of tumor cells treated by single radiotherapy or single chemotherapy (P<0.05).The sensitization enhancement ratios of TPT at 24 h and 48 h were 1.167 and 1.344,respectively,those of DDP were 1.314 and 1.538,respectively,and those of TAX were 1.076 and 1.316,respectively.Conclusio TPT,DDP and TAX have significant inhibition effects on cervical cancer cell line HeLa, which are time-dependant and dose-dependant. Besides,TPT,DDP and TAX have radiosensitizing effects, with DDP being the strongest and TAX being the weakest.
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PURPOSE: Selective inhibition of multiple molecular targets may improve the antitumor activity of radiation. Two specific inhibitors of selective cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) were combined with radiation on the HeLa cell line. To investigate cooperative mechanism with selective COX-2 inhibitor and EGFR blocker, in vitro experiments were done. MATERIASL AND METHODS: Antitumor effect was obtained by growth inhibition and apoptosis analysis by annexin V-Flous method. Radiation modulation effects were determined by the clonogenic cell survival assay. Surviving fractions at 2 Gy (SF2) and dose enhancement ratio at a surviving fraction of 0.25 were evaluated. To investigate the mechanism of the modulation of radiosensitivity, the cell cycle analyses were done by flow cytometry. The bcl-2 and bax expressions were analyzed by western blot. RESULTS: A cooperative effect were observed on the apoptosis of the HeLa cell line when combination of the two drugs, AG 1478 and NS 398 with radiation at the lowest doses, apoptosis of 22.70% compare with combination of the one drug with radiation, apoptosis of 8.49 %. In cell cycle analysis, accumulation of cell on G0/G1 phase and decrement of S phase fraction was observed from 24 hours to 72 hours after treatment with radiation, AG 1478 and NS 398. The combination of NS 398 and AG 1478 enhanced radiosensitivity in a concentration-dependent manner in HeLa cells with dose enhancement ratios of 3.00 and SF2 of 0.12 but the combination of one drug with radiation was not enhanced radiosensitivity with dose enhancement ratios of 1.12 and SF2 of 0.68 (p=0.005). The expression levels of bcl-2 and bax were reduced when combined with AG 1478 and NS 398. CONCLUSION: Our results indicate that the selective COX-2 inhibitor and EGFR blocker combined with radiation have potential additive or cooperative effects on radiation treatment and may act through various mechanisms including direct inhibition of tumor cell proliferation, suppression of tumor cell cycle progression and inhibition of anti-apoptotic proteins.
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Humans , Apoptosis , Apoptosis Regulatory Proteins , Blotting, Western , Cell Cycle , Cell Proliferation , Cell Survival , Cyclooxygenase 2 , Epidermal Growth Factor , Flow Cytometry , HeLa Cells , Radiation Tolerance , ErbB Receptors , S PhaseABSTRACT
Objective To investigate the effects of artesunate on human cervical cancer cell line HeLa.Methods The inhibitory effect on cell proliferation was assessed by MTT assay.Transmission electron microscopy and fluorescent staining were applied to demonstrate the presence of apoptosis.Cell cycle,reactive oxygen species(ROS) levels and mitochondrial membrane potential(??m) were examined by flow cytometry,respectively.Expression of caspase-3 was detected by immunohistochemical methods.Results Growth inhibition of ART on HeLa cells was time-and dose-dependent.Apoptotic feature was observed by transmission electron microscopy and fluorescent staining.The blocking of the cell cycle was in S-phase.The expression of caspase-3 in cytoplasm was positive.The generation of ROS increased obviously(P
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Objective To explore the chromosome aberration of cervical cancers induced by human papillomavirus(HPV16)virus.Methods The change of change of chromosomes of Hela cells induced by HPV-16 infection HPV-16 in fection was detected by FISH.Results The integration sites of HPV-16 were not observed in Hela cells.But HPV16 virus was integrated on chromosomes 3.Conclusion HPV16 could cause chromosome 3 aberration in Hela cells and result in the occurance of cervical cancers.
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Aim To inhibit the expression of human protection of telomere gene hPOT1 in HeLa cells using vector based on RNA interference(RNAi) technique and to investigate its effects on the activation of Caspase-3 in HeLa cell.Methods Three recombinant plasmids(our group constructed before) containing different hPOT1 target sequences(pmU6-shRNA1,pmU6-shRNA2 and pmU6-shRNA3)were transfected into HeLa cells by liposomal.The expression inhibition of hPOT1 was detected by RT-PCR and EMSA.The expression and activation of Caspase-3 were inspected by RT-PCR,Western blot analysis and Caspase-3 kit.Results After 48 hour′s transfecting,hPOT1 mRNA and protein level in HeLa cells reduced and the expression of Caspase-3 mRNA had no change.Caspase-3 zymogen decreased but its activity increased.Conclusions hPOT1 expression in HeLa cells can be significantly inhibited by using siRNAvectored RNAi and the down-regulation of hPOT1 expression can activate Caspase-3.