ABSTRACT
Objective:To investigate the effect and potential mechanism of miR-199a-5p on the radiosensitivity of cervical cancer CaSki cells.Methods:Cervical cancer CaSki cells were cultured in vitro. MiR-199a-5p mimics (miR-199a-5p mimics) were transfected into cervical cancer CaSki cells (miR-199a-5p group) with liposome by Lipofectamine 2000. CaSki cells transfected with mimics control were used as negative control (NC group) and non transfected CaSki cells were used as blank control (control group). After X-ray irradiation, real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-199a-5p in cells of each group. The effects of miR-199a-5p on radiosensitivity and apoptosis of CaSki cells were detected by clone formation assay and flow cytometry apoptosis assay. Bioinformatics software was used to predict the target gene of miR-199a-5p. Double luciferase reporter gene assay and Western blot were used to verify the targeting relationship between miR-199a-5p and thyroid hormone receptor interaction factor 4 (TRIP4). Results:The expression of miR-199a-5p in miR-199a-5p group was significantly higher than that in control group ( P<0.05). After X-ray irradiation, the expression of miR-199a-5p was more obvious ( P<0.05); Overexpression of miR-199a-5p could reduce the clonogenic ability and promote the apoptosis of CaSki cells ( P<0.05); Overexpression of miR-199a-5p could further reduce the clonal formation and promote the apoptosis of irradiated cells ( P<0.05); Double luciferase reporter gene experiment and Western blot confirmed that miR-199a-5p could target and negatively regulate TRIP4. Conclusions:miR-199a-5p can increase the radiosensitivity of cervical cancer CaSki cells by negatively regulating the expression of TRIP4.
ABSTRACT
@#[Abstract] Objective: To investigate the effect of long non-coding RNA (lncRNA)-CCAT2 on the proliferation and cell cycle of cervical cancer cells. Methods: The expression of CCAT2 in 3 cervical cancer cell lines (HeLa, C-33A, and CaSki) was detected by qPCR and the cell line with the highest expression level was selected for subsequent experiments. CCAT2 overexpression and interference vectors were designed and synthesized. After transfection, qPCR was performed to detect the transfection efficiency. The cells were divided into 5 groups: control, sh-EV (empty vector), overExp-EV , sh-CCAT2, and overExp-CCAT2. MTT assay was performed to evaluate cell viability. Flow cytometry was performed to measure cell cycle. WB was performed to detect the expressions of Ki67, cyclin D1, and cyclin dependent kinase 4 (CDK4). Results: Among HeLa, C-33A, and CaSki cells, the highest expression of CCAT2 was found in CaSki cells. CCAT2 overexpression and interference vectors were successfully transfected into the CaSki cells. Compared with the control group, the cells viability and proliferation in the sh-CCAT2 group was significantly decreased (all P<0.01), the proportion of cells in the G1 phase was significantly increased (P<0.01), and the expression levels of Ki67, cyclin D1, and CDK4 were significantly decreased (all P<0.01). However, in the overExp-CCAT2 group, the cell proliferation was enhanced and the expression levels of Ki67, cyclin D1, and CDK4 were significantly increased (all P<0.01). Conclusion: CCAT2 affects proliferation and cell cycle of cervical cancer cells by regulating the expressions of their associated proteins.
ABSTRACT
@# Objective: To investigate the effect of C-phycocyanin (C-PC) on the epithelial-mesenchymal transition (EMT) of cervical cancer Caski cells induced by transforming growth factor beta1 (TGF-β1). Methods: According to different treatment methods, Caski cells were divided into three groups: 10 ng/ml TGF-β1 treatment group, 10 ng/ml TGF-β1+300 μg/ml C-PC co-treatment group and control group (untreated). After 24 h of treatment, the morphological changes of Caski cells were observed, and the effects of TGF-β1 and C-PC on the migration and invasion of Caski cells were detected by Scratch test and Transwell test, respectively. Western blotting was used to detect the effect of C-PC on the expression of epithelial phenotypic marker protein E-cadherin and stromal phenotypic marker protein N-cadherin in TGF-β1-induced Caski cells, and qPCR was used to detect the mRNA expressions of EMT related factors Snail, Zeb1 and Twist. Results: Caski cells in the TGF-β1 treatment group lost the characteristics of the original epithelial phenotype, while the cells in the TGF-β1+C-PC co-treatment group maintained the characteristics of normal epithelial phenotype; the migration rate ([60.0±1.4]% vs [33.5±2.2]%, [40.0±2.8]%, both P<0.05) and the number of invasive transmembrane cells ([108.2±6.2] vs [25.2±3.1], [39.8±5.4], both P<0.01]) of Caski cells in the TGF- β1 treatment group were significantly higher than those in the co-treatment group and the control group. Compared with the control group, the expression of E-cadherin in Caski cells treated with TGF-β1 decreased significantly (P<0.05), while the mRNA expressions of Twist, Snail and Zeb1 increased significantly (all P<0.05); However, co-treatment with C-PC reversed above changes (P<0.05 or P<0.01), and significantly decreased the protein expression level of N-cadherin (P< 0.05). Conclusion: C-PC treatment can inhibit the invasion and metastasis ability of Caski cells induced by TGF-β1 and further affects the EMT process. The mechanism may be related to the decrease of mRNAexpressions of Twist, Snail and Zeb1 by C-PC treatment. ·
ABSTRACT
OBJECTIVE: To investigate the effect of genistein on the anticancer effects of chemotherapeutic agents, we examined the effect of a genistein and cisplatin combination on CaSki human cervical cancer cells. METHODS: After the cervical cancer cells (HeLa cells, CaSki cells) had been cultured, cisplatin and genistein were added to the culture medium, and the cell activity was measured using MTT assay. The CaSki cells were cultured in a medium containing cisplatin and genistein, and then, the cells were collected in order to measure p53, Bcl2, ERK, and caspase 3 levels by western blotting. RESULTS: Both the HeLa and CaSki cells had decreased cell viabilities when the cisplatin concentration was 10 μM or higher. When combined with genistein, the cell viabilities of the HeLa and CaSki cells decreased at cisplatin concentrations of 8 μM and 6 μM, respectively. The administration of genistein increased the toxicity of cisplatin in the HeLa and CaSki cells. In the CaSki cells, the p-ERK1/2 level decreased by 37%, the p53 expression level increased by 304%, and the cleaved caspase 3 level increased by 115% in the cisplatin+genistein group compared to that in the cisplatin group. Bcl2 expression was reduced by 69% in the cisplatin+genistein group compared to that in the cisplatin group. CONCLUSION: Genistein enhances the anticancer effect of cisplatin in CaSki cells, and can be used as a chemotherapeutic adjuvant to increase the activity of a chemotherapeutic agent.
Subject(s)
Humans , Blotting, Western , Caspase 3 , Cell Line , Cell Survival , Cisplatin , Genistein , HeLa Cells , Uterine Cervical NeoplasmsABSTRACT
@# Objective: To investigate whether miR-140 could increase the sensitivity of cervical cancer (CC) to oxaliplatin by downregulating the expression of programmed death-1 (PD-L1). Methods: qPCR was used to analyze miR-140 expression in normal human cervical cells, CC cells and oxaliplatin-resistant CC cells. Cells were transfected with miR-140 mimic, and then, the proliferation of CC cells and oxaliplatin-resistant CC cells was detected by using CCK-8 assay, and the colony formation rate of CC cells was obtained by using colony formation assay. Starbase and TargetScan were used to predict the targeted binding site of miR-140 and PD-L1, and the influence of miR-140 on the expression of PD-L1 was validated by dual luciferase reporter gene assay.Annexin V FITC/PI double staining and Wb assays were used to detect the effect of over-expression of miR-140 or both over-expression of PD-L1 and miR140 on the apoptosis, migration and expression of apoptosis-related proteins in CC cells after treatment with oxaliplatin. Moreover, transplantation tumor of CC cell lines was established in nude mice to assess the effects of miR-140 on enhancing the sensitivity of tumors to oxaliplatin. Results: The expression of miR-140 was significantly decreased in oxaliplatin-resistant CC cells (P<0.01). Over-expression of miR140 could significantly increase the sensitivity of oxaliplatin-resistant CC cells to oxaliplatin (P<0.05), and inhibit the CC cells proliferation and colony formation (P<0.01). miR-140 showed targeted binding to PD-L1 3'-UTR and inhibited its expression. Over-expression of miR-140 significantly promoted CC cell migration and apoptosis (P<0.01). However, co-transfection of PD-L1 counteracts the effects of miR-140 on cell metastasis and apoptosis (all P<0.05). In addition, xenograft tumor model in mice also verified that miR-140 could promote the sensitivity of tumors to oxaliplatin. Conclusion: miR-140 increases the sensitivity of CC to oxaliplatin through inhibition of PD-L1 expression. Therefore, up-regulation of miR-140 or down-regulation of PD-L1 in combination with oxaliplatin may be a novel strategy for the treatment of Oxaliplatin-resistant CC.
ABSTRACT
@# Objective: To investigate the influence of inhibiting expression of polyamine-modulated factor (PMF-1) on the antitumor effect of glucocorticoid dexamethasone (DEX) in human cervical cancer Caski cells. Methods: siRNAs which target human PMF-1 gene were designed and synthesized, and their effect on the expression of PMF-1 in Caski cells was evaluated by Western blotting. The PMF-1 down-regulated and control Caski cells were treated with DEX, and then the affect of PMF-1 down regulation on the sensitivity of the tumor cells to DEX was analyzed. MTT method was used to detect cell proliferation, flow cytometry was used to analyze cell cycle, Western blotting method was used to evaluate expression level of glucocorticoids receptor (GR), and HPLC was used to analyze intracellular polyamine content. Results: The transient transfection of Caski cells with siRNAwhich targets PMF-1 gene can significantly reduce the expression level of PMF-1 protein. Compared with the control cells, treating PMF-1 down-regulated Caski cells with DEX can more effectively inhibit cell proliferation（P<0.01), up regulate GR expression, arrest cell cycle at G2 stage（P<0.01), and also significantly reduce intracellular polyamine level（P<0.01). Conclusion：Inhibiting PMF-1 expression can enhance antitumor pharmacological activity of DEX against human cervical cancer cells, and the underlying mechanism may be related with enhanced cell cycle inhibition and decreased intracellular polyamine level.
ABSTRACT
@# Objective: To prepare a new type of phycocyanin/carboxymethyl chitosan-CD55 ligand peptide (CPC/CMC-CD55sp) nanospheres, and to study its targeted therapeutic effect on cervical cancer Caski cells. Methods: The novel CPC/CMC-CD55sp nanospheres (CPC/CMC-CD55sp) were synthesized by ionic cross-linking method, and the properties of nanospheres were observed by transmission electron microscopy (DLS) and fourier transform infrared spectroscopy (FTIR). The expression of CD55 on the surface of Caski and fibroblast (L-929) cells was detected by Western blotting and flow cytometry. The effect of nanospheres on the proliferation of Caski cells was detected by CCK-8. Flow cytometry and fluorescence microscopy were used to detect the uptake of microspheres by Caski cells; Western blotting and flow cytometry were used to detect the effect of CPC/CMC-CD55sp on expressions of apoptosis-related proteins and apoptosis rate in Caski cells; the hemolysis test was used to determine the biological safety of the drug. Results: CPC/ CMC-CD55sp was successfully prepared with good morphology and uniform diameter; and CD55 was highly expressed on the surface of Caski cells but low expressed on the surface of L-929 cells (P<0.01). CPC/CMC-CD55sp could targeted and efficiently reach Caski cells and be ingested into the cells. It exhibited weak hemolysis effect on human peripheral blood, which was in the safe range. CPC/ CMC-CD55sp displayed obvious inhibitory effect on Caski cell proliferation, and could induce cell apoptosis (P<0.05 or P<0.01). Conclusion: The new CPC/CMC-CD55sp can targeted inhibit the growth of cervical cancer Caski cells via inducing its apoptosis and has good bio-safety, which provides a new idea for the research and development of anti-tumor marine drugs.
ABSTRACT
OBJECTIVE: Etoposide is a potent and widely used antineoplastic agent. It is able to induce apoptosis in most cell types. However, very little is known about its mechanism of action. In this study, we demonstrate the cytotoxic signal that induced by etoposide and investigate how etoposide exerts antitumor activity in HPV-16 (+) CaSki cervical carcinoma cells. METHODS: Antiproliferation activity was measured in CaSki cell lines by using MTT assays, DNA fragmentation assay. Cell cycle distribution was analyzed using flow cytometry. Expression of proteins involved in the apoptotic pathway was analyzed by Western blotting (WB). Electron microscopic (EM) and biochemical studies (Western blotting, RT-PCR) revealed that non-apoptotic death was associated with autophagosomes/-autolysosomes. These parameters have also been measured in cells treated with 3-methyladenine (an autophagy inhibitor), zVAD-fmk (a pan-caspase inhibitor) and both. RESULTS: The etoposide induced apoptosis. In cell cycle analysis, etoposide-treated CaSki cells were few induced hypodiploid DNA content, suggesting that apoptotic cell death. EM study revealed that autophagic appearance in the presence of etoposide exhibited by autophagosomes/autolysosomes. It was confirmed by LysoTracker probe and WB against Beclin 1, APG 5, APG 12 and p53. When autophagy was blocked by 3-MA, not only the protein expression of Beclin 1, but also the antitumor effect of etoposide was suppressed. On the other hand, the addition of zVAD-fmk could induce a few etoposide-induced autophagy. And etoposide-treated CaSki cells were rescued by combination of 3-MA and zVAD-fmk. CONCLUSION: Our results suggest that etoposide not only initiated apoptosis but ultimately caused cell death through autophagy. In this study, we demonstrate novel features for the action of etoposide in HPV-16 (+) CaSki cervical carcinoma cells. Autophagic cell death induction by some anticancer agents underlines the potential utility of its induction as a new cancer treatment modality.