Journal: Current Issues in Molecular Biology

Article Title: The Therapeutic Effect of EZH2 Inhibitors in Targeting Human Papillomavirus Associated Cervical Cancer

doi: 10.3390/cimb47120990

Figure Lengend Snippet: MTT assay results for cytotoxicity of drug treatments. Drug dose–response curves after 48 h for HPV- C33a, HPV+ CaSki, and normal epithelial cell line CRL1790: ( A ) cisplatin, ( B ) EPZ6438, and ( C ) ZLD1039. Data is expressed as log of mean ± SD (n = 3). IC50 values, pinpointed on the graph, were calculated using GraphPad prism. ( D ) Table of mean IC50 value data (n = 3). The IC50 value for EPZ6438 could not be determined within the 0–80 µM range.

Article Snippet: According to human protein atlas [ ], the C33a cell line exhibits no expression of CDH1 gene, which encodes E-cadherin; however, mass spectrometry proteomics data could further confirm its protein expression.

Techniques: MTT Assay

Journal: Current Issues in Molecular Biology

Article Title: The Therapeutic Effect of EZH2 Inhibitors in Targeting Human Papillomavirus Associated Cervical Cancer

doi: 10.3390/cimb47120990

Figure Lengend Snippet: Migration rate assessment following the scratch wound–healing assay. ( A ) Representative images from the in vitro scratch wound–healing assays demonstrating cell migration into the cell-free region (outlined by black lines) following 48 h treatment (scale = 100 µm). ( B ) Summary plots showing the migration rates by C33a and CaSki cells after treatment (Mean ± SD, n = 3).

Article Snippet: According to human protein atlas [ ], the C33a cell line exhibits no expression of CDH1 gene, which encodes E-cadherin; however, mass spectrometry proteomics data could further confirm its protein expression.

Techniques: Migration, Wound Healing Assay, In Vitro

Journal: Current Issues in Molecular Biology

Article Title: The Therapeutic Effect of EZH2 Inhibitors in Targeting Human Papillomavirus Associated Cervical Cancer

doi: 10.3390/cimb47120990

Figure Lengend Snippet: Effect of EZH2 inhibitors on protein and mRNA expression of ZO-1 and E-cadherin on cervical cancer cells after 48 h treatment. ( A ) Immunofluorescence staining of ZO-1 and E-cadherin protein expression (pink) and nuclei (blue) under 400× microscope magnification. Scale = 50 µm. Mean fluorescence intensity of immunocytochemistry results were normalised to control cells (right) (n = 3). ( B ) Representative Western blots’ results of protein bands ZO-1 (250 kDa) and β-actin (42 kDa) for C33a and CaSki and E-cadherin (110 kDa) for CaSki are shown on the left. Bar charts on the right show the Western blot quantification of these protein expression levels following treatment. Values are expressed as a fold change of control (n = 3). ( C ) RT-qPCR analysis of relative ZO-1 (C33a and CaSki) and E-cadherin (CaSki) mRNA expression levels following 48 h treatment. Values are expressed as fold change of control values. Data is presented as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Bold asterisk above a single sample bar indicates the significant difference of that sample from the rest of the treatments.

Article Snippet: According to human protein atlas [ ], the C33a cell line exhibits no expression of CDH1 gene, which encodes E-cadherin; however, mass spectrometry proteomics data could further confirm its protein expression.

Techniques: Expressing, Immunofluorescence, Staining, Microscopy, Fluorescence, Immunocytochemistry, Control, Western Blot, Quantitative RT-PCR

Journal: Current Issues in Molecular Biology

Article Title: The Therapeutic Effect of EZH2 Inhibitors in Targeting Human Papillomavirus Associated Cervical Cancer

doi: 10.3390/cimb47120990

Figure Lengend Snippet: Immunohistochemical staining results of EZH2, p53, ZO-1, β-catenin, and HPV16 E6 from CAM assay tissue sections after 48 h treatment with control (DMSO 0.1%), cisplatin, and EPZ6438. Immunohistochemical staining of ( A ) C33a and ( B ) CaSki for expression of EZH2, p53, ZO-1, β-catenin, and HPV16 E6 from the CAM tissue model (n = 1). Scale = 50 µm. Chorionic (CE) and allantoic (AE) epithelial layers with sub-epithelial capillary network (SEC), tumour cells (TC), mesoderm (M), and blood vessels (BV) are displayed.

Article Snippet: According to human protein atlas [ ], the C33a cell line exhibits no expression of CDH1 gene, which encodes E-cadherin; however, mass spectrometry proteomics data could further confirm its protein expression.

Techniques: Immunohistochemical staining, Staining, Chick Chorioallantoic Membrane Assay, Control, Expressing

Journal: Molecular Medicine Reports

Article Title: hsa_circ_0101119 facilitates the progression of cervical cancer via an interaction with EIF4A3 to inhibit TCEAL6 expression

doi: 10.3892/mmr.2021.12293

Figure Lengend Snippet: hsa_circ_0101119 is highly expressed in CC tissues and cells. (A) Heatmap of differentially expressed circRNAs in CC tissues and normal tissues according to the online data set (GSE102686). (B) Expression level of hsa_circ_0101119 was detected via reverse transcription-quantitative PCR in CC cell lines (C-33A, SiHa, CaSki and HeLa) and normal human cervical epithelial cell line, HcerEpic. **P<0.01 vs. HcerEpic cells. (C) Expression level of hsa_circ_0101119 in CC tissues and normal tissues, according to the online data set (GSE102686). circRNA/circ, circular RNA.

Article Snippet: The four human CC cell lines (C-33A, SiHa, CaSki and HeLa) and the normal human cervical epithelial cell line, HcerEpic, were supplied by VCANBIO Cell & Gene Engineering Co., Ltd. All the cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), and cultured at 37°C in a humidified 5% CO 2 incubator.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: hsa_circ_0101119 facilitates the progression of cervical cancer via an interaction with EIF4A3 to inhibit TCEAL6 expression

doi: 10.3892/mmr.2021.12293

Figure Lengend Snippet: hsa_circ_0101119 recruits EIF4A3 to inhibit TCEAL6 expression in CC. (A) Bioinformatics was used to predict the interaction probabilities of the RNA-binding protein EIF4A3 with hsa_circ_0101119. Predictions with probabilities >0.5 were considered ‘positive’, suggesting that the corresponding RNA and protein are likely to interact. (B) RIP assay using anti-EIF4A3 showed that EIF4A3 precipitated hsa_circ_0101119 in SiHa and HeLa cell lysates. (C) Pull down assay indicated that biotin-labeled hsa_circ_0101119 interacted with EIF4A3. (D) Bioinformatics was used to predict the interaction probabilities of EIF4A3 with TCEAL6. (E) RIP assay using anti-EIF4A3 showed that EIF4A3 precipitated TCEAL6 in SiHa and HeLa cell lysates. (F) Expression levels of EIF4A3 and TCEAL6 were detected via RT-qPCR in CC cell lines (C-33A, SiHa, CaSki and HeLa) and a normal human cervical epithelial cell line, HcerEpic. (G) Expression levels of EIF4A3 and TCEAL6 in CC tissues and normal tissues, according to the analysis of TCGA. (H) Correlation between EIF4A3 and TCEAL6 in CC samples from TCGA. (I) After transfection with sh-EIF4A3, RT-qPCR was used to detect EIF4A3 expression in SiHa and HeLa cells. (J) After transfection with sh-EIF4A3, western blotting was performed to detect the expression level of TCEAL6 in SiHa and HeLa cells. (K) After co-transfection with si-hsa_circ_0101119 and sh-EIF4A3, western blotting was performed to measure the expression level of TCEAL6 in SiHa and HeLa cells. (L) A proposed model whereby hsa_circ_0101119 sequesters EIF4A3 away from TCEAL6 mRNA, in turn suppressing TCEAL6 mRNA translation. **P<0.01 vs. IgG group (B and E); *P<0.05, **P<0.01 vs. HcerEpic cells group (F); *P<0.05 vs. normal tissues group (G); **P<0.01, vs. sh-NC group (I and J); **P<0.01 vs. sh-NC group, ## P<0.01, vs. si-hsa_circ group. (K) RIP, RNA immunoprecipitation; RT-qPCR, reverse transcription-quantitative PCR; TCGA, The Cancer Genome Atlas; sh, short hairpin RNA; NC, negative control; si, small interfering RNA; circ, circular RNA; EIF4A3, eukaryotic initiation factor 4A-3; TCEAL6, transcription elongation factor A-like 6; T, tumor; N, normal; CC, cervical cancer.

Article Snippet: The four human CC cell lines (C-33A, SiHa, CaSki and HeLa) and the normal human cervical epithelial cell line, HcerEpic, were supplied by VCANBIO Cell & Gene Engineering Co., Ltd. All the cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), and cultured at 37°C in a humidified 5% CO 2 incubator.

Techniques: Expressing, RNA Binding Assay, Pull Down Assay, Labeling, Quantitative RT-PCR, Transfection, Western Blot, Cotransfection, Immunoprecipitation, Real-time Polymerase Chain Reaction, shRNA, Negative Control, Small Interfering RNA