Journal: Frontiers in Genetics

Article Title: Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing

doi: 10.3389/fgene.2021.798608

Figure Lengend Snippet: Nanopore sequencing found tandem HPV genomes in cervical cancer tissues and CaSki cells, respectively. (A) An 11.54 kb-long HPV 35 tandem genomic sequence was obtained by nanopore sequencing. The 11.54 kb-long sequence was aligned to HPV35 16B (NCBI: KX514416.1), which consists of three genomic segments, E1-E7-E6 (1567–10 nt), a whole genome frame (7894–10 nt), and URR-L1 (1561–10 nt. (B) A 21.18 kb-long HPV tandem sequence flanked with human genomic sequence at one end was obtained by nanopore sequencing from CaSki cells. The sequence was aligned to HPV16 (NCBI: U89348), in which a truncated genome (470–7905 nt) was connected to another truncated one (470–6905 nt) in a head-to-tail manner. (C) Another 15.07 kb-long HPV tandem sequence was obtained by nanopore sequencing from CaSki cells and was flanked at one end by the human gene; concatemers are formed by joining of incomplete HPV genomes with two spliced sites at 2032 nt and 4,586 nt.

Article Snippet: As a proof of concept, in this study, we completed whole genome sequencing from a cervical cancer tissue and a CaSki cell line with Oxford Nanopore Technologies.

Techniques: Nanopore Sequencing, Sequencing

Journal: Frontiers in Genetics

Article Title: Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing

doi: 10.3389/fgene.2021.798608

Figure Lengend Snippet: Physical state determination of the HPV genome by exonuclease V (ExoV)-qPCR–based assay.

Article Snippet: As a proof of concept, in this study, we completed whole genome sequencing from a cervical cancer tissue and a CaSki cell line with Oxford Nanopore Technologies.

Techniques:

Journal: Frontiers in Genetics

Article Title: Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing

doi: 10.3389/fgene.2021.798608

Figure Lengend Snippet: HPV integration sites identified by nanopore sequencing in CaSki cells and a cervical cancer tissue. (A) Distribution of HPV integration sites in different function regions of human genes identified from CaSki cells (HPV16) and a cervical cancer tissue (HPV35) by nanopore sequencing. (B) The HPV integration site was amplified by PCR from the CaSki cells, followed by agarose gel electrophoresis. C-33A cells (HPV-negative cervical cancer cells) was used as a negative control. (C) Sanger sequencing of the HPV integration site. PCR products were subjected to Sanger sequencing. Peaks of nucleotides at integration sites were shown. The cellular sequence from PRR30 was boxed with green color, and viral sequence was boxed with red color. (D) The sequence of the integration site located in PRR30 gene. The exon region of PRR30 gene was labeled with green color, and the URR region of HPV genome was labeled with red color. Three random nucleotides at the breakpoint (BP) were labeled with blue color.

Article Snippet: As a proof of concept, in this study, we completed whole genome sequencing from a cervical cancer tissue and a CaSki cell line with Oxford Nanopore Technologies.

Techniques: Nanopore Sequencing, Amplification, Agarose Gel Electrophoresis, Negative Control, Sequencing, Labeling

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

Journal: Cell Death & Disease

Article Title: Novel prosurvival function of Yip1A in human cervical cancer cells: constitutive activation of the IRE1 and PERK pathways of the unfolded protein response

doi: 10.1038/cddis.2017.147

Figure Lengend Snippet: Depletion of Yip1A induces apoptotic cell death in HeLa and CaSki cervical cancer cells. ( a ) HeLa and CaSki cells were transfected with control scramble siRNA (left panel) or Yip1A siRNA (right panel). Representative confocal micrographs show morphological differences between control and Yip1A-knockdown cells at the indicated time points. Scale bars are 50 μ m. ( b ) The viability of transfected cells was evaluated at the indicated time points using the MTT Cell Proliferation Assay. Data are means±S.D. from three independent experiments; * P <0.05, ** P <0.01. ( c ) Representative flow cytometric data for control (left panels) and Yip1A-knockdown (right panels) cells at the indicated time points after siRNA transfection. The percentages of apoptotic cells (Annexin V + /PI − + Annexin V + /PI + ) are shown in the bar graphs. Data are means±S.D. from three independent experiments; * P <0.05. ( d ) Representative confocal micrographs of control (upper panels) and Yip1A-knockdown (lower panels) cells at the indicated time points after siRNA transfection. Scale bars are 10 μ m. The percentages of TUNEL-positive cells are shown in the bar graphs. Data are means±S.D. from three independent experiments; * P <0.05, ** P <0.01. ( e ) Western blotting shows relative levels of cleaved caspase 3 and cleaved PARP protein in control and Yip1A-knockdown cells at the indicated time points after siRNA transfection. GAPDH was used for normalization. Data are means±S.D. from three independent experiments; * P <0.05, ** P <0.01

Article Snippet: A human cervical cancer cell line CaSki (IFO50007) was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan), and grown in RPMI 1640 Medium (Nissui) supplemented with 10% FBS (Sigma-Aldrich) and 2 mM l -glutamine (Gibco) under conditions of 5% CO 2 atmosphere at 37 °C.

Techniques: Transfection, Control, Knockdown, MTT Cell Proliferation, TUNEL Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Novel prosurvival function of Yip1A in human cervical cancer cells: constitutive activation of the IRE1 and PERK pathways of the unfolded protein response

doi: 10.1038/cddis.2017.147

Figure Lengend Snippet: Schematic representation of how Yip1A operates as a prosurvival modulator that coordinately activates the IRE1 and PERK pathways of the UPR to support the survival of HeLa and CaSki cervical cancer cells. See text for details. Ub, ubiquitination

Article Snippet: A human cervical cancer cell line CaSki (IFO50007) was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan), and grown in RPMI 1640 Medium (Nissui) supplemented with 10% FBS (Sigma-Aldrich) and 2 mM l -glutamine (Gibco) under conditions of 5% CO 2 atmosphere at 37 °C.

Techniques: Ubiquitin Proteomics