normal human cervical epithelial cells hucec  (ATCC)

Journal: Frontiers in Oncology

Article Title: Circular RNA circLMO1 Suppresses Cervical Cancer Growth and Metastasis by Triggering miR-4291/ ACSL4 -Mediated Ferroptosis

doi: 10.3389/fonc.2022.858598

Figure Lengend Snippet: Features of circLMO1. (A) Schematic diagram of the formation of circLMO1. The qPCR primers used to evaluate the linear sequence of circLMO1, the convergent primers, are represented by red triangles. The qPCR primers used to evaluate the reverse splice site of circLMO1, the divergent primers, are represented by purple triangles. (B) Sanger sequencing of the reverse splice site of circLMO1. (C) qPCR analysis of circLMO1 level in DNA and cDNA derived from C33A cells using convergent primers and divergent primers. (D) Oligo (dT)18 primers or random primers was used to synthesize first-strand cDNA, and then qPCR analysis was performed to evaluate the level of circLMO1 and mLMO1 in these cDNAs. The value of the random primer is used as a reference. (E) Total RNA was treated with RNase R, and then qPCR analysis were performed to evaluate circLMO1 and mLMO1 level. (F) qPCR analysis was performed to assess circLMO1 expression in various cervical cancer cell lines (SiHa, CaSki, C33A, and HeLa) and normal cervical epithelial cell lines (HUCEC). (G) qPCR analysis was performed to assess circLMO1 level in cervical cancer tissues (n = 31) and matched normal tissues. (H) qPCR analysis was performed to assess circLMO1 level in different FIGO stages of cervical cancer. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Normal human cervical epithelial cells (HUCEC), CaSki, and C33A cell lines were purchased from American Type Culture Collection (ATCC, VA, USA).

Techniques: Sequencing, Derivative Assay, Expressing

Journal: Frontiers in Oncology

Article Title: Circular RNA circLMO1 Suppresses Cervical Cancer Growth and Metastasis by Triggering miR-4291/ ACSL4 -Mediated Ferroptosis

doi: 10.3389/fonc.2022.858598

Figure Lengend Snippet: CircLMO1 acts as a sponge for miR-4291 in cervical cancer cells. (A) FISH assay were carried out to assess circLMO1 cell localization in C33A and CaSki cells. The RNA probe targeting circLMO1 was stained green and the nucleus was stained blue. (B) qPCR analysis of circLMO1 levels in the cytoplasm and nucleus of C33A cells. U6 was used as a positive control in the nucleus, and β-actin was used as a positive control in the cytoplasm. (C) AGO2 antibody was used to perform RIP assay to determine the combination of circLMO1 and AGO2 in CaSki cells. (D) qPCR analysis was performed to assess miRNAs expression in CaSki and HUCEC cells. (E) Schematic diagram of the predicted miR-4291-circLMO1 interaction. (F–H) After co-transfection with miR-4291 (or its mutant) and circLMO1-wt luciferase reporter gene (or its mutant), luciferase activity was assessed in CaSki and C33A cells. After transfection with biotin-labelled miR-4291, qPCR analysis was performed to assess circLMO1 level in the streptavidin precipitation complex from CaSki cells (I) or C33A cells (J) . (K) The co-localization of circLMO1 and miR-4291 analysed through double FISH assay in C33A cells. *p < 0.05, **p < 0.01.

Article Snippet: Normal human cervical epithelial cells (HUCEC), CaSki, and C33A cell lines were purchased from American Type Culture Collection (ATCC, VA, USA).

Techniques: Staining, Positive Control, Expressing, Cotransfection, Mutagenesis, Luciferase, Activity Assay, Transfection

Journal: Chinese Medical Journal

Article Title: LncRNA AFAP1-AS1/miR-27b-3p/VEGF-C axis modulates stemness characteristics in cervical cancer cells

doi: 10.1097/CM9.0000000000001665

Figure Lengend Snippet: Level changes of AFAP1-AS1 and stemness in CD44v6(+) CC cells. (A) Volcano plots showing expression profiles of lncRNAs. (B) qRT-PCR detected AFAP1-AS1 levels, ∗ P < 0.05; (C) Western blotting detected CD44v6 levels. ∗ P < 0.05. (D) CD44v6(+)/(−) cells sorting by FCM. (E) AFAP1-AS1 levels were increased in CD44v6(+) cells, ∗ P < 0.05; (F) and (G) Representative images of spheres for CC cells and number of spheres (100×). (H–J) qRT-PCR results showed that OCT4, OPN, CD133 mRNA levels were increased in CD44v6(+) cells. ∗ P < 0.05. (K–N) Western blotting results showed that OCT4, OPN, CD133 protein levels were increased in CD44v6(+) cells, ∗ P < 0.05. AFAP1-AS1: Actin filament-associated protein 1 antisense RNA 1; CD133: Cluster of differentiation 133; CC: Cervical cancer; CD44v6: Hyaluronic acid receptor cluster of differentiation 44 variant exon 6; HUCEC: Human cervical epithelial cell line; LncRNA: Long non-coding RNA; OCT4: Octamer-binding transcription factor 4; OPN: Osteopontin; qRT-PCR: Quantitative real-time PCR.

Article Snippet: We obtained SiHa and Hela, the human CC cells, and normal human cervical epithelial cell line (HUCEC) from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Variant Assay, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Cancer Cell International

Article Title: HAND2-AS1 inhibits invasion and metastasis of cervical cancer cells via microRNA-330-5p-mediated LDOC1

doi: 10.1186/s12935-019-1048-y

Figure Lengend Snippet: HAND2-AS1 is poorly expressed in cervical cancer and cells. a The heat map of some differentially expressed genes in the cervical cancer expression dataset GSE63678. The abscissa indicates the sample type and the ordinate indicates the gene expression level. The left dendrogram indicates the sample expression cluster, and the upper right histogram indicates color grade. b The expression of HAND2-AS1 in dataset GSE63678. The abscissa indicates the sample type, and the ordinate indicates the gene expression; the upper right indicates p value. c The expression of HAND2-AS1 in 68 cases of cervical cancer and adjacent tissues determined by RT-qPCR (N = 68), * p < 0.05 vs. the adjacent tissues. d The expression of HAND2-AS1 in human cervical cancer cell lines, * p < 0.05 vs. HUCEC cell line. All measurements were expressed by mean ± standard deviation. Paired t -test was used for comparison between cervical cancer tissues and adjacent tissues. The expression of each cell line was compared with one-way ANOVA, followed by Tukey’s post hoc test. The experiment was repeated three times. HAND2-AS1 HAND2 antisense RNA 1, RT-qPCR reverse transcription quantitative polymerase chain reaction, ANOVA analysis of variance, HUCEC human cervical epithelial cell

Article Snippet: Normal human cervical epithelial cell lines (HUCEC) (BSC-00166804, ATCC, Manassas, VA, USA) were cultured in RPMI 1640 medium (12633012, Shanghai Haoran Bio Technologies Co., Ltd., Shanghai, China) containing 10% fetal bovine serum.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Standard Deviation, Comparison, Reverse Transcription, Real-time Polymerase Chain Reaction