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
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