Journal: Molecular Therapy Oncolytics

Article Title: Potential for reversing miR-634 -mediated cytoprotective processes to improve efficacy of chemotherapy against oral squamous cell carcinoma

doi: 10.1016/j.omto.2022.02.002

Figure Lengend Snippet: Identification of the cIAP1 gene as a miR-634 direct target (A) Induction of apoptotic cell death by overexpression of miR-634 in SAS and Ca9-22 cells. Cells were evaluated at day 2 after transfection with 20 nM miR-NC or miR-634 . Upper: phase-contrast images in the miRNA-transfected cells. Scale bars: 20 μm. Lower: FACS analysis of the apoptotic cell population. (B) Western blot analysis of the miRNA-transfected cells. Cell lysates were prepared two days after transfection. (C) Identification of miR-634 target genes by RIP-Chip analysis in SAS and A431 cells. In total, 3,491 and 1,459 genes were identified as the candidates with a fold change (FC) > 2.0 by RIP-Chip analysis in SAS and A431 cells, respectively; 1,313 genes identified by both analyses were defined as the miR-634 target genes. (D) RIP-PCR analysis of the cIAP1 gene. Enrichment relative to IgG immunoprecipitation in miR-NC -transfected cells is presented. (E) Western blot analysis of cIAP1 in the miRNA-transfected cells. (F) The positions of two putative miR-634 binding sites (site 1; S1 and site 2; S2) within the coding region of the cIAP1 gene (accession No. NM_001166 ; 3,764 bp) are indicated. (G) Luciferase assay using reporter plasmids. Seed sequences of miR-634 and its mutant sequences are indicated. SAS cells were cotransfected with a reporter plasmid and an internal control vector, and after 5 h, either miR-NC or miR-634 was additionally transfected. Thirty-six hours after transfection, luciferase activity was measured. The luciferase activity in miR-634 -transfected cells relative to that in miR-NC -transfected cells is indicated on the vertical axis. In (A), (D), and (G), error bars indicate the SD of triplicate experiments, and data are presented as the mean ± SD. p values were calculated using the two-sided Student's t test (A and D) or one-way ANOVA (G). ∗p < 0.0001, ∗∗p < 0.005. NS, not significant.

Article Snippet: Among the 1,313 genes, we found cIAP1 , whose expression is involved in chemotherapeutic resistance, including CDDP (listed as BIRC2 in ; FC:5.22 in A431 cells and FC:10.01 in SAS cells).

Techniques: Over Expression, Transfection, Western Blot, RNA Immunoprecipitation - Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay, Luciferase, Mutagenesis, Plasmid Preparation, Control, Activity Assay

Journal: Molecular Therapy Oncolytics

Article Title: Potential for reversing miR-634 -mediated cytoprotective processes to improve efficacy of chemotherapy against oral squamous cell carcinoma

doi: 10.1016/j.omto.2022.02.002

Figure Lengend Snippet: Enhancement of CDDP-induced cytotoxicity by miR-634 in OSCC cells (A) Cell survival assay. Cells were transfected with increasing doses of miR-634 (2.5–10 nM) and treated with CDDP (2.5–10 μM) for two days from the following day. The results are reported as the relative rate compared with miR-NC -transfected and non-treated cells. (B) FACS analysis of the apoptotic cell population among cells with miRNAs and/or CDDP. Cells were transfected with miRNAs (2.5 nM) and treated with CDDP for two days from the following day. (C) Western blot analysis of cleaved caspase-3 (cCasp-3) and cleaved PARP (cPARP) as the apoptosis markers and the miR-634 target genes. (D) FACS analysis of the apoptotic cell population. Cells were transfected with siRNAs (10 nM) and treated with CDDP for two days from the following day or were concurrently treated with AT406 (10 μM) and CDDP for two days. (E) Western blot analysis of apoptosis markers and cIAP1. In (A), (B), and (D), error bars indicate the SD of triplicate experiments, and data are presented as the mean ± SD. p values were calculated using the two-way ANOVA (A and B) and two-sided Student's t test (D). ∗p < 0.0001, ∗∗p < 0.005.

Article Snippet: Among the 1,313 genes, we found cIAP1 , whose expression is involved in chemotherapeutic resistance, including CDDP (listed as BIRC2 in ; FC:5.22 in A431 cells and FC:10.01 in SAS cells).

Techniques: Clonogenic Cell Survival Assay, Transfection, Relative Rate, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Potential for reversing miR-634 -mediated cytoprotective processes to improve efficacy of chemotherapy against oral squamous cell carcinoma

doi: 10.1016/j.omto.2022.02.002

Figure Lengend Snippet: Overcoming CDDP resistance by miR-634 in OSCC cells (A) Western blot analysis of miR-634 target genes in RT7 cells and six OSCC cell lines. IC50 values (μM) for CDDP treatment for each cell line are indicated. (B) Quantitative genomic-PCR for the cIAP1 gene in OSCC cell lines. The copy number ratios are presented relative to the copy number in RT7 cells. (C) The correlation between copy number and the corresponding mRNA expression of the cIAP1 gene in 496 cases of HNSCC, including OSCC, from TCGA data. (D) FACS analysis of the apoptotic cell population among TSU cells. Cells were transfected with miRNAs (2.5 nM) and treated with CDDP for two days from the following day. (E) Western blot analysis of cleaved caspase-3 (cCasp-3) and cleaved PARP (cPARP) as the apoptosis markers, and the miR-634 target genes in TSU cells. (F) Generation of CDDP-acquired resistant cells (Ca9-22-R) from Ca9-22 cells. IC50 values are indicated. (G) FACS analysis of the apoptotic cell population among Ca9-22 and Ca9-22-R cells. Cells were transfected with miR-634 (2.5 nM) and treated with CDDP (10 uM) for two days from the following day. (H) Western blot analysis of miR-634 target genes in Ca9-22 and Ca9-22-R cells. In D and G, error bars indicate the SD of triplicate experiments, and data are presented as the mean ± SD. p values were calculated using the two-way ANOVA. ∗p < 0.0001.

Article Snippet: Among the 1,313 genes, we found cIAP1 , whose expression is involved in chemotherapeutic resistance, including CDDP (listed as BIRC2 in ; FC:5.22 in A431 cells and FC:10.01 in SAS cells).

Techniques: Western Blot, Expressing, Transfection