Journal: Current Issues in Molecular Biology

Article Title: Associating Patient Responses with Drug Sensitivity in Non-Small Cell Lung Carcinoma Using an Immunoassay on Patient-Derived Cell Cultures

doi: 10.3390/cimb47040281

Figure Lengend Snippet: Clinical parameters of NSCLC patients included in our study.

Article Snippet: Vinorelbine, pemetrexed, and osimertinib were purchased from Selleckchem (Houston, TX, USA).

Techniques: Mutagenesis

Journal: Molecular therapy. Nucleic acids

Article Title: miR-6077 promotes cisplatin/pemetrexed resistance in lung adenocarcinoma via CDKN1A/cell cycle arrest and KEAP1/ferroptosis pathways.

doi: 10.1016/j.omtn.2022.03.020

Figure Lengend Snippet: Figure 2. CDKN1A and KEAP1 are direct targets of miR-6077 (A) Venn plot showing the intersection of predicted targets of miR-6077. (B) The CRISPR-Cas 9 results of top eight predicted targets of miR-6077 whose depletion potentially confers resistance to CDDP/PEM. (C) Heatmap showing the relative expression levels of the eight predicted targets of miR-6077 in A549 cells transfected with miR-NC1, miR-NC2, and miR-6077 upon treatment with PBS or CDDP (10 mM)/PEM (1 mM) for 48 h. The results were obtained from RNA-seq. (D and E) Quantitative real-time PCR and western blotting assays showing the mRNA (D) and protein (E) levels of CDKN1A and KEAP1 in A549 cells transfected with miR-NC1, miR-NC2, and miR-6077 upon treat- ment with PBS or CDDP (10 mM)/PEM (1 mM) for 48 h. (F) Quantitative real-time PCR showing the relative expression levels of CDKN1A and KEAP1 in A549 cells upon treat- ment with PBS or CDDP (10 mM)/PEM (1 mM) for 48 h. (G) The predicted target sites of miR-6077 in the 30 UTR of CDKN1A and KEAP1. Normal and mutant seed regions are highlighted and underlined. (H) Luciferase reporter plasmids containing wild- or mutant-type CDKN1A (left) and KEAP1 (right) were co-transfected into HEK293T cells with miR-NC1, miR-NC2, or miR-6077. Bar plots exhibit the luciferase activity of the transfected cells. (I) Sanger sequencing confirming the G333C inactivating mutation within the first Kelch domain (KLD) of KEAP1 in A549 cells. Data are presented as the mean ± SD, n = 3 independent repeats. Unpaired, two-tailed t test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: CDDP (T1564), PEM (T6226), oxaliplatin (T0164), carboplatin (T1058), erastin (T1765), ferrostatin-1 (T6500), and deferoxamine (DFO; T1637) were purchased from Topscience (USA).

Techniques: CRISPR, Expressing, Transfection, RNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Mutagenesis, Luciferase, Activity Assay, Sequencing, Two Tailed Test

Journal: Molecular therapy. Nucleic acids

Article Title: miR-6077 promotes cisplatin/pemetrexed resistance in lung adenocarcinoma via CDKN1A/cell cycle arrest and KEAP1/ferroptosis pathways.

doi: 10.1016/j.omtn.2022.03.020

Figure Lengend Snippet: Figure 4. miR-6077 protects LUAD cells from CDDP/PEM-induced cell-cycle arrest by directly targeting CDKN1A (A) Quantitative real-time PCR assay confirming the ectopic overexpression of CDKN1A in A549 cells. (B) Dose-toxicity curves showing the viability of A549 cells (with or without CDKN1A overexpression) transfected with miR-NC1, miR-NC2, and miR-6077 upon CDDP/PEM treatment at the indicated concentrations for 48 h. (C–E) Colony formation ability (C), cell-cycle proportion (D), and protein levels of CDKN1A and its downstream cell-cycle regulators (E) in A549 cells (with or without CDKN1A overexpres- sion) transfected with miR-NC1, miR-NC2, and miR-6077 upon treatment with PBS or CDDP (10 mM)/PEM (1 mM) for 14 days (C) or 48 h (D and E). (F) Dose-toxicity curves showing the viability of A549 cells (with or without CDKN1A overexpression) transfected with miR-NC1, miR-NC2, and miR-6077 upon treatment with PEM and oxaliplatin or carboplatin at the indicated concentrations for 48 h. Data are presented as the mean ± SD, n = 3 independent repeats. Unpaired, two-tailed t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

Article Snippet: CDDP (T1564), PEM (T6226), oxaliplatin (T0164), carboplatin (T1058), erastin (T1765), ferrostatin-1 (T6500), and deferoxamine (DFO; T1637) were purchased from Topscience (USA).

Techniques: Real-time Polymerase Chain Reaction, Over Expression, Transfection, Two Tailed Test

Journal: Molecular therapy. Nucleic acids

Article Title: miR-6077 promotes cisplatin/pemetrexed resistance in lung adenocarcinoma via CDKN1A/cell cycle arrest and KEAP1/ferroptosis pathways.

doi: 10.1016/j.omtn.2022.03.020

Figure Lengend Snippet: Figure 5. miR-6077 protects LUAD cells from CDDP/PEM-induced ferroptosis by directly targeting KEAP1 (A) Quantitative real-time PCR assay confirming the ectopic overexpression of KEAP1 in H358 cells. (B) Dose-toxicity curves showing the viability of H358 cells with or without KEAP1 overexpression transfected with miR-NC1, miR-NC2, and miR-6077 upon CDDP/PEM treatment at the indicated concentrations for 48 h. (C–F) Colony formation ability (C), lipid peroxidation (D), relative glutathione levels (E), and protein levels of KEAP1 and its downstream ferroptosis regulators (F) in H358 cells (with or without KEAP1 overex- pression) transfected with miR-NC1, miR-NC2, and miR-6077 upon treatment with PBS or CDDP (20 mM)/PEM (2 mM) for 14 days (C) or 48 h (D–F). (G) Schematic outline of the CRISPR-Cas9-mediated homology-directed repair (HDR) in A549cells. The mutated KEAP1 was replaced by a wild-type sequence. Agarose gel electrophoresis confirming the successful HDR in A549 cells, and the introduced repair template containing homology arms was digested by ApaI enzyme as expected. (H and I) Lipid peroxidation (H) and relative glutathione levels (I) in A549-HDR cells transfected with miR-NC1, miR-NC2, and miR-6077 upon treatment with PBS or CDDP (20 mM)/PEM (2 mM) for 48 h. Data are presented as the mean ± SD, n = 3 independent repeats. Unpaired, two-tailed t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

Article Snippet: CDDP (T1564), PEM (T6226), oxaliplatin (T0164), carboplatin (T1058), erastin (T1765), ferrostatin-1 (T6500), and deferoxamine (DFO; T1637) were purchased from Topscience (USA).

Techniques: Real-time Polymerase Chain Reaction, Over Expression, Transfection, CRISPR, Sequencing, Agarose Gel Electrophoresis, Two Tailed Test

Journal: Molecular therapy. Nucleic acids

Article Title: miR-6077 promotes cisplatin/pemetrexed resistance in lung adenocarcinoma via CDKN1A/cell cycle arrest and KEAP1/ferroptosis pathways.

doi: 10.1016/j.omtn.2022.03.020

Figure Lengend Snippet: Figure 6. GMDS-AS1 and LINC01128 sensitize LUAD to CDDP/PEM and function as sponges for miR-6077 in LUAD (A) Flowchart displaying the screening process of lncRNAs potentially interacting with miR-6077. (B) Bar plot showing that the overexpression of selected lncRNAs leads to altered sensitivity to CDDP (20 mM)/PEM (2 mM) treatment in H1299. (C) Relative expression levels of selected lncRNAs in H1299 and H1299-CDDP/PEM-resistant cells. (D and E) Dose-toxicity curves showing the viability of H358 (D) and H1299 (E) cells (with or without GMDS-AS1/LINC01128 overexpression) transfected with miR-NC1, miR- NC2, and miR-6077 upon CDDP/PEM treatment at the indicated concentrations for 48 h. Blue asterisks represent the significance of comparison between GMDS-AS1- overexpressing H1299 transfected with miR-NC and with miR-6077, while red asterisks represent that of LINC01128-overexpressing H1299. (F) The predicted target sites of miR-6077 in GMDS-AS1 and LINC01128. (G) Luciferase reporter plasmids containing wild- or mutant-type GMDS-AS1 (left) and LINC01128 (right) were co-transfected into HEK293T cells with miR-NC1, miR-NC2, or miR-6077. Bar plots exhibit the luciferase activity of the transfected cells. (H) RNA pull-down followed by western blotting showing a possible interaction between Ago2 and GMDS-AS1 or LINC01128. (I) RIP assay of the enrichment of GMDS-AS1, LINC01128, miR-6077, CDKN1A, and KEAP1 transcripts on Ago2 relative to IgG in H1299 cells. (J) RIP assay showing that overexpression of GMDS-AS1 or LINC01128 disrupts the interactions between miR-6077 and CDKN1A or KEAP1. Data are presented as the mean ± SD, n = 3 independent repeats. (K and L) Quantitative real-time PCR assay confirming the ectopic overexpression of GMDS-AS1 and LINC01128 in H358 (K) and H1299 (L) cells. Unpaired, two-tailed t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

Article Snippet: CDDP (T1564), PEM (T6226), oxaliplatin (T0164), carboplatin (T1058), erastin (T1765), ferrostatin-1 (T6500), and deferoxamine (DFO; T1637) were purchased from Topscience (USA).

Techniques: Over Expression, Expressing, Transfection, Comparison, Luciferase, Mutagenesis, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test