Journal: Molecular Therapy. Nucleic Acids

Article Title: Mitochondrial Damage Mediated by miR-1 Overexpression in Cancer Stem Cells

doi: 10.1016/j.omtn.2019.10.016

Figure Lengend Snippet: The Influence of miR-1 on Mitochondrial Cristae Organization of Cancer Stem Cells (A) The sorting of ALDH1-positive cells. The baseline fluorescence was established by cells (P1 region) incubated with ALDEFLUOR substrate (BAAA) and ALDH1 inhibitor (DEAB). DEAB was used to block the background signal by inhibiting ALDH1 enzyme activity. Incubation of cells with ALDEFLUOR substrate in the absence of DEAB defined the ALDH1-positive population (P2 region). (B) Representative photographs of ALDH1-positive tumorspheres (top) and the percentages of tumor sphere formation of ALDH1-positive and ALDH1-negative cells (bottom). Scale bars, 10 μm. (C) Tumorigenicity of cancer stem cells (MDA-MB-435) in nude mice. Five mice were subcutaneously injected with the cells isolated from the spheres of tumorsphere formation assays (the ALDH1-positive cells). As controls, the ALDH1-negative cells were subcutaneously injected into five mice. Forty days later, the tumors were examined. The arrows indicate the tumors. (D) Differential expression of miR-1 in cancer stem cells and cancer non-stem cells. Quantitative real-time PCR was conducted to detect the expression level of miR-1 in melanoma stem cells (MSCs), melanoma non-stem cells (MNSCs), breast cancer stem cells (BCSCs), and breast cancer non-stem cells (BCNSCs) (**p < 0.01). U6 was used as an internal reference. (E) Overexpression of miR-1 in cancer stem cells. Cancer stem cells were transfected with miR-1 or control miRNA, followed by detection of miR-1 with quantitative real-time PCR (**p < 0.01). U6 was used as an internal reference. (F) Detection of stemness-associated genes in miR-1-overexpressing cancer stem cells. In the miR-1-transfected melanoma or breast cancer stem cells, the levels of stemness-associated genes’ expressions were examined by quantitative real-time PCR. (G) Influence of miR-1 overexpression on the viability of cancer stem cells. Cancer stem cells were transfected with miR-1. At different times after transfection, the viability of cancer stem cells was examined (**p < 0.01). (H) Effects of miR-1 overexpression on morphology of mitochondrial cristae of cancer stem cells. The mitochondrial cristae of miR-1-overexpressing cancer stem cells were examined under transmission electron microscopy (TEM) (left). The statistical data are indicated on the right (**p < 0.01). Scale bars, 0.5 μm. (I) Influence of miR-1 overexpression on mitochondrial transcripts of cancer stem cells. Mitochondrial transcripts of miR-1-overexpressing cancer stem cells were determined using quantitative real-time PCR (*p < 0.05; **p < 0.01). (J) Mitochondrial membrane potential analysis. At 36 h after miR-1 transfection, cancer stem cells were subjected to flow cytometry analysis and the value of mitochondrial membrane potential was calculated (**p < 0.01). Cancer stem cells transfected with control miRNA were used as controls. All assays were biologically repeated three times.

Article Snippet: Then, the cells were incubated with 1 μM mitochondrial tracker (Yeasen Biotech, Shanghai, China) that can label mitochondria at 37°C for 30 min. After fixing in 4% paraformaldehyde for 15 min, the cells were permeabilized with 0.1% Triton X-100 for 20 min.

Techniques: Fluorescence, Incubation, Blocking Assay, Activity Assay, Injection, Isolation, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Expressing, Over Expression, Transfection, Control, Transmission Assay, Electron Microscopy, Membrane, Flow Cytometry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Mitochondrial Damage Mediated by miR-1 Overexpression in Cancer Stem Cells

doi: 10.1016/j.omtn.2019.10.016

Figure Lengend Snippet: The Induction of miR-1-Mediated Mitochondrial Damage by Targeting GPD2 and MINOS1 Genes (A) Prediction of miR-1 target genes. As predicted, five genes ( MINOS1 , GPD2 , TMEM243 , TOMM70A , and CYB5B ) associated with mitochondria were the potential targets of miR-1. (B) The influence of miR-1 on the expressions of potential target genes. The melanoma stem cells (MSCs) were transfected with miR-1. At 48 h after transfection, the expression levels of potential target genes were examined using quantitative real-time PCR (**p < 0.01). (C) The direct interactions of miR-1 with its target genes. MDA-MB-435 cells were co-transfected with miR-1 and a luciferase reporter fused with MINOS1 or GPD2 3′ UTR. At 36 h after co-transfection, the firefly and renilla luciferase activities were analyzed. As controls, control miRNA, MINOS1 3′ UTR mutant, and GPD2 3′ UTR mutant were included in the co-transfections (**p < 0.01). (D) Western blot analysis of MINOS1 and GPD2 in miR-1-overexpressed melanoma stem cells (MSCs). At 36 h after miR-1 or control miRNA transfection, the protein level of MINOS1 or GPD2 was determined by western blot. Actin was used as a control. (E) Western blot analysis of MINOS1 and GPD2 in MSCs and melanoma non-stem cells (MNSCs). (F) Co-localization of MINOS1 and GPD2 with mitochondria of cancer stem cells. MSCs (MDA-MB-435) and breast cancer stem cells (MCF7) were respectively incubated with mitochondria tracker (MitoTracker). Then, the cells were fixed and stained with GPD2-specific or MINOS1-specific antibody. The nucleus was labeled with DAPI. The images were obtained using confocal microscopy. Scale bars, 10 μm. (G) The knockdown of GPD2 in cancer stem cells. The cancer stem cells were transfected with GPD2-siRNA. As a control, GPD2-siRNA-scrambled was included in the assays. The knockdown efficiency of GPD2 was investigated using quantitative real-time PCR (top) and western blot (bottom) (**p < 0.01). Actin was used as a negative control. (H) The silencing of MINOS1 in cancer stem cells. Quantitative real-time PCR (top) and western blot (bottom) were used to detect the expression level of MINOS1 in cancer stem cells (**p < 0.01). (I) The effects of GPD2 and MINOS1 silencing on the mitochondrial ultrastructure of cancer stem cells. Melanoma or breast cancer stem cells (MSCs and BCSCs) treated with sequence-specific siRNA were imaged under transmission electron microscopy. Scale bars, 0.5 μm.

Article Snippet: Then, the cells were incubated with 1 μM mitochondrial tracker (Yeasen Biotech, Shanghai, China) that can label mitochondria at 37°C for 30 min. After fixing in 4% paraformaldehyde for 15 min, the cells were permeabilized with 0.1% Triton X-100 for 20 min.

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Cotransfection, Control, Mutagenesis, Western Blot, Incubation, Staining, Labeling, Confocal Microscopy, Knockdown, Negative Control, Sequencing, Transmission Assay, Electron Microscopy

Journal: Molecular Therapy. Nucleic Acids

Article Title: Mitochondrial Damage Mediated by miR-1 Overexpression in Cancer Stem Cells

doi: 10.1016/j.omtn.2019.10.016

Figure Lengend Snippet: The Effects of miR-1-Protein Interactions on Mitochondrial Damage (A) The co-localization of LRPPRC protein (red) with MitoTracker (green). Melanoma stem cells were incubated with MitoTracker and fixed, followed by labeling with LRPPRC-specific antibody. Nuclei were stained with DAPI. Scale bar, 10 μm. (B) The detection of miR-1 existed in mitochondria. Mitochondria were isolated from cancer stem cells using ultracentrifugation (left). After RNase or/and Triton X-100 treatment, the level of miR-1 in the isolated mitochondria was investigated using quantitative real-time PCR (right) (**p < 0.01). As controls, the cytosolic β-tubulin mRNA and the mitochondrial 12S rRNA were also detected. (C) The silencing of LRPPRC in melanoma stem cells. Melanoma stem cells were transfected with LRPPRC-specific siRNA. The siRNA-scrambled was included as a control. At different times after transfection, the expression level of LRPPRC was examined with western blot using LRPPRC-specific antibody. Actin was used as a control. (D) The influence of LRPPRC silencing on mitochondrial damage. The siRNA-treated melanoma stem cells were observed under an electron microscope. Scale bars, 0.5 μm. (E) Influence of LRPPRC silencing on mitochondrial transcripts of melanoma stem cells (**p < 0.01). (F) The effects of LRPPRC silencing on the miR-1 level in mitochondria. In the presence of LRPPRC-siRNA, miR-1 was transfected into melanoma stem cells. The level of mitochondrial miR-1 was detected using quantitative real-time PCR (**p < 0.01). (G) The impact of miR-1 overexpression (miR-1 OE) on the stability of LRPPRC protein. Melanoma stem cells were transfected with miR-1. At 36 h after transfection, the mRNA level (left) and protein level (right) of LRPPRC were determined using quantitative real-time PCR and western blot, respectively.

Article Snippet: Then, the cells were incubated with 1 μM mitochondrial tracker (Yeasen Biotech, Shanghai, China) that can label mitochondria at 37°C for 30 min. After fixing in 4% paraformaldehyde for 15 min, the cells were permeabilized with 0.1% Triton X-100 for 20 min.

Techniques: Incubation, Labeling, Staining, Isolation, Real-time Polymerase Chain Reaction, Transfection, Control, Expressing, Western Blot, Microscopy, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: Mitochondrial Damage Mediated by miR-1 Overexpression in Cancer Stem Cells

doi: 10.1016/j.omtn.2019.10.016

Figure Lengend Snippet: The Rescue Effects Mediated by miR-1 Inhibition on Mitochondria (A) Expression level of miR-1 in miR-1-overexpressing melanoma stem cells. Melanoma stem cells were transfected with lentiviral particles containing miR-1. Lentiviral vector alone was used as a control. At 48 h after transfection, the expression level of miR-1 in cells was examined with quantitative real-time PCR (**p < 0.01). (B) Influence of miR-1 overexpression on the expression of target genes in melanoma stem cells. The expression levels of miR-1 target genes in miR-1-overexpressing melanoma stem cells were determined by qRT- PCR (**p < 0.01). (C) Impact of miR-1 overexpression on the tumorsphere formation capacity of melanoma stem cells. The tumorsphere formation capacity of miR-1-overexpressing melanoma stem cells was examined at different times. Scale bars, 10 μm. (D) Inhibition of miR-1 in stable miR-1-overexpressing melanoma stem cells. The miR-1 expression level was investigated using qRT-PCR after anti- miR-1 oligonuoleotide (AMO-miR-1) transfection (**p < 0.01). (E) Influence of miR-1 depletion on cell cycle of cancer stem cells. After AMO-miR-1 transfection, the cell cycle of stable miR-1-overexpressing cells (miR-1 OE) or control cells was evaluated with flow cytometry. (F) The rescued effects on mitophagy and target proteins. After AMO-miR-1 transfection, the protein levels of GPD2, MINOS1, LRPPRC, and mitophagy markers were investigated by western blot. Actin was used as a control. (G) The effects of miR-1 inhibition on the mitochondrial ultrastructure of melanoma stem cells. The stable miR-1 OE or control cells treated with specific AMO were imaged under transmission electron microscopy. Scale bars, 0.5 μm.

Article Snippet: Then, the cells were incubated with 1 μM mitochondrial tracker (Yeasen Biotech, Shanghai, China) that can label mitochondria at 37°C for 30 min. After fixing in 4% paraformaldehyde for 15 min, the cells were permeabilized with 0.1% Triton X-100 for 20 min.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Over Expression, Quantitative RT-PCR, Flow Cytometry, Western Blot, Transmission Assay, Electron Microscopy