Figure 1.

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.