mir-93-negative SUM159 cells have increased tumor-initiating capacity.

<p>A. ALDH⁺ cells from SUM159 cells shows lower mir-93 expression level in comparison to ALDH⁻ cells as accessed by qRT-PCR. P<0.05; Error bars represent mean ± STDEV. B. A schematic of mir-93-Sensor-GFP lentiviral construct; C. SUM159 cells were transduced with the mir-93-sensor-GFP lentivirus and selected with hygromycin B, and cells were sorted based on the GFP expression. A serial dilution of mir-93-negative (sensor/GFP-positive) SUM159 cells and mir-93-positive (sensor/GFP-negative) SUM159 cells were injected into the 4^th fatpads of NOD/SCID mouse. *p<0.05. D. mir-93-negative cells gave rise to tumors containing both mir-93-negative and mir-93-positive cell populations, but mir-93-positive cells only gave rise to tumors containing mir-93-positive cell populations.</p

mir-93 promotes tumor growth by increasing CSCs in MCF7 cells.

<p>A. Mir-93 is expressed equally in CD24⁻CD44⁺ and bulk (non-CD24⁻CD44⁺) populations of MCF7 cells. B. 1×10⁶ pTRIPZ-MCF7-mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. DOX alone, docetaxel alone or the combination increased the CD24⁻CD44⁺ population <i>in vitro</i>. C. 1000k pTRIPZ-MCF7-mir-93 cells were injected into the 4^th fatpads of NOD/SCID mice. Treatment was initiated as indicated by the red arrow. DOX alone (1 mg/ml in drinking water) promoted MCF7 tumor growth in vivo; docetaxel (10 mg/kg i.p. once weekly) alone or the combination inhibits MCF7 tumor growth in vivo. D. Tumors from each group were collected. Analysis for CD24 and CD44 was performed on dissociated cells. DOX alone, docetaxel alone, or the combination increased the CD24⁻CD44⁺ populations in MCF7. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4^th fatpads of secondary mice, which received no further treatment. Cells from DOX-, docetaxel-, or combination-treated tumors formed secondary tumors at all dilutions (50000, 5000, 500), whereas only higher numbers of cells (50000, 5000) obtained from control xenografts were able to generate tumors. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that tumor growth is significantly different between the control group and the group with the same colored curve.</p

mir-93 initiates MET in SUM159 cells.

<p>A. pTRIPZ-SUM159-mir-93 cells were plated in 2-well chamber slides with (DOX) or without (CTRL) Doxycycline for 7 days. E-Cadherin and Vimentin were deleted by immunofluorescence staining. Expression of mir-93 in SUM159 cells causes them to assume a more epithelial appearance associated with a decrease in Vimentin and an increase in membrane localized E-Cadherin expression. The phase micrographs for CTRL and DOX are also shown. E-Cadherin, Green; Vimentin, Red; DAPI, Blue. A representative sample from 3 independent samples is shown. B. The effect of mir-93 expression on a panel of epithelial and mesenchymal markers at the mRNA level as accessed by qPCR. pTRIPZ-SUM159-mir-93 cells were plated with or without DOX, and ALDH⁺ and ALDH⁻ cells were sorted at different times (12 hours, 1 day, 3 days, 8 days, 15 days) by Aldefluor assay. qRT-PCR was utilized to access the effects of mir-93 on mRNA expression of mesenchymal markers (Vimentin, N-Cadherin and Twist), epithelial markers (E-Cadherin and Claudin), and TGFβR2. *p<0.05; Error bars represent mean ± STDEV.</p

mir-93 regulates the cell cycle in SUM159 cells.

<p>A. SUM159 cells were stained with Aldefluor and Hoechst33342 and dead cells excluded by 7-AAD staining. Cells from G0/G1 and S/G2/M were sorted from ALDH⁺ or ALDH⁻ populations and mir-93 expression was measured with qRT-PCR. B. Cell cycle analysis of pTRIPZ-SUM159-mir-93 cells in the presence or absence of DOX for 7 days. Propidium iodide staining followed by flow cytometry was used to analyze cell cycle distribution. mir-93 induction with DOX resulted in a decreased proportion of cells in G0/G1 and an increased proportion of cells in S/G2/M. *p<0.05; Error bars represent mean ± STDEV.</p

mir-93 inhibits tumor growth and metastasis by decreasing CSCs in SUM159 cells.

<p>A. SUM159 cells were transduced with the pTRIPZ-mir-93 lentivirus and selected with Puromycin for 7 days. Tetracycline (DOX) induces mir-93 expression in suspension-cultured SUM159 cells by 10 hours; B. 1×10⁶ SUM159 cells or pTRIPZ-SUM159 -mir-93 cells were plated in T75 flasks and, after overnight, the cells were treated with Vehicle control, with (DOX) or without (CTRL) DOX (1 ug/ml), docetaxel (10 nM) or the combination for 7 days. Cells were utilized for Aldefluor assay and stained for Annexin V-APC and DAPI for apoptosis assay. C. 100 k pTRIPZ-SUM159-mir-93 cells were injected into the 4^th fatpads of NOD/SCID mice. The treatment started as indicated by the red arrow. DOX alone (1 mg/ml in drinking water), or docetaxel (10 mg/kg i.p. once weekly) alone, or the combination inhibits SUM159 tumor growth in vivo (note: The Y-axis is on a logarithmic scale). D. Tumors from each group were collected. ALDH was accessed by the Aldefluor assay on viable dissociated cells and by ALDH1 immunohistochemistry on fixed sections. E. Serial dilutions of cells obtained from these xenografts were implanted in the 4^th fatpads of secondary mice, which received no further treatment. F. 10k pTRIPZ-SUM159-mir-93 cells were injected into the 4^th fatpads of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. G. 200k pTRIPZ-SUM159-mir-93-Luc cells in 100 ul of PBS were injected into the left ventricle of NOD/SCID mice. The treatment started immediately after injection as indicated by the red arrow and stopped as indicated by the green arrow. Metastasis formation was monitored using bioluminescence imaging. Quantification of the normalized photon flux, measured at weekly intervals following inoculation. *p<0.05; Error bars represent mean ± STDEV. The colored “*” on the side of the tumor growth curve indicates that the tumor growth or metastasis is significantly different between the control group and the group with the same colored curve.</p

mir-93 targets stem cell regulatory genes.

<p>A. Schematic representation of the experimental design to identify the direct targets of mir-93 in SUM159 cells. B. Activity of the luciferase gene linked to the 3′UTR of AKT3, SOX4, or STAT3. The pMIR-REPORT firefly luciferase reporter plasmids with the wild-type 3′UTR sequences of AKT3, SOX4, or STAT3 were transiently transfected into pTRIPZ-mir-93-SUM159 cells and an internal control ACTB luciferase reporter was co-transfected for normalization. The cells were treated with or without DOX. Luciferase activities were measured after 48 hr. The relative luciferase activity was calculated as the ratio of (the results from the cells transfected by individual reporter)/(the results from the cells transfected by the internal control in the same cell group). The data are mean and standard deviation (SD) of separate transfections (n = 4). *p<0.05; Error bars represent mean ± STDEV.</p