Properties of CSCs obtained from docetaxel-resistant human lung adenocarcinoma cells (SPC-A1/DTX and H1299/DTX).

<p>(A₁, A₂, A₃) Percentage of CD133⁺/CD326⁺ CSCs as analyzed by Flow cytometry at the indicated time after cultivating CD133⁺/CD326⁺ CSCs (acquired by sorting SPC-A1/DTX and H1299/DTX cells, respectively) under differentiation conditions. (B) Mammosphere forming ability of the indicated cells (1000 cells) after 7 days under CSC-cultivating conditions. (C) qRT-PCR detection of relative mRNA levels of the CSC-related markers in the indicated cells. Data was calculated with the 2^−△△Ct method using GAPDH RNA as the reference gene. The expression level was measured relative to the fold change of the parental cells groups that were defined as 1.0. (D₁, D₂) The protein levels of the CSC-related markers in the indicated cells. GAPDH was used as an internal control. (E) Tumor incidence in nude mice that were subcutaneously injected with the indicated cells. Data were presented as mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

Identification of Suz-12 as a functional target of miR-200b in CSCs.

<p>(<b>A</b>) qRT-PCR detection of relative Suz-12 mRNA expression in the indicated cells. Data were normalized to GAPDH RNA and determined relative to the corresponding parental-cell groups. (<b>B</b>) The protein levels of Suz-12 as measured by Western blotting in the indicated cells. GAPDH was used as an internal control. (<b>C</b>) qRT-PCR detection of Suz-12 mRNA expression in CSCs after transfection of the indicated vectors. (<b>D</b>) Western blotting detection of Suz-12 protein expression in CSCs after transfection with the indicated vectors. GAPDH was used as an internal control. (<b>E</b>) Luciferase activity of the CSCs (SPC-A1/DTX) co-transfected with pcDNA/miR-200b (or pcDNA/miR-NC) or pLUC/Suz-12/3′-UTR-wt (or pLUC/Suz-12/3′-UTR-mut). Data were mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

Silencing of Suz-12 inhibits CSCs growth and tumorigenicity and reverses chemoresistance of CSCs.

<p>(<b>A</b>) The protein level of Suz-12 in docetaxel-resistant LAD cells after transfection with the indicated sh-RNA vectors (sh-RNA-Suz12#1, sh-RNA-Suz12#2, and sh-RNA-Suz12#3). (<b>B</b>) Mammosphere forming ability of docetaxel-resistant LAD cells (1000 cells) after transfection of the indicated vectors. (<b>C</b>) Flow cytometry analysis of percentage of CD133⁺/CD326⁺ CSCs as analyzed by in docetaxel-resistant LAD cells after transfection of the indicated vectors. (<b>D</b>) The protein level of Suz-12 in CSCs after transfection with the sh-RNA-control or sh-RNA-Suz12#3 vectors. (<b>E</b>) CCK-8 analysis of cell viability of the CSCs transfected with the indicated vectors at the indicated time. (<b>F</b>) Effect of Suz-12 inhibition on <i>in vivo</i> tumorigenicity of the CSCs from SPC-A1/DTX cells. (<b>G</b>) Effect of Suz-12 inhibition on chemoresistance of CSCs. Cell viability was measured by CCK-8 assay. GAPDH was used as an internal control. Data were presented as mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

MiR-200b has effects on CSCs by regulating Suz-12/E-cadherin.

<p>(<b>A</b>) Western blotting detection of E-cadherin protein expression in the indicated cells transfected with the indicated vectors. (<b>B</b>) qRT-PCR detection of E-cadherin mRNA expression as measured by in the CSCs transfected with the indicated vectors. (<b>C</b>) Western blotting detection of E-cadherin protein expression in the CSCs transfected with the indicated vectors. (<b>D</b>) and (<b>E</b>) Suz-12 overexpression rescued the increased level of both mRNA and protein expression of E-cadherin in CSCs (SPC-A1/DTX) mediated by miR-200b upregulation, while silencing of Suz-12 rescued the decreased expression of mRNA and protein of E-cadherin in CSCs (SPC-A1/DTX) induced by miR-200b repression. (<b>F</b>) qRT-PCR detection of miR-200b and Suz-12 mRNA expression at the indicated time after plating CD133⁺/CD326⁺ CSCs under differentiation conditions. (<b>G₁, G₂</b>) The protein levels of Suz-12 and E-cadherin at the indicated time after cultivating CD133⁺/CD326⁺ CSCs under differentiation conditions. (<b>H</b>) MiR-200b upregulation decreased the amount of Suz-12 binding to the promoter of E-cadherin in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. (<b>I</b>) MiR-200b overexpression reduced trimethylation of histone H3-lysine-27 (H3-K27) at E-cadherin promoter in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. GAPDH was used as an internal control. Data were presented as mean ± SD of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01; ^#<i>p</i><0.05, ^##<i>p</i><0.01, compared with corresponding 0 day groups.</p

HDAC1 repression or miR-200b overexpression reduces tumorigenicity and reverses chemoresistance of CSCs <i>in vivo</i>.

<p>(<b>A</b>) Tumorigenicity in nude mice subcutaneously injected with CSCs from SPC-A1/DTX cells (5000 cells/mouse, n = 5) that were stably transfected with the indicated vectors previously. (<b>B</b>) Tumor volume in nude mice injected with H1299/DTX cells that were stably transfected with the indicated vectors and were combined with DTX treatment. Data were presented as mean ± SD. (<b>C</b>) The protein level of E-cadherin and Suz-12 in tumors of the indicated groups that were provided at 5 weeks after the inoculation. GAPDH was used as an internal control. (<b>D</b>) Flow cytometry detection of percentage of CD133⁺/CD326⁺ CSCs in tumors of the indicated groups. (<b>E</b>) The mRNA level of miR-200b and Suz-12 in tumors of the indicated groups. Data were normalized to U6 RNA and GAPDH, respectively. Data were presented as mean ± SD of at least three independent experiments. **<i>p</i><0.01.</p

MicroRNA-650 Was a Prognostic Factor in Human Lung Adenocarcinoma and Confers the Docetaxel Chemoresistance of Lung Adenocarcinoma Cells via Regulating Bcl-2/Bax Expression

<div><p>Increasing evidence shows that dysregulation of microRNAs (miRNAs) is involved in malignant transformation. We investigated the clinical significance of miR-650 and its involvement in chemoresistance to docetaxel. Our results showed that the relative expression level of miR-650 was significantly higher in LAD tissues than in corresponding nontumor tissues and high level of miR-650 expression was found to be significantly associated with high incidence of lymph node metastasis, advanced clinical stage and poor prognosis of LAD patients. Univariate and multivariate analyses indicated that high miR-650 expression was an independent prognostic factor for survival. Also, we found that the level of miR-650 in LAD tissues was correlated with the response of patients to docetaxel-based chemotherapy. Silencing of miR-650 could increase the in vitro sensitivity of docetaxel-resistant LAD cells to docetaxel, while upregulation of miR-650 decreased the sensitivity of parental LAD cells to docetaxel both <i>in vitro</i> and <i>in vivo</i>. Additionally, silencing of miR-650 could enhance the caspase-3-dependent apoptosis, which might be correlated with the decreased ratio of Bcl-2/Bax. Further researches suggested that inhibitor of growth 4 (ING4) was a direct target of miR-650. Downregulated or upregulated ING4 expression could partially rescue the effects of miR-650 inhibitor or mimics in docetaxel-resistant or parental LAD cells. Furthermore, we found that ING4 was upregulated in docetaxel-responding LAD tissues, and its expression was inversely correlated with miR-650. Thus, miR-650 is a novel prognostic marker in LAD and its expression is a potential indicator of chemosensitivity to docetaxel-based chemotherapy regimen.</p> </div

siRNA/ING4 or pcDNA/ING4 could partially rescue the effects of anti-miR-650 or miR-650 mimics on the sensitivity of SPC-A1/DTX or SPC-A1 cells to docetaxel.

<p>(<b>A</b>) Western blot analysis of ING4 protein expression in anti-miR-NC or anti-miR650-transfected SPC-A1/DTX cells or SPC-A1/DTX cells co-transfected with anti-miR650 and siRNA/NC or siRNA/ING4. (<b>B</b>) MTT analysis of growth in anti-miR-NC or anti-miR650-transfected SPC-A1/DTX cells or SPC-A1/DTX cells co-transfected with anti-miR650 and siRNA/NC or siRNA/ING4. *<i>P</i><0.05, in comparison with anti-miR-650-transfected cells or cells co-transfected with anti-miR650 and siRNA/NC. (<b>C</b>) Analysis of the IC₅₀ values of docetaxel in anti-miR-NC or anti-miR650-transfected SPC-A1/DTX cells or SPC-A1/DTX cells co-transfected with anti-miR650 and siRNA/NC or siRNA/ING4. (<b>D</b>) Western Blot analysis of ING4 protein expression in miR-NC or miR-650 mimics-transfected SPC-A1 cells or SPC-A1 cells co-transfected with miR-650 mimics and pcDNA/control or pcDNA/ING4. (<b>E</b>) MTT analysis of growth in miR-NC or miR-650 mimics-transfected SPC-A1 cells or SPC-A1 cells co-transfected with miR-650 mimics and pcDNA/control or pcDNA/ING4. *<i>P</i><0.05, in comparison with miR-650 mimics-transfected cells or cells co-transfected with miR-650 mimics and pcDNA/control. (<b>F</b>) Analysis of the IC₅₀ values of docetaxel in miR-NC or miR-650 mimics-transfected SPC-A1 cells or SPC-A1 cells co-transfected with miR-650 mimics and pcDNA/control or pcDNA/ING4 using MTT assay. The data are expressed as the mean value ± SEM of the results obtained from three independent experiments.</p

Effect of anti-miR-650 on the in vitro sensitivity of docetaxel-resistant LAD cells to docetaxel.

<p>(<b>A</b>) 48h after SPC-A1/DTX or H1299/DTX cells were transfected with anti-miR-650 (or anti-miR-NC), qRT-PCR assay was performed to detect the expression of miR-650. (<b>B</b>) MTT analysis of growth in anti-miR-650 or anti-miR-NC-transfected SPC-A1/DTX or H1299/DTX cells. (<b>C</b>) Representative results of colony formation of SPC-A1/DTX or H1299/DTX cells transfected with anti-miR-650 or anti-miR-NC. (<b>D</b>) Analysis of the IC₅₀ values of docetaxel in SPC-A1/DTX or H1299/DTX cells transfected with anti-miR-NC or anti-miR-650 using MTT assays. Results represent the average of three independent experiments (mean±SD). *<i>P</i><0.05 or **<i>P</i><0.01, in comparison with anti-miR-NC-transfected cells.</p

Correlation between miR-650 expression and the responses of LAD patients to docetaxel.

<p>(<b>A</b>) Relative expression levels of miR-650 was detected in docetaxel-sensitive (n=25) and insensitive (n=19) LAD tissues via qRT-PCR. (<b>B</b>) Kaplan-Meier survival curve indicates that patients with high miR-650 expression have shorter progress-free survival (PFS) than those with low miR-650 expression (log-rank test, <i>P</i>=0.0024). (<b>C</b>) Detection of miR-650 expression in docetaxel-resistant LAD cells (SPC-A1/DTX and H1299/DTX) and parental LAD cells (SPC-A1 and H1299) via qRT-PCR. (<b>D</b>) Analysis of the expression of miR-650 in docetaxel-resistant or parental LAD cells after treatment with docetaxel (5.0 µg/L) via qRT-PCR. Abundance of miRNA was normalized to U6 RNA. Each experiment was performed at least in triplicate.</p

Effect of miR-650 mimics on the in vitro sensitivity of parental LAD cells to docetaxel.

<p>(<b>A</b>) 48h after SPC-A1 or H1299 cells were transfected with miR-650 mimics or miR-NC mimics, qRT-PCR assay was performed to detect the expression of miR-650. (<b>B</b>) MTT analysis of growth in SPC-A1 or H1299 cells transfected with miR-650 mimics or miR-NC mimics. (<b>C</b>) Representative results of colony formation of SPC-A1 or H1299 cells transfected with miR-650 mimics or miR-NC mimics. (<b>D</b>) Analysis of the IC₅₀ values of docetaxel in SPC-A1 or H1299 cells transfected with miR-650 mimics or miR-NC mimics using MTT assay. Results represent the average of three independent experiments (mean±SD). *<i>P</i><0.05 or **<i>P</i><0.01, in comparison with anti-miR-NC-transfected cells.</p