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

Detection of miR-650 expression in LAD tissues and corresponding nontumor tissues.

<p>(<b>A</b>) Upregulation and downregulation of miR-650 expression. qRT-PCR for miR-650 was performed by using 26 human LAD tissues and corresponding adjacent normal tissues. The mean and standard deviation of expression levels relative to U6 expression levels are shown and are normalized to the expression in the normal tissue of each matched pair. RU, relative units. (<b>B</b>) The mean level of miR-650 expression in LAD tissues (n=26) was significantly higher than that in corresponding adjacent normal tissues (n=26) (<i>P</i><0.001). The data represent at least triplicate measurements from single RNA samples. (<b>C</b>) Kaplan-Meier survival analysis for LAD patients. Comparison of the overall survival (OS) between low-miR-650(n=44) and high-miR-650(n=52) patients (<i>P</i>=0.008). Comparison of the OS between patients with tumor well and moderate differentiation (n=41) and patients with tumor poor differentiation (n=55; <i>P</i>= 0.042). Comparison of the OS between patients with tumor metastasis (n=47) and patients with no tumor metastasis (n=49; <i>P</i>=0.012). Comparison of the OS between clinical stage I+II (n=53) and III+IV patients (n=43; <i>P</i>=0.011). <i>P</i><0.05 indicates a significant difference between groups. Corresponding <i>P</i> values analyzed by log-rank tests are indicated.</p

ING4 was significantly downregulated in docetaxel-non-responding LAD tissues and inversely correlated with miR-650 expression.

<p>(<b>A</b>) Immunohistochemical staining for ING4 protein in non-responding or responding LAD tissues. Isotype IgG at the same concentration was used as a negative control for immunohistochemical staining. Positive ING4 protein staining was mainly located in the nucleus of tumor cells. IN4 expression was higher in responding tumors than in nonresponding tumors. <i>Bars</i>: 100µm. (<b>B</b>) Relative expression levels of ING4 protein was detected in docetaxel-responding (n=25) and non-responding (n=19) LAD tissues via Western blot assay. GAPDH was used as an internal control. (<b>C</b>) A statistically significant inverse correlation between miR-650 and ING4 protein levels in 44 cases of LAD tissues (Spearman’s correlation analysis, r = -0.0645; <i>P</i>=0.018). Results represent the average of three independent experiments (mean±SD). Corresponding <i>P</i> values analyzed by Spearman correlation test are indicated.</p

Effects of miR-650 expression on the survival pathway in SPC-A1/DTX cells.

<p>(<b>A</b>) Flow cytometric analysis of apoptosis in anti-miR-NC or anti-miR-650-transfected SPC-A1/DTX cells. (<b>B</b>) Hoechst staining analysis of apoptosis in anti-miR-NC or anti-miR-650-transfected SPC-A1/DTX cells. (<b>C</b>) Western blot detection of the expression of pro-caspase-3 and cleaved caspase-3 proteins in anti-miR-650 or pcDNA/ING4-transfected SPC-A1/DTX cells (anti-miR-NC or pcDNA/control was used as control). (<b>D</b>) Detection of caspase-3 activity in anti-miR-650 or pcDNA/ING4-transfected SPC-A1/DTX cells (anti-miR-NC or pcDNA/control was used as control). (<b>E</b>) Western blot analysis of the expression of Bcl-2 and Bax proteins in anti-miR-650 or pcDNA/ING4-transfected SPC-A1/DTX cells (anti-miR-NC or pcDNA/control was used as control). Equal loading was confirmed by showing equal GAPDH levels. 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 or pcDNA/control-transfected cells.</p