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. 2014 Oct 6:4:6527.
doi: 10.1038/srep06527.

Reduced miR-3127-5p expression promotes NSCLC proliferation/invasion and contributes to dasatinib sensitivity via the c-Abl/Ras/ERK pathway

Yifeng Sun  1 , Chang Chen  2 , Peng Zhang  2 , Huikang Xie  3 , Likun Hou  4 , Zheng Hui  5 , Yongjie Xu  5 , Qiaoling Du  6 , Xiao Zhou  5 , Bo Su  3 , Wen Gao  7
Affiliations

Reduced miR-3127-5p expression promotes NSCLC proliferation/invasion and contributes to dasatinib sensitivity via the c-Abl/Ras/ERK pathway

Yifeng Sun et al. Sci Rep. .

"V体育2025版" Abstract

miR-3127-5p is a primate-specific miRNA which is down-regulated in recurrent NSCLC tissue vs. matched primary tumor tissue (N = 15) and in tumor tissue vs. normal lung tissue (N = 177). Reduced miR-3127-5p expression is associated with a higher Ki-67 proliferation index and unfavorable prognosis in NSCLC. Overexpression of miR-3127-5p significantly reduced NSCLC cells proliferation, migration, and motility in vitro and in vivo. The oncogene ABL1 was a direct miR-3127-5p target, and miR-3127-5p regulated the activation of the Abl/Ras/ERK pathway and transactivated downstream proliferation/metastasis-associated molecules. Overexpression of miR-3127-5p in A549 or H292 cells resulted in enhanced resistance to dasatinib, an Abl/src tyrosine kinase inhibitor. miR-3127-5p expression levels were correlated with dasatinib sensitivity in NSCLC cell lines without K-Ras G12 mutation. In conclusion, miR-3127-5p acts as a tumor suppressor gene and is a potential biomarker for dasatinib sensitivity in the non-mutated Ras subset of NSCLC. VSports手机版.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. miR-3127-5p expression is down-regulated in NSCLC tumor tissues, and low miR-3127-5p levels correlate with unfavorable prognosis in patients with NSCLC.
(A): miRNA expression in primary tumor tissues and tissues from relapse tumors were evaluated by microarray analysis. (N = 3, P: primary tumor; R: recurrent tumor, miRNAs with log2 R/P<-0.8 or log2R/P>0.8 were shown. miR-3127-5p, miR-3940-5p, miR-4294 and miR-4447 for validation were indicated). (B): The expression of miR-3127-5p was significantly down-regulated in relapsed tumor tissue compared to paired primary tumor tissue. Paired Student's t-test (N = 15). (C): The expression of miR-3127-5p in normal, tumor-adjacent, and tumor tissues of 177 matched NSCLC samples. N: normal; TN: tumor adjacent normal; T: tumor. Data were analyzed using Wilcoxon Mann Whitney U test; middle line: median; upper and bottom line: min and max; upper and bottom edge of box: 25% and 75% IQR. (D): Reduced miR-3127-5p expression is associated with shorter overall survival in patients with NSCLC (Low: n = 59; Middle: n = 58; High: n = 58; log-rank test; 2YS: 2-year overall survival). (E): Left panel: miR-3127-5p expression was significantly down-regulated in Ki-67-positive NSCLC tumors (N = 177, Wilcoxon Mann Whitney U test). Right panel: Representative photographs of Ki-67 IHC positive and negative NSCLC tissues (ADC: adenocarcinoma; SQC: squamous carcinoma; AD-SQC: adeno-squamous carcinoma; Numbers in parentheses: -ΔΔCT for miR-3127-5p).
Figure 2
Figure 2. miR-3127-5p affects proliferation, migration, and motility of A549 and H292 cells.
(A) and (B): Left panel: Q-PCR determination of miR-3127-5p expression in A549 cells and H292 cells stably transfected with Lv-GFP, Lv-3127, or Lv-3127-off. Right panel: Growth curves of (A) A549 cells and (B) H292 cells stably transduced with control Lv-GFP; Lv-3127, a vector for overexpression of miR-3127-5p; or Lv-3127-off, a vector for expression of an miR-3127-5p inhibitor. Cells (1000/well) were cultured in a 96-well plate, and cell growth was monitored every 24 h for 7 days using a CCK-8 assay, each type of cell was analyzed in quadruplicate (p < 0.01 for A549 cells and H292 cells, repeated measure ANOVA). (C): Cell cycle analysis. The percentage of cells in G0/G1 phase increased and that in S phase decreased in Lv-3127 transduced A549 (left panel) and H292 (right panel) cells; the opposite was observed in Lv-3127-off transduced cells. (D): miR-3127-5p overexpression reduced cell motility, and miR-3127-5p knockdown increased cell motility in the wound scratch assay in both A549 (left panel) and H292 (right panel) cells. A uniform scratch was made in each confluent layer culture; the extent of the wound closure was monitored under a phase-contrast microscope, and photographs were taken at 0, 12, and 24 h. Triplicate experimentation generated similar results. (E): A549 (upper panels) and H292 (lower panels) cells were loaded onto the top well of the transwell inserts in triplicate for cell migration assay. Photographs are representative of cells that had migrated to the bottom chamber after 48 h. Cells were stained with hematoxylin and observed under a microscope (×200). Invasion was quantified by determining the total number of cells that had migrated through the membrane.
Figure 3
Figure 3. miR-3127-5p knockdown promotes the tumorigenicity and metastasis in mouse xenograft model.
(A): Growth curve of subcutaneous tumors from A549 (upper panels) or H292 (bottom panels) cells transduced with Lv-3127, Lv-3127-off, or Lv-GFP in the first 5 weeks after inoculation. (B): Xenografted tumors were observed using a whole-body fluorescent imaging system at 30 days after inoculation. (C): After 13 weeks, animals were sacrificed, and the number of visible neoplastic nodules (metastases) on each lung was counted. Representative photographs of the whole lungs and HE staining for lung metastasis lesion sections (100×) are shown. (D): Representative photographs of c-Abl, Ki-67, cyclin D1, MMP-9, and VEGF IHC of A549 or H292 xenograft tumors (200×).
Figure 4
Figure 4. Oncogene c-Abl is a direct target of miR-3127-5p.
(A): Two sites in the ABL1 3′-UTR are complementary to miR-3127-5p as analyzed by Targetscan. (B): 293T cells were co-transfected with pmiR-RB-Report-ABL1_3′UTR or pmiR-ABL1_3′UTR-Mut plasmids and pL-GFP, pL-miR-3127-5p, or pL-miR-3127-5p-off plasmids. A Renilla-TK luciferase reporter (5 ng) was added as an internal control. The luciferase activity of the cells was determined after 48 h. (C): A549 or H292 cells transduced with Lv-3127, Lv-3127-off, or Lv-GFP were subjected to immunoblotting for c-Abl (The full-length blots were presented in the supplementary Figure S4). (D): H292-3127 was transducted with pGV142-CMV-ABL1 and control vectors, the cell cycle analysis and immunoblotting of c-abl were performed at 36hrs after eletroporation. (E): Wound scratch assay (left panel) and transwell invasion assay (right panel) of H292-3127 transduced with pGV142-CMV-ABL1 (Magnification, ×200). Triplicate experiments were done. (F): Left panels: Representative photographs of c-Abl IHC staining grades in NSCLC tumor samples (200×). Right panels: miR-3127-5p expression was low in c-Abl IHC positive staining samples (mean ± SEM, ANOVA).
Figure 5
Figure 5. miR-3127-5p regulates c-Abl/Ras/ERK pathway activation by targeting oncogene c-Abl.
(A): A549 or H292 cells transduced with Lv-3127, Lv-3127-off, or Lv-GFP were lysed. Lysates (1.0 mg) were incubated with 50 μL of GST-Raf1-RBD beads for 30 min to pull down active Ras. Samples were immunoblotted using an anti-K-Ras antibody, thereby allowing levels of GTP-bound Ras within the cells to be assessed. Whole cell lysates (50 µg) were also subjected to western blot for detection of total K-Ras. The numbers under bands indicate the ratio of integrated optical density to GAPDH normalized by GFP control (The full-length blots were presented in the supplementary Figure S5). (B): A549 or H292 cells transduced with Lv-3127, Lv-3127-off, or Lv-GFP were subjected to immunoblotting with antibodies to ERK, p-ERK, and downstream factors MMP-9, VEGF, and cyclin D1 (The full-length blots were presented in the supplementary Figure S4, 5). (C): Schematic diagram illustrating the effect of reduced miR-3127-5p levels on activation of c-Abl/Ras/ERK pathway and promotion of tumor proliferation and metastasis.
Figure 6
Figure 6. between expression of miR-3127-5p and response to dasatinib in NSCLC cell lines.
(A): Dasatinib sensitivity of A549 (left) and H292 (right) cells transduced with Lv-3127, Lv-3127-off, or Lv-GFP as determined in a CCK8 cell viability assay. (p < 0.01, repeated measure ANOVA). (B): miR-3127-5p expression in the 14 NSCLC cell lines as determined using an miRNA poly (A) tailing-based Sybr Green Q-PCR assay. (C): Immunoblotting of c-Abl protein for NSCLC cell lines. The number under each band refers to the integrated band density (The full-length blots were presented in the supplementary Figure S6). (D): Negative relationship between c-Abl protein and miR-3127-5p expression in the NSCLC cell lines without K-Ras condon 12 mutation (Pearson's correlation, p < 0.01, r2 = 0.443). (E): IC50 values for dasatinib in NSCLC cell lines determined by CCK8 cell viability assay. (F): miR-3127-5p expression level was linearly associated with dasatinib IC50 for the 11 NSCLC cell lines after exclusion of the three cell lines with a K-Ras codon 12 mutation (K-Ras mutation status: H23(G12C), H358(G12C), A549(G12S), H460(Q61H), SPC-A1(Q61H). Pearson's correlation, p < 0.01, r2 = 0.895).
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