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. 2017 Dec 8;8(69):113558-113570.
doi: 10.18632/oncotarget.23060. eCollection 2017 Dec 26.

miR-769-5p suppressed cell proliferation, migration and invasion by targeting TGFBR1 in non-small cell lung carcinoma

Zhao Yang  1 , Jin He  2 , Peng Gao  1 , Yi Niu  1 , Jie Zhang  3 , Lei Wang  3 , Meiyue Liu  1 , Xiaomei Wei  1 , Chunling Liu  3 , Chao Zhang  1 , Wei Wang  1 , Jiayi Du  3 , Hongmin Li  3 , Wanning Hu  1 , Guogui Sun  1
Affiliations

"VSports app下载" miR-769-5p suppressed cell proliferation, migration and invasion by targeting TGFBR1 in non-small cell lung carcinoma

Zhao Yang et al. Oncotarget. .

Abstract

MicroRNAs (miRNAs) are key regulators of multiple cancers, including non-small cell lung carcinoma (NSCLC). The aim of this study was to determine the expression pattern of miR-769-5p in NSCLC and to investigate its biological role during tumorigenesis. We showed that miR-769-5p was significantly downregulated and predicted poor prognosis in NSCLC compared with corresponding normal tissues. We then investigated its function and found that miR-769-5p significantly inhibited cell proliferation, migration and invasion in vitro and reduced tumor growth and metastasis in vivo. Furthermore, we explored the molecular mechanisms by which miR-769-5p contributes to NSCLC suppression and identified TGFBR1 as a direct target gene of miR-769-5p. Finally, we showed that TGFBR1 had opposite effects to those of miR-769-5p on lung cancer cells, suggesting that miR-769-5p might inhibit lung tumorigenesis by silencing TGFBR1 VSports手机版. Taken together, our results demonstrated that miR-769-5p plays a pivotal role in NSCLC by inhibiting cell proliferation, migration and invasion by targeting TGFBR1. .

Keywords: gene therapy; invasion and metastasis; lung cancer; microRNAs. V体育安卓版.

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Conflict of interest statement (V体育官网入口)

CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1. miR-769-5p expression in NSCLC and association with clinical factors
(A) miR-769-5p levels were measured in 70 NSCLC samples and pair-matched lung tissues and normalized against an endogenous U6 RNA control by qRT-PCR analysis; (B and C) The expression of miR-769-5p in NSCLC was associated with clinical stages (B) and lymph node metastasis (C); (D) Kaplan–Meier curves depicting overall survival according to the expression of miR-769-5p. **p < 0.01.
Figure 2
Figure 2. Elevated miR-769-5p inhibits cell proliferation, colony formation and migration
(A) RNA level of miR-769-5p in 5 NSCLC cell lines; (B and C) Quantitative analysis of miR-769-5p level after the transfection of miR-769-5p mimic in A549 and H157 cell lines; (D and E) Cell growth curve was measured by MTS after the transfection of miR-769-5p mimic in A549 and H157 cell lines, and the OD 570 values were normalized to the start point (0 hour); (F and G) Representative images and quantitative analysis of colony formation was performed after the transfection of miR-769-5p mimic in A549 and H157 cell lines; (H and I) Representative images and quantitative analysis of transwell assays was performed after the transfection of miR-769-5p mimic in A549 and H157 cell lines. Data are presented as the mean value ± SD from triplicate experiments. *p < 0.05; **p < 0.01.
Figure 3
Figure 3
Repression of miR-769-5p in A973 and GLC82 cells significantly promoted cell growth, colony formation and migration; (A and B) Quantitative analysis of miR-769-5p level after the transfection of miR-769-5p inhibitor in A973 and GLC82 cell lines; (C and D) Cell growth curve was measured by MTS after the transfection of miR-769-5p inhibitor in A973 and GLC82 cell lines, and the OD 570 values were normalized to the start point (0 hour); (E and F) Representative images and quantitative analysis of colony formation was performed after the transfection of miR-769-5p inhibitor in A973 and GLC82 cell lines; (G and H) Representative images and quantitative analysis of transwell assays was performed after the transfection of miR-769-5p inhibitor in A973 and GLC82 cell lines. Data are presented as the mean value ± SD from triplicate experiments. *p < 0.05; **p < 0.01.
Figure 4
Figure 4. TGFBR1 is a direct target gene of miR-769-5p
(A) TGFBR1 was identified as potential regulatory target of miR-769-5p by analysis of down-regulated genes using prediction tools; (B–D) The expression levels of the TGFBR1 mRNA and protein were measured by qRT-PCR and western blot analysis using GAPDH as the loading control after transfection of miR-769-5p mimic in A549 and H157 cell lines; (E–G) The expression levels of the TGFBR1 mRNA and protein were measured by qRT-PCR and western blot analysis using GAPDH as the loading control after transfection of miR-769-5p inhibitors in A973 and GLC82 cell lines; (H) Dual-luciferase reporter assay. The relative luciferase activity was normalized to the Renilla luciferase activity after co-transfection of cells with miR-769-5p mimic and pmiR-RB-REPORT™ construct containing the WT or MUT TGFBR1 3′-UTR region in A549 and H157 cell lines. Data are presented as the mean value ± SD from triplicate experiments. **p < 0.01.
Figure 5
Figure 5. Rescue assays were further performed to confirm that TGFBR1 is a functional target of miR-769-5p
(A and B) The mRNA and protein level of TGFBR1 in A549 and H157 cell lines with miR-769-5p mimic and pEGFP-C1 plasmid containing TGFBR1 CDS sequence. (C and D) Transwell assays of co-transfected cells with miR-769-5p mimic and TGFBR1 plasmid. Data are presented as the mean value ± SD from triplicate experiments. **p < 0.01.
Figure 6
Figure 6. miR-769-5p suppressed tumor growth and metastasis in vivo and targeted TGFBR1
(A–C) The A973 cells were subcutaneously injected into nude mice to form solid and metastatic tumors and synchronously treated with miR-769-5p antagomir or miR antagomir NC (n = 5 for each group); (D) The number of metastatic nodules was observed and quantified in the lungs of mice treated with miR-769-5p antagomir or miR antagomir NC by the vein injection method. (E) Immunohistochemical staining of Ki67, PCNA and TGFBR1 in tumor tissues dissected from nude mice treated with miR-769-5p antagomir miR or antagomir NC. (F) Representative IHC images of TGFBR1 in NSCLCtissues and paired normal lung tissues are shown. (G) Spearman correlation analysis of the negative correlation between the expression of miR-769-5p and TGFBR1. *p < 0.05; **p < 0.01.
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