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MicroRNA-34a inhibits the proliferation and promotes the apoptosis of non-small cell lung cancer H1299 cell line by targeting TGFβR2

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Tumor Biology

Abstract

MicroRNAs (MiRNAs) are small non-coding RNA molecules which act as important regulators of post-transcriptional gene expression by binding 3′-untranslated region (3′-UTR) of target messenger RNA (mRNA). In this study, we analyzed miRNA-34a (miR-34a) as a tumor suppressor in non-small cell lung cancer (NSCLC) H1299 cell line. The expression level of miR-34a in four different NSCLC cell lines, H1299, A549, SPCA-1, and HCC827, was significantly lower than that in the non-tumorigenic bronchial epithelium cell line BEAS-2B. In human NSCLC tissues, miR-34a expression level was also significantly decreased in pT2-4 compared with the pT1 group. Moreover, miR-34a mimic could inhibit the proliferation and triggered apoptosis in H1299 cells. Luciferase assays revealed that miR-34a inhibited TGFβR2 expression by targeting one binding site in the 3′-UTR of TGFβR2 mRNA. Quantitative real-time PCR (qRT-PCR) and Western blot assays verified that miR-34a reduced TGFβR2 expression at both mRNA and protein levels. Furthermore, downregulation of TGFβR2 by siRNA showed the same effects on the proliferation and apoptosis as miR-34a mimic in H1299 cells. Our results demonstrated that miR-34a could inhibit the proliferation and promote the apoptosis of H1299 cells partially through the downregulation of its target gene TGFβR2.

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Abbreviations

ATCC:

American Type Culture Collection

CCK-8:

Cell Counting Kit-8

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

Mad:

Mothers against decapentaplegic

miR-34a:

miRNA-34a

MiRNAs:

microRNAs

miRNA-NC:

miRNA negative control

NSCLC:

Non-small cell lung cancer

PCR:

Polymerase chain reaction

qRT-PCR:

Quantitative real-time PCR

SCLC:

Small cell lung cancer

SDS-PAGE:

Sodium dodecyl sulfate polyacryl-amide gel electrophoresis

TGFβR2:

Transforming growth factor beta receptor II

3′-UTR:

3′-untranslated region

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Acknowledgments

This work was supported in part by grants from the National Natural Science Foundation of China (31170750, 31100570), the National Basic Research Program of China (2011CBA01105), and Shanghai Municipal Commission of Economy and Information (11CH-08) and a foundation from state key laboratory of molecular biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Life Sciences, Chinese Academy of Sciences.

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Authors and Affiliations

  1. School of Life Sciences, Shanghai University, Shanghai, 200444, China

    Zhong-Liang Ma, Pin-Pin Hou, Yan-Li Li, De-Tao Wang, Tian-Wei Yuan, Jia-Li Wei, Bo-Tao Zhao & You-Xin Jin

  2. Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology Chinese Academy of Sciences, Shenzhen, 518055, China

    Yan Jin

  3. Department of Laboratory Medicine, Shanghai Chest Hospital affiliated to Shanghai Jiaotong University, Shanghai, 200030, China

    Jia-Tao Lou

  4. Shanghai Shines Pharmaceuticals Co., Ltd., Shanghai, 200032, China

    Xin-Tai Zhao

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  1. Zhong-Liang Ma
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Correspondence to Yan Jin or You-Xin Jin.

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Zhong-Liang Ma and Pin-Pin Hou contributed equally to this work.

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Fig. S1

MiR-34a level was increased or decreased in H1299 cells transfected with miR-34a mimic and pcDNA3.1-miR-34a or miR-34a inhibitor. a MiR-34a mimic transfection could significantly increase miR-34a level, while miR-34a inhibitor transfection significantly decreased miR-34a level in H1299 cells. b MiR-34a expression level was assayed after transfection with pcDNA3.1 and pcDNA3.1-miR-34a in H1299 cells. Data were performed through qRT-PCR. U6 was used as an internal control. Each assay was performed in triplicate. *** P < 0.001 (GIF 16 kb)

High resolution image (TIFF 1284 kb)

Fig. S2

Six target genes of miR-34a were verified by the luciferase assay. * P < 0.05, ** P < 0.01 and *** P < 0.001 (GIF 56 kb)

High resolution image (TIFF 3292 kb)

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Ma, ZL., Hou, PP., Li, YL. et al. MicroRNA-34a inhibits the proliferation and promotes the apoptosis of non-small cell lung cancer H1299 cell line by targeting TGFβR2. Tumor Biol. 36, 2481–2490 (2015). https://doi.org/10.1007/s13277-014-2861-5

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