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. 2019 Apr 13;9(9):2460-2474.
doi: 10.7150/thno.31097. eCollection 2019.

HCP5 is a SMAD3-responsive long non-coding RNA that promotes lung adenocarcinoma metastasis via miR-203/SNAI axis

Lin Jiang  1   2 , Ranran Wang  3 , Li Fang  4 , Xiaolu Ge  1   2 , Lingna Chen  1   2 , Ming Zhou  1   2 , Yanhong Zhou  1   2 , Wei Xiong  1   2 , Yerong Hu  3 , Xianming Tang  3 , Guiyuan Li  1   2 , Zheng Li  1   2   5
Affiliations

HCP5 is a SMAD3-responsive long non-coding RNA that promotes lung adenocarcinoma metastasis via miR-203/SNAI axis

Lin Jiang et al. Theranostics. .

Abstract

Introduction: Transforming growth factor-beta (TGFβ) signaling plays a vital role in lung adenocarcinoma (LUAD) progression. However, the involvement of TGFβ-regulated long non-coding RNAs (lncRNAs) in metastasis of LUAD remains poorly understood. Methods: We performed bioinformatic analyses to identify putative lncRNAs regulated by TGF-β/SMAD3 and validated the results by quantitative PCR in LUAD cells. We performed luciferase reporter and chromatin immunoprecipitation assays to demonstrate the transcriptional regulation of the lncRNA histocompatibility leukocyte antigen complex P5 (HCP5) we decided to focus on. Stable HCP5 knockdown and HCP5-overexpressing A549 cell variants were generated respectively, to study HCP5 function and understand its mechanism of action. We also confirmed our findings in mouse xenografts and metastasis models. We analyzed the correlation between the level of lncRNA expression with EGFR, KRAS mutations, smoke state and prognostic of LUAD patients. Results: We found that the lncRNA HCP5 is induced by TGFβ and transcriptionally regulated by SMAD3, which promotes LUAD tumor growth and metastasis. Moreover, HCP5 is overexpressed in tumor tissues of patients with LUAD, specifically in patients with EGFR and KRAS mutations and current smoker. HCP5 high expression level is positively correlated with poor prognosis of patients with LUAD. Finally, we demonstrated that upregulation of HCP5 increases the expression of Snail and Slug by sponging the microRNA-203 (miR-203) and promoting epithelial-mesenchymal transition (EMT) in LUAD cells. Conclusions: Our work demonstrates that the lncRNA HCP5 is transcriptionally regulated by SMAD3 and acts as a new regulator in the TGFβ/SMAD signaling pathway. Therefore, HCP5 can serve as a potential therapeutic target in LUAD.

Keywords: HCP5; Long non-coding RNA; SMAD3; lung adenocarcinoma; metastasis..

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
HCP5 is a TGFβ/SMAD3 induced lncRNA. (A) A549 cells were treated with or without 10 μM SIS3 for 30 min and subsequently treated with or without 5 ng/mL TGF-β for 1 h. The levels of E-cadherin, N-cadherin, SMAD3 and p-SMAD3 were determined by western blotting. (B) The levels of ten lncRNA were detected by qRT-PCR in A549 cells treated as indicated above. The three lncRNAs with the most obvious changes are in a red frame. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (C-E) HCP5 (C), HAGLR (D) and MIR181A2HG (E) expression in human LUAD tissues and normal lung tissues analyzed in the GSE31210 dataset. (F-H) The expression level of HCP5, HAGLR and MIR181A2HG was verified by qRT-PCR in four LUAD lines, human bronchial epithelial (HBE) and human renal epithelial cell line 293 (HEK293). (I) The relative expression of HCP5 in 30 paired LUAD tissues and adjacent lung tissues. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (J) Kaplan-Meier analysis of the overall survival in patients with LUAD based on the levels of HCP5 expression using the GSE19188 dataset (p < 0.05). (K) HCP5 expression in EGFR and KRAS mutations and EML4-ALK rearrangement LUAD patients was analyzed in GSE31210 dataset. *P < 0.05, **P < 0.01, ***P< 0.001, NS: no statistical significance.
Figure 2
Figure 2
HCP5 is a direct transcriptional target of SMAD3. (A, B) The expression of HCP5 was detected by qRT-PCR in SMAD3-silenced (A) or -overexpressing (B) A549 cells. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (C) Schematic representation of five HCP5 promoter reporters. The red dots represent the three binding sites of SMAD3 to the HCP5 promoter as predicted by the JASPAR and meme-FIMO databases. (D) Dual-luciferase reporter assays detected the activity of the five HCP5 promoter fragments described in (C) in SMAD3-overexpressing A549 cells; luciferase activity was normalized to Renilla. The H3 promoter fragment exhibits the highest activity. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (E) Schematic illustrations showing the predicted locations of two SMAD3-binding sites (SMAD3-BR) in HCP5 promoter and the amplified regions (R1, including H3) of PCR for ChIP assays. (F) ChIP-PCR assays using antibodies specific for SMAD3 to prove that SMAD3 binds to HCP5 promoter. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
HCP5 regulates LUAD cell proliferation and metastasis. (A) Transwell assays to investigate the invasion ability of HCP5-silenced and control A549 cells. Scale bar: 100 μm. (B, C) Wound-healing assay investigate the migratory ability of HCP5 knockdown and control A549 cells. Scale bar: 200 μm. (D) Silencing of HCP5 inhibits the colony formation of A549 cells. (E) Cell Counting Kit-8 (CCK-8) assays were performed in A549 cells silenced for HCP5. (F) Transwell assay to investigate the invasion ability of HCP5-overexpressing and control A549 cells. Scale bar: 100 μm. (G) Overexpression of HCP5 promotes the colony formation of cells. (H, I) Wound-healing assay investigate the migratory ability of HCP5 overexpression and control A549 cells. Scale bar: 200 μm. (J) Cell Counting Kit-8 (CCK-8) assays were performed in A549 cells overexpressing HCP5. All data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). *P< 0.05, **P < 0.01, ***P< 0.001.
Figure 4
Figure 4
HCP5 knockdown inhibits cell proliferation and metastasis in nude mice. (A) Mice were subcutaneously injected with cells stably silenced for HCP5 (shHCP5) or control cells (shCtrl). Images of the tumor lumps from the indicated groups at the endpoint of the experiment. n = 6 mice per group. (B) Tumor formation in mice treated as in (A) was monitored at the indicated time points. Data are shown as the mean ± SEM (two-tailed Student's t-test); n = 6 mice per group. (C) Tumor weights in mice treated as in (A) were recorded. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (D) QRT-PCR for HCP5 expression in xenograft tumor tissues of shHCP5 or shCtrl group. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (E) shHCP5 or shCtrl A549 cells were intravenously injected into nude mice and lung tissues were isolated after eight weeks. Representative lung images showing metastases in the lung. Arrows indicate the metastatic nodules. (F) Microscopic images of lung tissue sections stained by hematoxylin and eosin. Left: Scale bar = 200 μm. Right: Scale bar = 50 μm. (G) The number of metastatic nodules in the lungs is shown as the mean ± S.E.M.; n=4 mice per group (two-tailed Student's t-test). *P< 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
HCP5 promotes EMT of A549 cells. (A, B) Phase-contrast microscopy images of A549 silenced for or overexpressing HCP5. (C, D) The mRNA levels of EMT markers (CDH1, VIM) and transcription factors (SNAI1, SNAI2, TWIST and ZEB1) in indicated A549 silenced for or overexpressing HCP5. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (E, F) Western-blot analysis of the EMT markers (E-cadherin, Vimentin), Snail and Slug expression in A549 cells silenced for or overexpressing HCP5. *P < 0.05, **P < 0.01, ***P< 0.001.
Figure 6
Figure 6
HCP5 positively regulates EMT via the miR-203/SNAI axis. (A, B) The expression of HCP5 in A549 cells transfected with miR-203 mimics or inhibitors or negative control oligonucleotides. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (C, D) miR-203 mimics inhibit (C), whereas miR-203 inhibitors promote (D) Luciferase activity in A549 cells cotransfected with miR-203 mimics or inhibitors and luciferase reporters containing HCP5 or mutant transcript. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (E) MiR-203 was specifically pulled down by biotin-labelled HCP5 compared with HCP5-antisense in A549 cells. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (F) FISH assay HCP5 (red) is co-localized with miR-203 (green). The arrow indicates the approximate location. (G-I) The mRNA (G, H) or protein levels(I) of Slug, Snail, vimentin and E-cadherin when HCP5 knockdown A549 cells transfected with miR-203 inhibitors or HCP5-overexpressing A549 cells transfected with miR-203 mimics. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (J) Transwell assays to investigate the invasion ability of HCP5 knockdown A549 cells transfected with miR-203 inhibitors or HCP5-overexpressing A549 cells transfected with miR-203 mimics. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). *P< 0.05, **P< 0.01, ***P < 0.001.
Figure 7
Figure 7
HCP5 is required for the TGF-β/SMAD3-induced invasive phenotype in A549 cells. (A) Transwell assays to investigate the invasion ability of SMAD3 knockdown A549 cells transfected with HCP5 or SMAD3-overexpressing A549 cells transfected with siHCP5. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). (B-E) The mRNA (B, C) or protein levels (D, E) of Slug, Snail, vimentin and E-cadherin when SMAD3 knockdown A549 cells transfected with HCP5 or SMAD3-overexpressing A549 cells transfected with siHCP5. Data are shown as the mean ± S.E.M. of three independent experiments (two-tailed Student's t-test). *P< 0.05, **P < 0.01, ***P< 0.001.
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References

    1. Eser PO, Janne PA. TGFbeta pathway inhibition in the treatment of non-small cell lung cancer. Pharmacol Ther. 2018;184:112–30. - PubMed
    1. Yu JR, Tai Y, Jin Y, Hammell MC, Wilkinson JE, Roe JS. et al. TGF-beta/Smad signaling through DOCK4 facilitates lung adenocarcinoma metastasis. Genes Dev. 2015;29:250–61. - PMC - PubMed
    1. Herbertz S, Sawyer JS, Stauber AJ, Gueorguieva I, Driscoll KE, Estrem ST. et al. Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway. Drug Des Devel Ther. 2015;9:4479–99. - PMC - PubMed
    1. Feng XH, Derynck R. Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol. 2005;21:659–93. - PubMed
    1. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003;425:577–84. - PubMed

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