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. 2022 Sep 28;21(1):186.
doi: 10.1186/s12943-022-01651-4.

The reversion of DNA methylation-induced miRNA silence via biomimetic nanoparticles-mediated gene delivery for efficient lung adenocarcinoma therapy

Lu Liang #  1 , Huiyu Cen #  1 , Jionghua Huang #  1   2 , Aiping Qin #  1 , Wenyan Xu  1 , Siran Wang  3 , Zhijun Chen  4 , Lin Tan  1 , Qiqi Zhang  1 , Xiyong Yu  5 , Xin Yang  6 , Lingmin Zhang  7
Affiliations

The reversion of DNA methylation-induced miRNA silence via biomimetic nanoparticles-mediated gene delivery for efficient lung adenocarcinoma therapy

Lu Liang et al. Mol Cancer. .

Abstract

Background: Lung cancer is one of the fatal cancers worldwide, and over 60% of patients are lung adenocarcinoma (LUAD). Our clinical data demonstrated that DNA methylation of the promoter region of miR-126-3p was upregulated, which led to the decreased expression of miR-126-3p in 67 cases of lung cancer tissues, implying that miR-126-3p acted as a tumor suppressor. Transduction of miR-126-3p is a potential therapeutic strategy for treating LUAD, yet the physiological environment and properties of miRNA challenge current transduction approaches.

Methods: We evaluated the expression of miR-126-3p in 67 pairs of lung cancer tissues and the corresponding adjacent non-tumorous tissues by Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The relationship between the overall survival of lung cancer patients and miR-126-3p was analyzed by the Cancer Genome Atlas cohort database (Oncolnc, http://www.oncolnc.org ). We analyzed DNA methylation Methylation-specific PCR (MSP) analysis. To determine whether ADAM9 is the direct target of miR-126-3p, we performed the 3'-UTR luciferase reporter assay. The protein levels in the cells or tissues were evaluated with western blotting (WB) analysis. The biodistribution of nanoparticles were monitored by in vivo tracking system.

Results: We describe the development of novel stealth and matrix metalloproteinase 2 (MMP2)-activated biomimetic nanoparticles, which are constructed using MMP2-responsive peptides to bind the miR-126-3p (known as MAIN), and further camouflaged with red blood cell (RBC) membranes (hence named REMAIN). REMAIN was able to effectively transduce miRNA into lung cancer cells and release them via MMP2 responsiveness. Additionally, REMAIN possessed the advantages of the natural RBC membrane, including extended circulation time, lower toxicity, better biocompatibility, and immune escape. Moreover, in vitro and in vivo results demonstrated that REMAIN effectively induced apoptosis of lung cancer cells and inhibited LUAD development and progression by targeting ADAM9.

Conclusion: The novel style of stealth and MMP2-activated biomimetic nanoparticles show great potential in miRNA delivery.

Keywords: Biomimetic nanoparticles; DNA methylation; Lung adenocarcinoma; MMP2; MicroRNA.

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Conflict of interest statement

The authors declare no competing interests.

Figures

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Scheme 1
Fig. 1
Fig. 1
miR-126-3p expression is silenced by hypermethylation of its promoter in lung cancer cells. A The miR-126-3p levels were measured by RT-qPCR in 67 pairs of lung adenocarcinoma tissues and adjacent non-tumor tissues. B RT-qPCR detected the miR-126-3p expression levels in BEAS-2B, A549, NCI-H1299, and H460 cells. C The overall survival of lung cancer patients with low or high miR-126-3p expression. D The relative expression levels of methylation of miR-126-3p were detected in tumor samples and adjacent lung samples by qRT-PCR. E MSP for methylation or demethylation of the miRNA-126-3p gene promoter in different LUAD and BEAS-2B cells. F The relative expression levels of miR-126-3p in different LUAD and BEAS-2B cells (treated with or without 5-Aza-dC for 24 h) were measured by RT-qPCR. *, P < 0.05; **, P < 0.01; ***, P < 0.001
Fig. 2
Fig. 2
miR-126-3p is a negative regulator of ADAM9 in LUAD. A The target genes of miR-126-3p screened by mirDB database. B Diagram of putative miR-126-3p binding sequence in ADAM9 3′-UTR and its mutant in luciferase reporter assay. A luciferase reporter assay was performed to evaluate luciferase activity in A549 and NCI-H1299 cells. C ADAM9 mRNA levels were evaluated in lung adenocarcinoma tissues and adjacent lung tissues. D The correlation of miR-126-3p expression to mRNA expression in lung adenocarcinoma tissues. The mRNA (E) and protein (F) levels of ADAM9 in miR-126-3p mimic-transfected A549 and NCI-H1299 cells. The mRNA (G) and protein (H) levels of ADAM9 in A549 and NCI-H1299 cells treatment with 5-Aza, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001
Fig. 3
Fig. 3
The properties of RBCM coated nanoparticles. A TEM image analysis and (B) the size distribution of MAIN. C TEM analysis and (D) the size distribution of REMAIN. E Nuclease protection assay of miR-126-3p. F SDS-PAGE analysis of REMAIN. G WB analysis of MMP2 levels. H The cumulative release of miR-126-3p in different conditions
Fig. 4
Fig. 4
The cellular uptake in different conditions. A The cellular uptake of different formulations containing 150 nM miR-126-3p. B The lysosome escape of Cy5-miR-126-3p. C Scheme illustration of lysosome escape. NCI-H1299 cells were incubated with REMAIN for 1 and 9 h, respectively. Excitation/Emission: FITC-Phalloidin (488 nm/525 nm); Cy5 (633 nm/670 nm); DAPI (358 nm/461 nm)
Fig. 5
Fig. 5
The effect of different formulations of nanoparticles on NCI-H1299 cells in vitro. A miR-126-3p levels, (B) Live/dead staining, (C) Tube formation of HUVECs, (D) Cell invasion and (E) Cell migration was analyzed after treatment with PBS, Lipo3000/miR-126-3p, REMAIN, MAIN, REMAIN-NC or free miR-126-3p, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001
Fig. 6
Fig. 6
The inhibition of LUAD cell migration, invasion, and endothelial-to-mesenchymal transition. A549 and NCI-H1299 cells were treated with REMAIN or si-ADAM9 for 48 h. The mRNA (A) and protein (B) levels of ADAM9 in A549 and NCI-H1299 cells treatment with REMAIN, respectively. (C) Wound healing assays measured cell migration. (D) Transwell assays measured cell invasion. (E) Western blot assays determined the expression of EMT markers E-cadherin, N-cadherin, and Vimentin. *, P < 0.05; **, P < 0.01; ***, P < 0.001
Fig. 7
Fig. 7
Biodistribution and circulation of different nanoparticles in vivo. A Biodistribution of nanoparticles in vivo. B Ex-vivo images of the major organs. C The quantitative analysis of the fluorescence in the major organs and tumors. D Circulation lifetime of nanoparticles. *, P < 0.05; **, P < 0.01; ***, P < 0.001
Fig. 8
Fig. 8
The effect of different formulations of nanoparticles on tumor growth in vivo (n = 6). A The excised tumors were imaged. B The excised tumors were weighed. C and D The changes in the tumor volumes were determined. The mRNA levels of ADAM9 (E) and miR-126-3p (F) were detected in tumors. G The protein levels of ADAM9 were measured in tumors. *, P < 0.05; **, P < 0.01; ***, P < 0.001
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References

    1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–249. doi: 10.3322/caac.21660. - DOI - PubMed
    1. Crawford M, Brawner E, Batte K, Yu L, Hunter MG, Otterson GA, et al. MicroRNA-126 inhibits invasion in non-small cell lung carcinoma cell lines. Biochem Biophys Res Commun. 2008;373(4):607–612. doi: 10.1016/j.bbrc.2008.06.090. - DOI - PubMed
    1. Liu Y, Zhou Y, Feng X, An P, Quan X, Wang H, et al. MicroRNA-126 functions as a tumor suppressor in colorectal cancer cells by targeting CXCR4 via the AKT and ERK1/2 signaling pathways. Int J Oncol. 2014;44(1):203–210. doi: 10.3892/ijo.2013.2168. - DOI - PubMed
    1. Zhang Y, Yang P, Sun T, Li D, Xu X, Rui Y, et al. miR-126 and miR-126* repress recruitment of mesenchymal stem cells and inflammatory monocytes to inhibit breast cancer metastasis. Nat Cell Biol. 2013;15(3):284–294. doi: 10.1038/ncb2690. - DOI - PMC - PubMed
    1. Kesharwani P, Gajbhiye V, Jain NK. A review of nanocarriers for the delivery of small interfering RNA. Biomaterials. 2012;33(29):7138–7150. doi: 10.1016/j.biomaterials.2012.06.068. - DOI - PubMed

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