Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Nov;22(5):798.
doi: 10.3892/ol.2021.13059. Epub 2021 Sep 20.

Knockdown of lncRNA NUTM2A-AS1 inhibits lung adenocarcinoma cell viability by regulating the miR-590-5p/METTL3 axis

Jie Wang  1 , Jingyun Zha  2 , Xiaolin Wang  1
Affiliations

Knockdown of lncRNA NUTM2A-AS1 inhibits lung adenocarcinoma cell viability by regulating the miR-590-5p/METTL3 axis

Jie Wang et al. Oncol Lett. 2021 Nov.

Abstract

Lung adenocarcinoma (LUAD) is the leading cause of cancer-related mortality worldwide. Long non-coding RNA (lncRNA) NUT family member 2A antisense RNA 1 (NUTM2A-AS1) is dysregulated in LUAD; however, its role in this disease remains unclear. The present study aimed to identify the underlying molecular mechanism of the effect of lncRNA NUTM2A-AS1 in LUAD by exploring whether lncRNA NUTM2A-AS1 could affect LUAD cell proliferation and apoptosis through the microRNA (miR)-590-5p/methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3) axis. miR-590-5p was predicted and verified as the direct target of NUTM2A-AS1 using bioinformatics analysis and a dual luciferase reporter assay. The expression levels of NUTM2A-AS1 and miR-590-5p in lung cancer cells, and the effects of NUTM2A-AS1 on cell viability and apoptosis were determined using MTT assays and flow cytometry, respectively. Reverse transcription-quantitative PCR analysis revealed that the expression levels of NUTM2A-AS1 were significantly upregulated, while those of miR-590-5p were significantly downregulated, in lung cancer cells compared with the control epithelial cells. NUTM2A-AS1 knockdown inhibited NCI-H23 cell viability and induced apoptosis by upregulating miR-590-5p expression. Moreover, the function and regulatory mechanism of miR-590-5p in LUAD were also investigated. It was determined that miR-590-5p could interact with METTL3, and further analysis of the expression levels of METTL3 in lung cancer cells demonstrated that METTL3 was significantly upregulated in NCI-H23 and A549 cells compared with the control cells. In addition, miR-590-5p inhibited NCI-H23 cell viability and induced apoptosis by downregulating METTL3 expression. In conclusion, the findings of the present study suggested that NUTM2A-AS1 knockdown may inhibit LUAD progression by regulating the miR-590-5p/METTL3 axis. These results may provide insight into the mechanisms underlying the tumorigenesis of LUAD and offer a new treatment strategy for the disease.

Keywords: N6-adenosine-methyltransferase complex catalytic subunit; NUT family member 2A antisense RNA 1; long non-coding RNA; lung adenocarcinoma; methyltransferase 3; microRNA-590-5p.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
miR-590-5p is a direct target of the long non-coding RNA NUTM2A-AS1. (A) miR-590-5p was predicted as a potential target of NUTM2A-AS1 using StarBase. (B) miR-590-5p expression levels in 293T cells transfected with the mimic control or miR-590-5p mimic. (C) Dual luciferase reporter assay was performed to confirm the interaction between miR-590-5p and NUTM2A-AS1. **P<0.01 vs. mimic control group. miR/miRNA, microRNA; NUTM2A-AS1, NUT family member 2A antisense RNA 1; WT, wild-type; MUT, mutant; 3′-UTR.
Figure 2.
Figure 2.
Expression levels of NUTM2A-AS1 and miR-590-5p in lung adenocarcinoma cell lines. (A) RT-qPCR was used to determine that the expression levels of NUTM2A-AS1 were upregulated in A549 and NCI-H23 cells. (B) RT-qPCR was used to determine that the expression levels of miR-590-5p were downregulated in A549 and NCI-H23 cells. **P<0.01 vs. BEAS2B cells. lncRNA, long non-coding RNA; NUTM2A-AS1, NUT family member 2A antisense RNA 1; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR.
Figure 3.
Figure 3.
Long non-coding RNA NUTM2A-AS1 negatively regulates miR-590-5p expression in NCI-H23 cells. (A) NUTM2A-AS1 expression levels in NCI-H23 cells transfected with control-siRNA, or NUTM2A-AS1-siRNA. (B) miR-590-5p expression levels in NCI-H23 cells transfected with inhibitor control or miR-590-5p inhibitor. (C) miR-590-5p expression levels in NCI-H23 cells transfected with control-siRNA, NUTM2A-AS1-siRNA, NUTM2A-AS1-siRNA + inhibitor control or NUTM2A-AS1-siRNA + miR-590-5p inhibitor. **P<0.01 vs. control-siRNA; ##P<0.01 vs. inhibitor control; &&P<0.01 vs. NUTM2A-AS1-siRNA + inhibitor control. miR, microRNA; siRNA, small interfering RNA; NUTM2A-AS1, NUT family member 2A antisense RNA 1.
Figure 4.
Figure 4.
Long non-coding RNA NUTM2A-AS1 knockdown inhibits NCI-H23 cell viability and induces apoptosis by upregulating miR-590-5p. (A) Viability of NCI-H23 cells was assessed using an MTT assay after 24, 48 and 72 h of incubation. (B) mRNA and (C) protein expression levels of PCNA were assessed using reverse transcription-quantitative PCR and western blotting, respectively. (D and E) Apoptosis was detected using flow cytometry. (F) Cleaved caspase-3 and caspase-3 protein expression levels were measured using western blotting. (G) Cleaved caspase-3/caspase-3 ratio. **P<0.01 vs. control-siRNA; ##P<0.01 vs. NUTM2A-AS1-siRNA + inhibitor control. NUTM2A-AS1, NUT family member 2A antisense RNA 1; miR, microRNA; siRNA, small interfering RNA; PCNA, proliferating cell nuclear antigen.
Figure 5.
Figure 5.
METTL3 is a direct target of miR-590-5p. (A) METTL3 was predicted as a potential target of miR-590-5p using StarBase. (B) Dual luciferase reporter assay was performed to confirm the interaction between miR-590-5p and METTL3. (C and D) METTL3 expression levels were measured in A549 and NCI-H23 cells using western blotting and reverse transcription-quantitative PCR. **P<0.01 vs. mimic control; ##P<0.01 vs. BEAS2B cells. METTL3, methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit; miR, microRNA; WT, wild-type; MUT, mutant.
Figure 6.
Figure 6.
miR-590-5p negatively regulates METTL3 in NCI-H23 cells. (A) miR-590-5p and (B) METTL3 expression levels were measured using RT-qPCR. METTL3 (C) mRNA and (D) protein expression levels were measured using RT-qPCR and western blotting, respectively. **P<0.01 vs. mimic control; ##P<0.01 vs. control-plasmid; &&P<0.01 vs. miR-590-5p mimic + control-plasmid. miR, microRNA; METTL3, methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit; RT-qPCR, reverse transcription-quantitative PCR.
Figure 7.
Figure 7.
miR-590-5p inhibits NCI-H23 cell viability and induces apoptosis by downregulating METTL3. (A) Viability of NCI-H23 cells was assessed using an MTT assay after 24, 48 or 72 h of incubation. (B) mRNA and (C) protein expression levels of PCNA were assessed using reverse transcription-quantitative PCR and western blotting, respectively. (D and E) Apoptosis was detected using flow cytometry. (F) Cleaved caspase-3 and caspase-3 protein expression levels were measured using western blotting. (G) Cleaved caspase-3/caspase-3 ratio. **P<0.01 vs. mimic control; ##P<0.01 vs. miR-590-5p mimic + control-plasmid. miR, microRNA; METTL3, methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit; PCNA, proliferating cell nuclear antigen.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chang JT, Lee YM, Huang RS. The impact of the cancer genome atlas on lung cancer. Transl Res. 2015;166:568–585. doi: 10.1016/j.trsl.2015.08.001. - DOI - PMC - PubMed
    1. Girard L, Rodriguez-Canales J, Behrens C, Thompson DM, Botros IW, Tang H, Xie Y, Rekhtman N, Travis WD, Wistuba II, et al. An expression signature as an aid to the histologic classification of non-small cell lung cancer. Clin Cancer Res. 2016;22:4880–4889. doi: 10.1158/1078-0432.CCR-15-2900. - DOI - PMC - PubMed
    1. Sardenberg RA, Pinto C, Bueno CA, Younes RN. Non-small cell lung cancer stage IV long-term survival with isolated spleen metastasis. Ann Thorac Surg. 2013;95:1432–1434. doi: 10.1016/j.athoracsur.2012.08.086. - DOI - PubMed
    1. Travis WD. Pathology of lung cancer. Clin Chest Med. 2011;32:669–692. doi: 10.1016/j.ccm.2011.08.005. - DOI - PubMed
    1. Cancer Genome Atlas Research Network, corp-author. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511:543–550. doi: 10.1038/nature13385. - DOI - PMC - PubMed