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Article Open Access

MicroRNA‑199a‑3p suppresses non‑small cell lung cancer progression by targeting FTO to enhance m6A‑mediated downregulation of MZF1 and its transcriptional activation of CLDND1

  • Authors:
    • Yuzhen Cui
    • Xiaoqian Li
    • Hongkui Zhang
    • Weiqiang Yuan
    • Enbo Zhu
  • View Affiliations / Copyright

    Affiliations: Department of Oncology, Yanbian University Affiliated Hospital, Yanji, Jilin 133000, P.R. China, Department of Nephrology, Yanbian University Affiliated Hospital, Yanji, Jilin 133000, P.R. China, Department of Neurology, Yanbian University Affiliated Hospital, Yanji, Jilin 133000, P.R. China
    Copyright: © Cui et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 26
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    Published online on: November 3, 2025
       https://doi.org/10.3892/mmr.2025.13736
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Abstract

The present study aimed to investigate the effect of microRNA (miR)-199a-3p on the biological function of non-small cell lung cancer (NSCLC) adenocarcinoma cells by targeting the fat mass and obesity‑associated protein (FTO)/myeloid zinc finger 1 (MZF1)/claudin domain‑containing 1 (CLDND1) axis. Human NSCLC cell lines, primarily A549 cells, were used for in vitro assays. Reverse transcription‑quantitative PCR and western blotting were performed to assess the expression of relevant genes and proteins. Dual‑luciferase reporter assays were used to verify the relationship between miR‑199a‑3p and FTO, as well as the transcriptional regulation of CLDND1 by MZF1. Methylated RNA immunoprecipitation was used to evaluate the N6‑methyladenosine (m6A) modification levels of MZF1, whereas photoactivatable ribonucleoside‑enhanced crosslinking and immunoprecipitation supported the binding of FTO to MZF1 mRNA. Cell proliferation, migration, invasion and apoptosis were assessed using Cell Counting Kit‑8, Transwell and flow cytometry assays. miR‑199a‑3p was downregulated in NSCLC tissues and cells. Overexpression of miR‑199a‑3p inhibited A549 cell proliferation, invasion and migration. Mechanistically, miR‑199a‑3p directly targeted and suppressed FTO, an m6A demethylase, leading to enhanced m6A modification of MZF1 mRNA and a subsequent decrease in MZF1 expression. Knockdown of MZF1 attenuated the oncogenic effects mediated by FTO, confirming that MZF1 served as a downstream effector of the miR‑199a‑3p/FTO axis. Moreover, MZF1 transcriptionally activated CLDND1, thereby facilitating the malignant phenotype of NSCLC cells. Collectively, these findings demonstrate that miR‑199a‑3p suppresses NSCLC progression by targeting FTO, promoting m6A methylation‑dependent downregulation of MZF1, and consequently decreasing CLDND1 expression. Thus, the miR‑199a‑3p/FTO/MZF1/CLDND1 axis may serve as a promising therapeutic target in NSCLC.
View Figures

Figure 1

Expression levels of miR-199a-3p in
NSCLC tissues and cells. (A) RT-qPCR was utilized to test the
expression level of miR-199a-3p in NSCLC tissues, and paracancerous
tissues were regarded as the control (n=15). (B) RT-qPCR was
performed to detect miR-199a-3p expression in normal bronchial
epithelial cells (BEAS-2B) and NSCLC cell lines (A549, H1299, H460
and H1975). Data are presented as the mean ± SD from three
independent experiments (n=3). All cell experiments were repeated
three times. RT-qPCR, reverse transcription-quantitative PCR; miR,
microRNA; NSCLC, non-small cell lung cancer.

Figure 2

Effect of miR-199a-3p on the
biological function of A549 cells by downregulating FTO. (A)
Bioinformatics tool ‘miRwalk’ predicted the possible downstream
target gene of miR-199a-3p: FTO. (B) RT-qPCR and western blot
analysis were performed to detect the expression levels of FTO in
non-small cell lung cancer tissues, with paracancerous tissues as
the control (n=15). (C) RT-qPCR was performed to detect the
expression levels of FTO in lung cancer cells. (D) After
miR-199a-3p mimic transfection, RT-qPCR was used to detect the
expression levels of miR-199a-3p and FTO. (E) A dual-luciferase
reporter assay verified the targeting relationship between
miR-199a-3p and FTO. (F) RT-qPCR was used to assess the expression
levels of FTO in A549 cells in each miR mimic + oe plasmid
combination group. (G) Cell Counting Kit-8 assay assessing the
proliferation of A549 cells in each miR mimic + oe plasmid
combination group. (H) Transwell assays indicated the migration and
invasion of A549 cells in each miR mimic + oe plasmid combination
group. Scale bar, 200 µm. (I) Flow cytometry was performed to
detect the apoptosis of A549 cells in each miR mimic + oe plasmid
combination group. Data are presented as the mean ± SEM, n=3
independent experiments. oe, overexpression; RT-qPCR, reverse
transcription-quantitative PCR; miR, microRNA; FTO, fat mass and
obesity-associated protein; NC, negative control; Wt, wild-type;
Mut, mutant; OD, optical density.

Figure 3

Mechanism of m6A
modification regulation of MZF1 by FTO. (A) The SRAMP online tool
(http://www.cuilab.cn/sramp) predicted
potential m6A modification sites on MZF1 mRNA. (B)
RT-qPCR and western blot analysis were conducted to assess the
expression levels of MZF1 in NSCLC tissues, with paracancerous
tissues as the control (n=15). (C) RT-qPCR analysis of MZF1 mRNA
expression in normal bronchial epithelial (BEAS-2B) and NSCLC cell
lines (CAL12T, HCC44, NCIH1993 and A549). (D) RT-qPCR was used to
determine MZF1 expression after overexpression of FTO. (E)
Methylated RNA immunoprecipitation assay detecting the
m6A modification level of MZF1 after overexpression of
FTO. (F) PAR-CLIP assay detecting the direct interaction between
FTO and MZF1 mRNA. All cell experiments were repeated three times.
oe, overexpression; m6A, N6-methyladenosine;
MZF1, myeloid zinc finger 1; FTO, fat mass and obesity-associated
protein; RT-qPCR, reverse transcription-quantitative PCR; NSCLC,
non-small cell lung cancer; NC, negative control; PAR-CLIP,
photoactivatable ribonucleoside-enhanced crosslinking and
immunoprecipitation.

Figure 4

Effect of FTO on the biological
function of A549 cells via regulation of MZF1. (A) Reverse
transcription-quantitative PCR was performed to detect the
expression levels of FTO and MZF1 in A549 cells in each oe plasmid
+ shRNA combination group. (B) Cell Counting Kit-8 assay examining
the proliferation of A549 cells in each oe plasmid + shRNA
combination group. (C) Transwell assay assessing the migration and
invasion of A549 cells in each oe plasmid + shRNA combination
group. Scale bar, 200 µm. (D) Flow cytometry was conducted to
detect the apoptosis of A549 cells. All cell experiments were
repeated three times. oe, overexpression; NC, negative control; sh,
short hairpin RNA; FTO, fat mass and obesity-associated protein;
MZF1, myeloid zinc finger 1; OD, optical density.

Figure 5

Mechanism of the transcriptional
regulation of CLDND1 by MZF1. (A) The JASPAR website (https://jaspar.genereg.net/) was used to retrieve the
binding site of the transcription factor MZF1. (B) RT-qPCR and
western blot analysis were performed to detect the expression
levels of CLDND1 in non-small cell lung cancer tissues, using
paracancerous tissues as the control (n=15). (C) RT-qPCR analysis
of CLDND1 mRNA expression in normal bronchial epithelial (BEAS-2B)
and NSCLC cell lines (CAL12T, HCC44, NCIH1993 and A549). (D) After
overexpression of MZF1, RT-qPCR was performed to measure MZF1 and
CLDND1 expression. (E) Dual-luciferase reporter assay examining the
effect of MZF1 on CLDND1 promoter activity. (F) Chromatin
immunoprecipitation assays determining the enrichment of MZF1 in
the CLDND1 promoter region [Chr3:167, 450, 703–167, 450, 714
(hg38)]. All cell experiments were repeated three times. oe,
overexpression; CLDND1, claudin domain-containing 1; MZF1, myeloid
zinc finger 1; RT-qPCR, reverse transcription-quantitative PCR; NC,
negative control; Wt, wild-type; Mut, mutant.

Figure 6

Effect of MZF1 on the biological
function of lung cancer cells via regulation of CLDND1. (A) Reverse
transcription-quantitative PCR was performed to detect the
expression levels of MZF1 and CLDND1 in A549 cells in each group.
(B) Cell Counting Kit-8 assay of the proliferation of A549 cells in
each group. (C) Transwell assay examining the migration and
invasion of A549 cells in each group. Scale bar, 200 µm. (D) Flow
cytometry was performed to detect the apoptosis of A549 cells in
each group. comparisons. All cell experiments were repeated three
times. MZF1, myeloid zinc finger 1; CLDND1, claudin
domain-containing 1; oe, overexpression; NC, negative control; sh,
short hairpin RNA; OD, optical density.
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Copy and paste a formatted citation
Spandidos Publications style
Cui Y, Li X, Zhang H, Yuan W and Zhu E: MicroRNA‑199a‑3p suppresses non‑small cell lung cancer progression by targeting FTO to enhance m6A‑mediated downregulation of MZF1 and its transcriptional activation of CLDND1. Mol Med Rep 33: 26, 2026.
APA
Cui, Y., Li, X., Zhang, H., Yuan, W., & Zhu, E. (2026). MicroRNA‑199a‑3p suppresses non‑small cell lung cancer progression by targeting FTO to enhance m6A‑mediated downregulation of MZF1 and its transcriptional activation of CLDND1. Molecular Medicine Reports, 33, 26. https://doi.org/10.3892/mmr.2025.13736
MLA
Cui, Y., Li, X., Zhang, H., Yuan, W., Zhu, E."MicroRNA‑199a‑3p suppresses non‑small cell lung cancer progression by targeting FTO to enhance m6A‑mediated downregulation of MZF1 and its transcriptional activation of CLDND1". Molecular Medicine Reports 33.1 (2026): 26.
Chicago
Cui, Y., Li, X., Zhang, H., Yuan, W., Zhu, E."MicroRNA‑199a‑3p suppresses non‑small cell lung cancer progression by targeting FTO to enhance m6A‑mediated downregulation of MZF1 and its transcriptional activation of CLDND1". Molecular Medicine Reports 33, no. 1 (2026): 26. https://doi.org/10.3892/mmr.2025.13736
Copy and paste a formatted citation
x
Spandidos Publications style
Cui Y, Li X, Zhang H, Yuan W and Zhu E: MicroRNA‑199a‑3p suppresses non‑small cell lung cancer progression by targeting FTO to enhance m6A‑mediated downregulation of MZF1 and its transcriptional activation of CLDND1. Mol Med Rep 33: 26, 2026.
APA
Cui, Y., Li, X., Zhang, H., Yuan, W., & Zhu, E. (2026). MicroRNA‑199a‑3p suppresses non‑small cell lung cancer progression by targeting FTO to enhance m6A‑mediated downregulation of MZF1 and its transcriptional activation of CLDND1. Molecular Medicine Reports, 33, 26. https://doi.org/10.3892/mmr.2025.13736
MLA
Cui, Y., Li, X., Zhang, H., Yuan, W., Zhu, E."MicroRNA‑199a‑3p suppresses non‑small cell lung cancer progression by targeting FTO to enhance m6A‑mediated downregulation of MZF1 and its transcriptional activation of CLDND1". Molecular Medicine Reports 33.1 (2026): 26.
Chicago
Cui, Y., Li, X., Zhang, H., Yuan, W., Zhu, E."MicroRNA‑199a‑3p suppresses non‑small cell lung cancer progression by targeting FTO to enhance m6A‑mediated downregulation of MZF1 and its transcriptional activation of CLDND1". Molecular Medicine Reports 33, no. 1 (2026): 26. https://doi.org/10.3892/mmr.2025.13736
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