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. 2019 Feb;19(2):831-840.
doi: 10.3892/mmr.2018.9723. Epub 2018 Dec 4.

MicroRNA‑29a enhances cisplatin sensitivity in non‑small cell lung cancer through the regulation of REV3L

Xialin Chen #  1 , Hong Zhu #  2 , Wanli Ye  1 , Yayun Cui  3 , Ming Chen  4
Affiliations

MicroRNA‑29a enhances cisplatin sensitivity in non‑small cell lung cancer through the regulation of REV3L

Xialin Chen et al. Mol Med Rep. 2019 Feb.

Abstract

Cisplatin‑based chemotherapy may greatly enhance patient prognosis; however, chemotherapy resistance remains an obstacle to curing patients with non‑small cell lung cancer (NSCLC). The aim of the present study was to explore the microRNAs (miRs) that could regulate cisplatin sensitivity and provide a potential treatment method for cisplatin resistance in clinical. Results from the present study revealed that miR‑29a overexpression enhanced and miR‑29a inhibition reduced the sensitivity of two NSCLC cell lines, A549 and H1650, to cisplatin treatment. In addition, reduced miR‑29a expression levels were observed in cisplatin‑resistant A549 cells (A549rCDDP), and increased expression of miR‑29a augmented cisplatin‑induced inhibition of proliferation and apoptosis in A549rCDDP cells. These data indicated that miR‑29a expression may be involved in the development of cisplatin resistance. miR‑29a was revealed to negatively regulate REV3‑like DNA‑directed polymerase ζ catalytic subunit (REV3L) expression in both A549 and H1650 cells; elevated expression of REV3L in A549rCDDP cells was also detected. REV3L encodes the catalytic subunit of DNA polymerase ζ and was hypothesized, based on results from the online tool TargetScan 7.1, to be a target gene of miR‑29a; this was confirmed with a dual luciferase assay. Cells treated with a very low concentration of cisplatin exhibited a significant reduction in proliferation and cell cycle arrest at the G2/M phase in REV3L‑knockdown as well as in miR‑29a‑upregulated A549 cells. Notably, reduced miR‑29a expression and an increase in REV3L mRNA expression were observed in tumor tissues from patients with NSCLC. Additionally, a negative correlation between miR‑29a and REV3L mRNA expression levels in tumor tissues from patients with NSCLC was observed; low expression of miR‑29a and high expression of REV3L were closely associated with an advanced tumor‑node‑metastasis classification. The results of the present study suggested a pivotal role of miR‑29a in mediating NSCLC cell sensitivity towards cisplatin through the regulation of REV3L.

Keywords: microRNA-29a; cisplatin sensitivity; non-small-cell lung cancer; REV3-like DNA-directed polymerase ζ catalytic subunit.

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Figures

Figure 1.
Figure 1.
miR-29a regulates cisplatin sensitivity of non-small cell lung cancer cell lines A549 and H1650. (A) Transfection of miR-29a mimics increased miR-29a expression levels in A549 cells, as determined by RT-qPCR. (B and C) Overexpression of miR-29a enhanced the sensitivity of (B) A549 and (C) H650 cells towards cisplatin treatment, as determined by the reduced viability of transfected cells. (D) Transfection of miR-29a inhibitor decreased miR-29a expression levels in A549 cells, as determined by RT-qPCR. Downregulation of miR-29a desensitized (E) A549 and (F) H1650 cells towards cisplatin treatment, as determined by increased viability in treated sells. *P<0.05, **P<0.01 and ***P<0.0001 vs. miR-NC. miR, microRNA; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Figure 2.
Figure 2.
Upregulation of REV3L contributed to cisplatin resistance of A549rCDDP cells. (A) Compared with A549 cells, cisplatin-resistant A549rCDDP cells were relatively insensitive towards cisplatin treatment, as indicated by the significantly higher cell viability; ***P<0.001 vs. A549. (B) miR-29a expression levels were significantly lower in A549rCDDP cells compared with A549 cells, as determined by reverse transcription-quantitative polymerase chain reaction. (C) Overexpression of miR-29a increased sensitivity of A549rCDDP cells towards cisplatin treatment, as indicated by the reduction in viability; **P<0.01 and ***P<0.001 vs. miR-NC. (D) miR-29a overexpression reduced cell proliferation in the presence of cisplatin in A549rCDDP cells; **P<0.01 and ***P<0.001 vs. miR-NC + cisplatin. (E) miR-29a overexpression significantly increased cell apoptosis rate in the presence of cisplatin in A549rCDDP cells. miR, microRNA; NC, negative control; OD, optical density; PI, propidium iodide; REV3L, REV3-like DNA-directed polymerase ζ catalytic subunit.
Figure 3.
Figure 3.
REV3L is negatively regulated by miR-29a in non-small cell lung cancer cell lines cells. (A and B) Overexpression of miR-29a decreased REV3L (A) mRNA and (B) protein expression levels in A549 and H1650 cells, as detected by RT-qPCR and western blotting, respectively. (C) mRNA and (D) protein expression levels of REV3L were elevated in A549rCDDP cells in comparison with A549 cells. (E and F) Overexpression of miR-29a decreased REV3L (E) mRNA and (F) protein expression levels in A549rCDDP cells. ***P<0.001. miR, microRNA; NC, negative control; REV3L, REV3-like DNA-directed polymerase ζ catalytic subunit; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Figure 4.
Figure 4.
REV3L is a direct target of miR-29a. (A) Sequence alignment of REV3L 3′UTR and miR-29a. (B) 293 cells were co-transfected with miR-29a mimics or miR-NC mimics and REV3L 3′UTR-WT or REV3L 3′UTR-Mut. Transfection of miR-29a mimics significantly reduced luciferase activity in cells transfected with REV3L 3′UTR-WT. ***P<0.001. miR, microRNA; Mut, mutant; NC, negative control; REV3L, REV3-like DNA-directed polymerase ζ catalytic subunit; UTR, untranslated region; WT, wild-type.
Figure 5.
Figure 5.
REV3L is involved in miR-29a-regulated cisplatin sensitivity through regulation of the cell cycle. (A) mRNA and (B) protein expression levels of REV3L were decreased following transfection of REV3L siRNA1 or siRNA2 in A549 cells, as detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. (C) Silencing of REV3L or overexpression of miR-29a induced G2/M arrest in A549 cells co-treated with low concentration of cisplatin (2 µg/ml). (D) Quantitative analysis of cell cycle distribution from (C). (E) Proliferation was inhibited in A549 cells transfected with REV3L siRNAs or miR-29a mimics and co-treated with low concentration of cisplatin (2 µg/ml). **P<0.01 and ***P<0.001 vs. Control siRNA + miR-NC mimics + cisplatin (2 µg/ml). miR, microRNA; NC, negative control; REV3L, REV3-like DNA-directed polymerase ζ catalytic subunit; siRNA, small interfering RNA.
Figure 6.
Figure 6.
miR-29a expression is negatively correlated with REV3L mRNA levels in NSCLC tumor tissues. (A) miR-29a expression levels were significantly lower in tumoral tissues from patients with NSCLC compared with adjacent normal tissues. (B) REV3L mRNA levels were significantly elevated in tumoral tissues from patients with NSCLC compared with adjacent normal tissues. (C) A negative correlation was identified between miR-29a and REV3L mRNA expression levels in NSCLC tumor tissues, as determined by Pearson correlation analysis. ***P<0.001. miR, microRNA; NSCLC, non-small cell lung cancer; REV3L, REV3-like DNA-directed polymerase ζ catalytic subunit.
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