TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis

Abstract

Background: The acquisition of drug resistance has been considered as a main obstacle for cancer chemotherapy. Tumor protein 53 target gene 1 (TP53TG1), a p53-induced lncRNA, plays a vital role in the progression of human cancers. However, little is known about the detailed function and molecular mechanism of TP53TG1 in cisplatin resistance of NSCLC.

Methods: qRT-PCR analysis was used to detect the expression of TP53TG1, miR-18a and PTEN mRNA in NSCLC tissues and cells. Western blot analysis was performed to determine the protein level of PTEN and cleaved caspase-3. Cell viability and IC50 value were measured by MTT assay. Cell apoptosis was confirmed by flow cytometry assay. Subcellular fractionation assay was used to identify the subcellular location of TP53TG1. Dual-luciferase reporter assay, RNA pull down assay and RNA immunoprecipitation assay were carried out to verify the interaction between TP53TG1 and miR-18a. Xenografts in nude mice were established to verify the effect of TP53TG1 on cisplatin sensitivity of NSCLC cells in vivo.

Results: TP53TG1 level was downregulated in NSCLC tissues and cell lines. Upregulated TP53TG1 enhanced cisplatin sensitivity and apoptosis of A549/DDP cells, while TP53TG1 depletion inhibited cisplatin sensitivity and apoptosis of A549 cells. TP53TG1 suppressed miR-18a expression in A549 cells. Moreover, TP53TG1-mediated enhancement effect on cisplatin sensitivity was abated following the restoration of miR-18a expression in A549/DDP cells, while si-TP53TG1-induced decrease of cisplatin sensitivity and apoptosis was counteracted by miR-18a inhibitor in A549 cells. Furthermore, TP53TG1 promoted PTEN expression via inhibiting miR-18a. Finally, TP53TG1 sensitized NSCLC cells to cisplatin in vivo.

Conclusion: TP53TG1 increased the sensitivity of NSCLC cells to cisplatin by modulating miR-18a/PTEN axis, elucidating a novel approach to boost the effectiveness of chemotherapy for NSCLC.

Keywords: Cisplatin; Drug sensitivity; MiR-18a; Non-small cell lung cancer (NSCLC); PTEN; Tumor protein 53 target gene 1 (TP53TG1).

Fig. 1

TP53TG1 expression levels in NSCLC tissues and cells. TP53TG1 levels were assessed by qRT-PCR assay in 40 paired NSCLC tissues and adjacent normal tissues (a), in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples (b), in NSCLC cell lines (SK-MES-1, H1299, A549) and normal bronchial epithelial cell line HBE (c), as well as in A549 cells and its cisplatin-resistant cells A549/DDP (d). qRT-PCR assay of miR-18a expression (e) and PTEN expression pattern (f) in HBE, A549 and A549/DDP cells. Each experiment is repeated at least three times. *P < 0.05 vs. respective control

Fig. 2

TP53TG1 was associated with cisplatin sensitivity in NSCLC cells. a A549 and A549/DDP cells were exposed to different concentrations of cisplatin (1, 10, 20, 40, 80, 160 μM) for 48 h, followed by the determination of cell viability and the calculation of IC50 of cisplatin by MTT assay. b A549/DDP cells were transfected with pcDNA-TP53TG1 and A549 cells were introduced with two individual TP53TG1 siRNAs (si-TP53TG1#1 and si-TP53TG1#2), followed by the detection of TP53TG1 expression by qRT-PCR assay. c pcDNA-TP53TG1-transfected A549/DDP cells were treated with various concentrations of cisplatin for 48 h, and IC50 of cisplatin and cell proliferation capacity were measured by MTT. d si-TP53TG1#1- or si-TP53TG1#2-transfected A549 cells were treated with different doses of cisplatin for 48 h, and IC50 of cisplatin and cell proliferation capacity were monitored by MTT. e Cell apoptosis was evaluated by flow cytometry in pcDNA-TP53TG1-transfected A549/DDP cells after exposed to 60 μM of cisplatin for 48 h. f The apoptotic rate was analyzed by flow cytometry in si-TP53TG1#1- or si-TP53TG1#2-transfected A549 cells after treated with 20 μM of cisplatin for 48 h. Each experiment is repeated three times. *P < 0.05 vs. respective control

Fig. 3

TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c, d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. *P < 0.05 vs. corresponding control

Fig. 4

TP53TG1-induced cisplatin sensitivity of NSCLC cells was decreased following miR-18a upregulation. A549/DDP cells were transfected with pcDNA-TP53TG1 alone or together with miR-18a mimics, followed by qRT-PCR assay of miR-18a expression (a), MTT analysis of IC50 of cisplatin (c) and flow cytometry analysis of apoptotic rate (e). A549 cells were introduced with si-TP53TG1#1 alone or together with anti-miR-18a, followed by measurement of miR-18a expression by qRT-PCR (b), determination of IC50 of cisplatin by MTT (d), detection of apoptotic rate by flow cytometry (f). Each experiment is repeated three times. *P < 0.05 vs. corresponding control

Fig. 5

TP53TG1 regulated PTEN expression through miR-18a in NSCLC cells. a PTEN expression was assessed by western blot in A549 cells transfected with miR-18a mimics or anti-miR-18a. b Western blot assay of PTEN expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1. Dual-luciferase reporter assay was performed by transfecting PTEN-WT vector into A549 cells together with miR-18 mimics or miR-18a mimics + pcDNA-TP53TG1 (c), and anti-miR-18a or anti-miR-18a + si-TP53TG1#1 (d). e qRT-PCR assay of PTEN expression in 40 pairs of NSCLC tumor samples. f qRT-PCR assay of PTEN expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. g The correlation analysis between TP53TG1 and PTEN expression in NSCLC tumor specimen. Each experiment is repeated three times. *P < 0.05 vs. respective control

Fig. 6

Overexpression of TP53TG1 sensitized NSCLC cells to cisplatin in vivo. About 2.0 × 10⁷ SUNE2 cells stably transfected with lenti-control or lenti-TP53TG1 were subcutaneously inoculated into the nude mice, followed by intraperitoneal injection of PBS or cisplatin. Mice were euthanized to remove tumor masses at 32 days after inoculation. a The tumor volumes were measured with a caliper at indicated time points. b The representative photographs and average weights of resected tumors. c qRT-PCR analysis of TP53TG1, miR-18a and PTEN mRNA levels in excised tumor tissues. d Western blot assay of PTEN and cleaved caspase-3 levels in excised tumor tissues. Each experiment is repeated three times. *P < 0.05 vs. corresponding control