LncRNA MEG3 enhances cisplatin sensitivity in non-small cell lung cancer by regulating miR-21-5p/SOX7 axis

Abstract

Background: Long noncoding RNAs (lncRNAs) have been revealed to play essential role in drug resistance of multiple cancers. LncRNA MEG3 was previously reported to be associated with cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC) cells. However, the molecular mechanism of MEG3 affecting DDP resistance in NSCLC remains to be further illustrated. In this study, we attempted to discuss whether MEG3 also could function as a competing endogenous RNA to regulate DDP resistance in NSCLC.

Materials and methods: The expression of MEG3, miR-21-5p, and sex-determining region Y-box 7 (SOX7) in NSCLC tissues or cells was examined by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and caspase-3 activity analysis were applied to assess the DDP sensitivity of NSCLC cells. The interaction between MEG3, miR-21-5p, and SOX7 was explored by luciferase reporter assay, RNA immunoprecipitation (RIP) assay, qRT-PCR, and Western blot. Mouse NSCLC transplanted tumor was established to verify the functional role of MEG3 in DDP resistance in vivo.

Results: MEG3 was downregulated in DDP-resistant NSCLC cells. Overexpression of MEG3 enhanced DDP sensitivity of NSCLC cells in vitro. MEG3 directly interacted with miR-21-5p and suppressed its expression. miR-21-5p significantly abolished the effects of MEG3 on DDP resistance via modulating cell proliferation and apoptosis. SOX7 was identified as a direct target of miR-21-5p and MEG3 positively regulated SOX7 expression by suppressing miR-21-5p. Moreover, MEG3 knockdown-induced pro-proliferative and anti-apoptotic effects were reversed in DDP-resistant NSCLC cells by upregulating SOX7. Furthermore, upregulation of MEG3 induced sensitivity of NSCLC cells to DDP in vivo.

Conclusion: MEG3 overexpression induced DDP sensitivity of NSCLC cells by regulating miR-21-5p/SOX7 axis, shedding light on the molecular mechanism of MEG3 involved in the development of DDP resistance of NSCLC cells.

Keywords: DDP resistance; MEG3; NSCLC; miR-21-5p/SOX7.

Figure 1

Upregulation of MEG3 increased the sensitivity of DDP-resistant NSCLC cells to DDP. Notes: (A) qRT-PCR analysis of MEG3 expression in the tumor tissues of 25 DDP-sensitive and 23 DDP-resistant NSCLC patients. (B) IC₅₀ value was determined by MTT assay after parental (A549, H1299) and DDP-resistant (A549-DDP and H1299-DDP) NSCLC cells were exposed to different concentrations of DDP (0, 2, 4, 8, 16, 32, and 64 μM) for 48 h. (C) qRT-PCR analysis of MEG3 expression in A549, A549-DDP, H1299, and H1299-DDP cells. (D) qRT-PCR analysis of MEG3 expression in MEG3-transfected A549-DDP cells and si-MEG3-introduced H1299-DDP cells. (E) IC₅₀ value was assessed by MTT assay in MEG3-transfected A549-DDP cells and si-MEG3-introduced H1299-DDP cells following treatment with different concentrations of DDP (0, 2, 4, 8, 16, 32, and 64 μM) for 48 h. *P<0.05. Abbreviations: MTT, 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction; si-NC, nonspecific oligonucleotides.

Figure 2

MEG3 acted as a molecular sponge to suppress miR-21-5p expression. Notes: (A) Sequence alignment between miR-21-5p and MEG3. The WT binding sites of miR-21-5p on MEG3 and its mutated binding sequences are shown. (B) Luciferase activity was measured in 293T cells cotransfected with WT-MEG3 or MUT-MEG3 and miR-21-5p or miR-NC. (C) Anti-Ago2 RIP assay was conducted in A549-DDP cell extracts, followed by qRT-PCR analysis of RIP. (D) MEG3 expression was detected by qRT-PCR in MEG3-transfected A549-DDP cells and si-MEG3-transfected H1299-DDP cells. *P<0.05. Abbreviations: Ago2, argonaute 2; miR-NC, scrambled oligonucleotides; MUT, mutated miR-21-5p binding sites; qRT-PCR, quantitative real-time polymerase chain reaction; RIP, RNA immunoprecipitation; WT, wild-type.

Figure 3

miR-21-5p counteracted MEG3-induced DDP sensitivity of DDP-resistant NSCLC cells. Notes: A549-DDP cells were transfected with vector, MEG3, MEG3 + miR-NC, or MEG3 + miR-21-5p, and H1299-DDP cells were introduced with si-NC, si-MEG3, si-MEG3 + anti-miR-NC, or si-MEG3 + anti-miR-21-5p. MTT assay was carried out to assess cell viability at 0, 24, 48, and 72 h in transfected A549-DDP (A) and H1299-DDP (B) cells after treatment with DDP. Flow cytometry analysis was applied to detect apoptosis of transfected A549-DDP (C) and H1299-DDP (D) cells with DDP treatment. Caspase-3 activity analysis was conducted to measure caspase-3 activity in transfected A549-DDP (E) and H1299-DDP (F) cells following DDP treatment. *P<0.05. Abbreviations: miR-NC, scrambled oligonucleotides; MTT, 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide; NSCLC, non-small cell lung cancer; si-NC, nonspecific oligonucleotides.

Figure 4

miR-21-5p targets SOX7 to suppress its expression. Notes: (A) The WT or mutated miR-21-5p binding sites in the 3′UTR of SOX7. (B) Luciferase reporter assay was carried out to measure luciferase activity in A549 and H1299 cells cotransfected with WT-SOX7-3′UTR or MUT-SOX7-3′UTR and miR-21-5p or miR-NC. (C) Western blot analysis was carried out to determine the protein level of SOX7 in A549 cells transfected with miR-21-5p or miR-NC and H1299 cells introduced with anti-miR-21-5p or anti-miR-NC. qRT-PCR analysis of miR-21-5p (D) and SOX7 (E) expressions in 25 DDP-sensitive and 23 DDP-resistant NSCLC patients. *P<0.05. Abbreviations: miR-NC, scrambled oligonucleotides; MUT, mutated miR-21-5p binding sites; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction; SOX7, sex-determining region Y-box 7; UTR, untranslated region; WT, wild-type.

Figure 5

MEG3 improved SOX7 expression via modulating miR-21-5p. Notes: (A) Western blot analysis of protein expression levels of SOX7 in A549-DDP cells with transfection of vector, MEG3, MEG3 + miR-NC, MEG3 + miR-21-5p. (B) Western blot analysis of protein expression levels of SOX7 in H1299-DDP cells with transfection of si-NC, si-MEG3, si-MEG3 + anti-miR-NC, si-MEG3 + anti-miR-21-5p. *P<0.05. Abbreviations: miR-NC, scrambled oligonucleotides; SOX7, sex-determining region Y-box 7; si-NC, nonspecific oligonucleotides.

Figure 6

MEG3 knockdown increased DDP resistance of DDP-resistant NSCLC cells by inhibiting SOX7 expression. Notes: A549-DDP and H1299-DDP cells were introduced with si-NC, si-MEG3, si-MEG3 + vector, or si-MEG3 + SOX7. MTT assay was performed to detect cell proliferation in transfected A549-DDP (A) and H1299-DDP (B) cells after treatment with DDP. Flow cytometry analysis was conducted to assess apoptosis of transfected A549-DDP (C) and H1299-DDP (D) cells following administration with DDP. *P<0.05. Abbreviations: MTT, 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide; NSCLC, non-small cell lung cancer; SOX7, sex-determining region Y-box 7; si-NC, nonspecific oligonucleotides.

Figure 7

Overexpression of MEG3 increased the sensitivity of DDP-resistant NSCLC cells to DDP in vivo. Notes: A549-DDP cells transfected with MEG3 or vector were subcutaneously inoculated into the right flank of nude mice, followed by intraperitoneal injection with 4 mg/kg DDP every 4 days. (A) Changes of tumor volume in nude mice bearing A549-DDP cells with MEG3 or vector. (B) Measurement of excised tumor weight. (C) qRT-PCR analysis for MEG3, miR-21-5p, and SOX7 in removed tumor masses. *P<0.05. Abbreviations: NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction; SOX7, sex-determining region Y-box 7.