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

Figures & data

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.