miR-124 and miR-142 enhance cisplatin sensitivity of non-small cell lung cancer cells through repressing autophagy via directly targeting SIRT1

Abstract

Background: Drug resistance is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Recently, miRNAs are reported to be involved in the drug resistance of NSCLC. The roles of miR-124 and miR-142 in the multidrug resistance of NSCLC cells have been reported. However, the underlying mechanism by which miR-124 and miR-142 regulate resistance to cisplatin (CDDP) remains unknown. Methods: The expressions of miR-124, miR-142 and sirtuin 1 (SIRT1) in CDDP-sensitive and CDDP-resistant NSCLC tissues and cells were detected by qRT-PCR and western blot. IC50 value and cell proliferation were determined by MTT assay. Apoptosis was assessed by flow cytometry analysis. Autophagy was evaluated by western blot analysis of the protein levels of LC3-I, LC3-II and p62, and FITC-LC3 punctate formation assay. The interaction between miR-124 or miR-142 and SIRT1 was determined by luciferase reporter, RNA immunoprecipitation (RIP) and western blot assays. A tumor xenograft was performed to further validate the role of miR-124 and miR-142 in the sensitivity of CDDP-resistant NSCLC to cisplatin. Results: miR-124 and miR-142 were downregulated, while SIRT1 was upregulated in CDDP-resistant NSCLC tissues and cells compared to CDDP-sensitive groups. Functionally, overexpression of miR-124 and miR-142 or SIRT1 silencing enhanced the CDDP sensitivity of H1299/CDDP cells via suppressing autophagy, as evidenced by the reduced LC3-II/LC3-I radio, elevated p62 protein, and suppressed FITC-LC3 punctate formation in H1299/CDDP cells. miR-124 and miR-142 were demonstrated to co-target SIRT1. Re-expression of SIRT1 overturned miR-124 and miR-142-mediated chemosensitivity in H1299/CDDP cells via triggering autophagy. Conclusion: miR-124 and miR-142 enhance the cytotoxic effect of CDDP through repressing autophagy via targeting SIRT1 in CDDP-resistant NSCLC cells.

Fig. 1. The expressions of miR-124 and miR-142 in CDDP-sensitive and CDDP-resistant NSCLC. (A and B) qRT-PCR analysis was performed to determine the expressions of miR-124 and miR-142 in adjacent normal tissues, CDDP-sensitive and CDDP-resistant NSCLC patients. (C) MTT assay was performed to determine the IC50 value of CDDP in H1299 and H1299/CDDP cells. (D) The enrichments of miR-124 and miR-142 in H1299 and H1299/CDDP cells. *P < 0.05.

Fig. 2. The effects of miR-124 or miR-142 on the chemosensitivity of CDDP-resistant NSCLC cells. H1299/CDDP cells were transfected with miR-NC, miR-124, miR-nc, or miR-142. (A) The expressions of miR-124 and miR-142 in H1299 and H1299/CDDP cells were measured by qRT-PCR. (B–D) MTT and flow cytometry assays were performed to determine IC50 value, cell proliferation, and apoptosis in H1299/CDDP cells. (E and F) Western blot and FITC-LC3 punctate formation were carried out to analyze cell autophagy. *P < 0.05.

Fig. 3. The interaction between miR-124, miR-142 and SIRT1 in CDDP-resistant NSCLC cells. (A) The predicted miR-124 or miR-142 binding sites in the 3′UTR of SIRT1. (B) After H1299/CDDP cells were cotransfected with miR-124, miR-142, or respective controls and luciferase reporter plasmids carrying the wild-type or mutated 3′UTR of SIRT1, luciferase reporter assay was performed to detect luciferase activity. (C) The enrichments of miR-124, miR-142, and SIRT1 were detected by RIP assay. (D) The protein level of SIRT1 in H1299/CDDP cells transfected with miR-124, miR-142, or respective control was evaluated by western blot. *P < 0.05.

Fig. 4. The effects of SIRT1 silencing on the chemoresistance of CDDP-resistant NSCLC cells. (A) The mRNA expression of SIRT1 in CDDP-sensitive and CDDP-resistant NSCLC patients was detected by qRT-PCR. (B) The protein level of SIRT1 in H1299 or H1299/CDDP cells was measured by western blot. (C) The protein level of SIRT1 in H1299/CDDP cells transfected with si-SIRT1 or si-NC was examined by western blot. (D–F) H1299/CDDP cells were transfected with si-NC or si-SIRT1. MTT and flow cytometry assays were carried out to determine IC50 value, cell proliferation, and apoptosis. (G and H) Western blot and FITC-LC3 punctate formation assays were performed to assess cell autophagy. *P < 0.05.

Fig. 5. miR-124 and miR-142 inhibited autophagy and facilitated apoptosis in CDDP-resistant NSCLC cells by targeting SIRT1. H1299/CDDP cells were transfected with miR-124, miR-142, or matched controls or combined with SIRT1 or pcDNA. (A) The protein levels of SIRT1 in H1299/CDDP cells were detected by western blot assay. (B–D) MTT and flow cytometry assays were performed to measure the IC50 value, cell proliferation, and apoptosis. (E and F) Western blot and FITC-LC3 punctate formation were performed to assess cell autophagy. *P < 0.05.

Fig. 6. miR-124 and miR-142 enhanced chemosensitivity in vivo. BABL/c nude mice were injected subcutaneously with miR-NC, miR-124, miR-nc, or miR-142-transfected H1299/CDDP cells, and then treated with 10 mg kg⁻¹ CDDP when tumor volume was about 60 mm³. (A) Tumor volume was measured at different periods after treatment. (B) Tumor weight was detected at end point. (C) miR-124 and miR-142 expressions in tumor tissues were detected by qRT-PCR. (D) The protein expression of SIRT1 in tumor was determined by western blot assay. *P < 0.05.