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Research Paper

Knock-down of microRNA miR-556-5p increases cisplatin-sensitivity in non-small cell lung cancer (NSCLC) via activating NLR family pyrin domain containing 3 (NLRP3)-mediated pyroptotic cell death

ORCID Icon, , &
Pages 6332-6342 | Received 06 Jul 2021, Accepted 18 Aug 2021, Published online: 07 Sep 2021

Figures & data

Table 1. The correlations of miR-556-5p levels with the clinicopathological characteristics of NSCLC patients

Figure 1. Upregulated miR-556-5p predicted cisplatin-resistance in NSCLC. (a) The cancer tissues were collected from NSCLC patients with cisplatin-sensitive and resistant properties, and miR-556-5p levels in the above tissues were examined by Real-Time qPCR analysis. (b, c) The CS-NSCLC and CR-NSCLC cells were exposed to high-dose cisplatin for 0 h, 6 h, 12 h, 18 h, 24 h and 48 h, and MTT assay was performed to evaluate cell viability. (d) The NSCLC cells were respectively stained with Annexin V-FITC and PI, and a flow cytometer was used to examine cell apoptosis ratio. (f) The expression levels of miR-556-5p in CS-NSCLC and CR-NSCLC cells were examined by Real-Time qPCR. Each experiment had 3 repetitions, and *P < 0.05

Figure 1. Upregulated miR-556-5p predicted cisplatin-resistance in NSCLC. (a) The cancer tissues were collected from NSCLC patients with cisplatin-sensitive and resistant properties, and miR-556-5p levels in the above tissues were examined by Real-Time qPCR analysis. (b, c) The CS-NSCLC and CR-NSCLC cells were exposed to high-dose cisplatin for 0 h, 6 h, 12 h, 18 h, 24 h and 48 h, and MTT assay was performed to evaluate cell viability. (d) The NSCLC cells were respectively stained with Annexin V-FITC and PI, and a flow cytometer was used to examine cell apoptosis ratio. (f) The expression levels of miR-556-5p in CS-NSCLC and CR-NSCLC cells were examined by Real-Time qPCR. Each experiment had 3 repetitions, and *P < 0.05

Figure 2. Downregulation of miR-556-5p increased cisplatin-sensitivity in CR-NSCLC cells. (a, b) The inhibitor for miR-556-5p was delivered into the A549/DDP and H1299/DDP cells for its downregulation, and the transfection efficiency was examined by Real-Time qPCR. (c, d) Cell viability in the CR-NSCLC cells were measured by performing the MTT assay. The in vivo xenograft tumor bearing mice models were established, and (e, f) tumor weight and (g) volume were monitored, each group had 3 mice. Each experiment had 3 repetitions, and *P < 0.05

Figure 2. Downregulation of miR-556-5p increased cisplatin-sensitivity in CR-NSCLC cells. (a, b) The inhibitor for miR-556-5p was delivered into the A549/DDP and H1299/DDP cells for its downregulation, and the transfection efficiency was examined by Real-Time qPCR. (c, d) Cell viability in the CR-NSCLC cells were measured by performing the MTT assay. The in vivo xenograft tumor bearing mice models were established, and (e, f) tumor weight and (g) volume were monitored, each group had 3 mice. Each experiment had 3 repetitions, and *P < 0.05

Figure 3. Overexpression of miR-556-5p promoted cisplatin-resistance in CS-NSCLC cells. (a, b) The miR-556-5p mimic was transfected into the A549 and H1299 cells for its upregulation. (c, d) Upregulated miR-556-5p increased cell viability in high-dose cisplatin-treated CS-NSCLC cells, as determined by MTT assay. (e, f) Cell apoptosis in A549 and H1299 cells was determined by Annexin V-FITC and PI double staining assay. Each experiment had 3 repetitions, and *P < 0.05

Figure 3. Overexpression of miR-556-5p promoted cisplatin-resistance in CS-NSCLC cells. (a, b) The miR-556-5p mimic was transfected into the A549 and H1299 cells for its upregulation. (c, d) Upregulated miR-556-5p increased cell viability in high-dose cisplatin-treated CS-NSCLC cells, as determined by MTT assay. (e, f) Cell apoptosis in A549 and H1299 cells was determined by Annexin V-FITC and PI double staining assay. Each experiment had 3 repetitions, and *P < 0.05

Figure 4. Knock-down of miR-556-5p promoted NLRP3-mediated cell pyroptosis in high-dose cisplatin treated CR-NSCLC cells. (a) The targeting sites between miR-556-5p and 3ʹUTR of NLRP3 mRNA were predicted, which were validated by performing the following (b, c) dual-luciferase reporter gene system assay. (d) The mRNA and (e) protein level of NLRP3 in NSCLC cells were determined by Real-Time qPCR and Western Blot analysis (The uncropped WB images could be found in Figure S1). (f) NLRP3 mRNA was downregulated in the cisplatin-resistant NSCLC tissues, and (g) correlations between NLRP3 mRNA and miR-556-5p in clinical tissues were analyzed by Pearson correlation analysis. (h, i) The pyroptosis associated signatures were detected by Western Blot analysis (The uncropped WB images were shown in Figure S2-S3), and (j, k) ELISA was performed to evaluate IL-1β and IL-18 secretion in the CR-NSCLC cells’ supernatants. Each experiment had 3 repetitions, and *P < 0.05

Figure 4. Knock-down of miR-556-5p promoted NLRP3-mediated cell pyroptosis in high-dose cisplatin treated CR-NSCLC cells. (a) The targeting sites between miR-556-5p and 3ʹUTR of NLRP3 mRNA were predicted, which were validated by performing the following (b, c) dual-luciferase reporter gene system assay. (d) The mRNA and (e) protein level of NLRP3 in NSCLC cells were determined by Real-Time qPCR and Western Blot analysis (The uncropped WB images could be found in Figure S1). (f) NLRP3 mRNA was downregulated in the cisplatin-resistant NSCLC tissues, and (g) correlations between NLRP3 mRNA and miR-556-5p in clinical tissues were analyzed by Pearson correlation analysis. (h, i) The pyroptosis associated signatures were detected by Western Blot analysis (The uncropped WB images were shown in Figure S2-S3), and (j, k) ELISA was performed to evaluate IL-1β and IL-18 secretion in the CR-NSCLC cells’ supernatants. Each experiment had 3 repetitions, and *P < 0.05

Figure 5. Silence of miR-556-5p increased cisplatin-sensitivity in CR-NSCLC cells via inducing NLRP3-mediated pyroptotic cell death. (a, b) Cell viability was determined by MTT assay, (c–e) and flow cytometer was employed to measure cell apoptosis ratio in A549/DDP and H1299/DDP cells. Each experiment had 3 repetitions, and *P < 0.05

Figure 5. Silence of miR-556-5p increased cisplatin-sensitivity in CR-NSCLC cells via inducing NLRP3-mediated pyroptotic cell death. (a, b) Cell viability was determined by MTT assay, (c–e) and flow cytometer was employed to measure cell apoptosis ratio in A549/DDP and H1299/DDP cells. Each experiment had 3 repetitions, and *P < 0.05
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