miR-130a targets MET and induces TRAIL-sensitivity in NSCLC by downregulating miR-221 and 222

Abstract

Non-small cell lung cancer (NSCLC) accounts for ∼80% of all lung cancers. Although some advances in lung cancer therapy have been made, patient survival is still quite poor. Two microRNAs, miR-221 and miR-222, upregulated by the MET proto-oncogene, have been already described to enhance cell survival and to induce TNF-related apoptosis-inducing ligand (TRAIL) resistance in NSCLC cell lines, through the downregulation of p27(kip1), PTEN and TIMP3. Here, we further investigated this pathway and showed that miR-130a, expressed at low level in lung cancer cell lines, by targeting MET was able to reduce TRAIL resistance in NSCLC cells through the c-Jun-mediated downregulation of miR-221 and miR-222. Moreover, we found that miR-130a reduced migratory capacity of NSCLC. A better understanding of MET-miR-221 and 222 axis regulation in drug resistance is the key in developing new strategies in NSCLC therapy.

Figure 1. miRNA 130a targets the MET protoncogene

a) MET contains one predicted miR-130a binding site. In the figure the alignment of the seed region of miR-130a with MET 3′ UTR is shown. The sites of target delection mutagenesis are indicated in red. b) qRT-PCR in A549 after enforced expression of miR-130a. c) MET 3′UTR is target of miR-130a. pGL3-MET luciferase construct containing a wild type (left side of histogram) or mutated (right side of histogram) MET 3′ UTR, was trasfected in A549 cells. * Significance values of P< 0.05 relative to A549 miR scrambled trasfected. d) miR-130a enforced expression decreases endogenous levels of MET protein in A549 NSCLC. A549 cells were trasfected with scrambled and miR-130a for 72h. MET expression was assessed by western blot. Loading control was obtained using anti-β-actin antibody. e) qRT-PCR showing miR-130a overexpression in A549 cells after miR-130a enforced expression related to Fig. 1d. f) qRT-PCR of MET mRNA after miR-130a forced expression in A549 cells. MET mRNA is downregulated by miR-130a. Error bars indicate ± SD.

Figure 2. miRNA 130a downregulates miRNA 221/222 expression through AP1

a) Western blots after miR-130a forced expression. MiR-130a forced expression induces the inhibition of the Akt pathway comparable with the inhibition that results by the downregulation of MET expression by siRNA-MET. b) qRT-PCR showing miR-130a expression in A549 cells after miR-130a enforced expression related to Fig. 2a. c) AP1 activity is modulated by miR-130a. pGL3-AP1 responsive element luciferase construct was trasfected with scrambled and miR-130a for 24, 48 and 72h. * Significance values of P< 0.005 relative to A549 miR scrambled trasfected. d) qRT-PCR showing miR-221&222 levels after enforced expression of a scrambled miR and miR-130a. * Significance values of P< 0.05 relative to A549 miR scrambled trasfected. e) ChIP analysis was performed with chromatin from A549 c-Jun positive cells. A549 cells were non-trasfected or trasfected with miR-control, miR-130a, siRNA-control and siRNA-MET. Chromatin was immunoprecipited with c-Jun antibody. f) Western blot after enforced expression of miR-130a and siRNA-MET related to Fig. 2 e showing the MET is downregulation as control. Error bars indicate ± SD.

Figure 3. miRNA 130a activates apoptosis in NSCLC through miR-221 and miR-222 downregulation

a) Western blot after scrambled, miR-130a and miR-221 enforced expression in A549 cells treated with (400ng/ml) TRAIL for 40 minutes. miR-130a induces PARP activation after TRAIL treatment. Co-trasfection of miR-130a and miR-221induces only partially the PARP cleavage. b) qRT-PCR showing miR-130a and miR-221 after their enforced expression, data related to Fig.3a. c) qRT-PCR showing miR-221/222 endogenous expression levels after miR-130a trasfection in A549 cell treated with (400ng/ml) TRAIL for 40 min. d) Proliferation assay on A549 cells. Cells were incubated with Super-Killer TRAIL (400ng/ml) for 48hr and viability was evaluated as described in the experimental procedures. * Significance values of P < 0.05 relative to untreated A549 cells. e) Apoptosis was evaluated by Annexin-V. A549 were trasfected with miRNA control, miR-130a, miR-221 and both miR-130a/221. Cells were incubated for 3hr with Super-Killer TRAIL (400ng/ml). The percentage of apoptotic cells increase after miR-130a overexpression. The introduction of both miR-130a and miR-221 partially restore TRAIL resistance. f) Apoptosis was evaluated by caspase 3/7. A549 were trasfected with miRNA control, miR-130a, miR-221 and both miR-130a/221.Cells were incubated for 3hr with Super-Killer TRAIL (400ng/ml). Apoptosis was evaluated by caspase 3/7 activity as described in the experimental procedeures. * Significance values of P < 0.05 relative to untreated A549 cells. Error bars indicate ± SD.

Figure 4. miRNA 130a modulates migration in NSCLC

a) A549 monolayer cultures were scratched and incubated in RPMI 5% FBS. Directly after scratching (0h), 8hs and 24hs later images were acquired. Dashed lines indicate the front of migration. b) Quantization of migration distance using Image J software. * Significance values of P < 0.05 relative to miR-scramble trasfected A549 cells. c) qRT-PCR showing miR-221&222 after enforced expression of scrambled miR and miR-130a related to Fig. 4a and 4b. d) A549 cells were trasfected with miR-control and miR-130a and then plated on a pore membrane lying on RPMI 10% FBS media. After 24h of incubation migrated cells were stained and images were acquired. Representative fields of migrated cells (magnification, ×100). e) Quantization of migration by migrated cell counting. * Significance values of P < 0.05 relative to miR-scramble trasfected A549 cells. f) qRT-PCR showing miR-221&222 after enforced expression of scrambled miR and miR-130a related to Fig. 4d and 4e. * Significance values of P < 0.05 relative to miR-scramble trasfected A549 cells. Error bars indicate ± SD.