Down-regulation of miR-214 reverses erlotinib resistance in non-small-cell lung cancer through up-regulating LHX6 expression

Abstract

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are standard treatments for advanced non-small-cell lung cancer (NSCLC) patients. However, acquired resistance to EGFR-TKIs is widely detected across the world, and the exact mechanisms have not been fully demonstrated until now. This study aimed to examine the role of miR-214 in the acquired resistance to erlotinib in NSCLC, and elucidate the underlying mechanisms. qRT-PCR assay detected higher miR-214 expression in the plasma of NSCLC patients with acquired EGFR-TKI resistance than prior to EGFR-TKI therapy, and in the generated erlotinib-resistant HCC827 (HCC827/ER) cells than in HCC827 cells. Bioinformatics analysis and dual-luciferase reporter assay indentified LHX6 as a direct target gene of miR-214, and LHX6 expression was detected to be down-regulated in erlotinib-resistant HCC827 cells. Transwell invasion assay revealed that overexpressing LHX6 reversed the increase in the invasive ability of HCC827 cells induced by miR-214 overexpression, and the CRISPR-Cas9 system-mediated LHX6 knockdown reversed the reduction in the invasion of erlotinib-resistant HCC827 cells caused by miR-214 down-regulation. The results of the present study demonstrate that down-regulation of miR-214 may reverse acquired resistance to erlotinib in NSCLC through mediating its direct target gene LHX6 expression.

Figure 1

miR-214 expression in HCC827 and HCC827/ER cells and in the plasma of NSCLC patients pre- and post-treatment with EGFR-TKIs. (a) qRT-PCR assay revealed an increase in the miR-214 expression in the plasma of NSCLC patients with acquired EGFR-TKI resistance than in NSCLC patients prior of EGFR-TKI therapy (P = 0.398). (b) MTS assay showed a 289 nM IC₅₀ for HCC827 cells and a 1,843 nM erlotinib IC₅₀ for HCC827/ER cells. *P < 0.05; (c) qRT-PCR assay showed significant up-regulation of miR-214 expression in HCC827/ER cells than in HCC827 cells. *P < 0.05; All data are expressed as mean ± SD from three independent experiments.

Figure 2

LHX6 is a direct target gene of miR-214. (a) Following 48 h transfection, qRT-PCR assay shows up-regulation of miR-214 expression in HCC827 cells transfected with miR-214 agomir than in cells transfected with miR-NC (P < 0.01); (b) qRT-PCR analysis reveals down-regulation of LHX6 expression in HCC827 cells transfected with miR-214 agomir than in cells transfected with miR-NC agomir (P < 0.01); (c and d) Western blotting assay reveals down-regulation of LHX6 protein expression in HCC827 cells transfected with miR-214 agomir than in cells transfected with miR-NC agomir (P < 0.0001); (e) Sequences of the 3′ UTR region of the LHX6 gene matching to miR-214. The 5′ terminus of miR-214, matched by the seed sequences of the LHX6 3′ UTR region, is ligated to the dual-luciferase reporter vector psi-check2 to construct wild-type (LHX6-3′ UTR/wt) and mutant (LHX6-3′ UTR/mut1 and LHX6-3′ UTR/mut2) dual-luciferase reporter genes; (f and g) Following co-transfection in 293 T (F) and HCC827 cells (G) with 50 nM miR-NC or miR-214 agomir in combination with 0.1 μg LHX6-3′ UTR-wt, LHX6-3′ UTR-mut1 or LHX6-3′ UTR-mut2 for 24 h, dual-luciferase reporter assay reveals a significant lower relative luciferase activity of LHX6-3′ UTR/wt in cells transfected with miR-214 agomir than in cells transfected with miR-NC, while significantly up-regulated relative luciferase activities of LHX6-3′ UTR/mut1 (P < 0.001) and LHX6-3′ UTR/mut2 (P < 0.001) were measured than that of LHX6-3′ UTR/wt in HCC827 and 293 T cells transfected with miR-214 agomir. All data are expressed as mean ± SD from three independent experiments. *P < 0.01; **P < 0.001; ***P < 0.01; ****P < 0.01.

Figure 3

LHX6 expression is down-regulated in HCC827/ER cells at both translational and transcriptional levels. (a) qRT-PCR assay reveals down-regulation of LHX6 mRNA expression in HCC827/ER cells than in HCC827 cells (P < 0.0001); (b and c) Western blotting analysis shows down-regulation of LHX6 protein expression in HCC827/ER cells than in HCC827 cells (P < 0.0001). All data are expressed as mean ± SD from three independent experiments. ****P < 0.01.

Figure 4

Effect of miR-214 expression on the sensitivity to EGFR-TKI in HCC827 and HCC827/ER cells. (a and b) Western blotting analysis determines LHX6 protein expression in HCC827 cells, HCC827 cells transfected with miR-214 agomir for 48 h, and HCC827 cells co-transfected with miR-214 agomir and pLV-LHX6 for 48 h, respectively; (c) MTS assay measures 72 h erlotinib IC₅₀ values in HCC827 cells, HCC827 cells transfected with miR-214 agomir for 24 h, and HCC827 cells co-transfected with miR-214 agomir and pLV-LHX6 for 24 h, respectively; (d) Transwell invasion assay measures the counts of membrane-penetrating cells in HCC827 cells, HCC827 cells transfected with miR-214 agomir for 48 h, and HCC827 cells co-transfected with miR-214 agomir and pLV-LHX6 for 48 h, respectively; (e) Following miR-214 knockdown using the CRISPR/Cas9 system, qRT-PCR assay shows down-regulation of miR-214 expression in HCC827/ER/miR-214-CRI cells; (f and g) In the stably transfected HCC827/ER/mir-214-CRI cell line, sg1 and sg2 targeting LHX6 were designed and linked with the vector lentiCRISPRv2 to establish stably transfected HCC827/ER/miR-214-CRI/LHX6-sg1 and HCC827/ER/miR-214-CRI/LHX6-sg2 cell lines with miR-214 knockdown, and then, LHX6 protein expression is determined in these cell lines using Western blotting assay; (h) MTS assay reveals 72 h erlotinib IC₅₀ values in these cell lines; (i) Transwell invasion assay measures the counts of membrane-penetrating cells. All data are expressed as mean ± SD from three independent experiments.