Silencing of Long Non-coding RNA MIAT Sensitizes Lung Cancer Cells to Gefitinib by Epigenetically Regulating miR-34a

Abstract

Long non-coding RNA (lncRNA) myocardial infarction associated transcript (MIAT) was recently identified as oncogene in several cancers. However, the role of MIAT on acquired resistance in lung cancer and the underlying mechanisms remain unclear. Here, we showed that the expression of MIAT in lung cancer tissues was upregulated compared with adjacent tissues. LncRNA MIAT expression was associated with tumor size, lymph node metastasis, distant metastasis and TNM stage. Univariate analysis and multivariate analysis revealed that the lncRNA MIAT to be an independent factor for predicating the prognosis of lung cancer patients. Low lncRNA MIAT have longer overall survival time and progression-free survival time than patients with high lncRNA MIAT expression. Moreover, the knockdown of MIAT significantly sensitized PC9 and gefitinib-resistant PC9 cells to gefitinib in vitro and in vivo, and increased the expression of miR-34a and inactivated PI3K/Akt signaling. MIAT interacted with miR-34a and epigenetically controlled the miR-34a expression by hyper-methylating its promotor. Taken together, our findings demonstrated that knockdown of MIAT by siRNA enhances lung cancer cells to gefitinib through the PI3K/Akt signaling pathway by epigenetically regulating miR-34a. Thus, MIAT may be a useful prognostic marker and therapeutic target for lung cancer patients.

Keywords: acquired resistance; c-Met; epigenetic; long non-coding RNA; lung cancer.

FIGURE 1

The expression of MIAT in lung cancer tissues. (A) qRT-PCR was used to determine the expression of MIAT in NSCLC tissues (N = 212) and adjacent control (N = 186). (B) The expression of MIAT in adjacent tissues and grade I+II and grade III+IV lung cancer tissues. (C) The expression of MIAT in patients who sensitive (N = 45) or primary resistant (N = 25) to EGFR-TKI. (D) Overall survival analysis in NSCLC patients with low or high MIAT expression. (E) Progress-free survival analysis in NSCLC patients with low or high MIAT expression. (F) The expression of MIAT in BEAS-2B and lung cancer cell lines. ^∗P < 0.05.

FIGURE 2

Knockdown of MIAT sensitizes PC9 and PC9/R cells to gefitinib. (A) qRT-PCR analysis for MIAT in PC9 and PC9/R cells after transfection with MIAT siRNA or negative control. (B) CCK-8 assay was used to determine the IC50 in PC9 and PC9/R cells (PC9 vs. PC9/R: 29.05 vs. 62.61 μM). (C) CCK-8 assay was used to determine the change of IC50 in PC9/R cells after knockdown of MIAT (siMIAT vs.NC: 38.2 vs. 60.2 μM). (D) Colony formation assay was used to determine the cell growth after knockdown of MIAT and gefitinib treatment. (E) Flow cytometry was used to measure cell apoptosis after knockdown of MIAT and gefitinib treatment. (F) Western blot analysis for apoptosis marker Bcl-2, Bax and cleaved caspase 3 after knockdown of MIAT and gefitinib treatment. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 3

MIAT epigenetically regulates miR-34a. (A) The relative luciferase activities were inhibited in the PC9 cells co-transfected with wild-type MIAT 3′UTR vector and miR-34a, but not with the mutant-type vector. Firefly luciferase activity was normalized to Renilla luciferase. (B) Association of MIAT and miR-34a between with Ago2 in PC9 cells. Cellular lysates from PC9 cells were used for RIP with antibody against Ago2. MIAT and miR-34a expression levels were detected using qRT-PCR. (C) qRT-PCR analysis for miR-34a in PC9 and PC9/R cells after transfection with MIAT siRNA or negative control. (D) MSP test results indicated that methylation of miR-34a promoter was higher in lung cancer tissues than that of in matched adjacent tissues. H1870 cells were used as positive control and H1048 cells were used as negative control. M, methylation; U, unmethylation. (E) qRT-PCR analysis for miR-34a in lung cancer tissues and matched adjacent tissues in (D). N = 6. (F) The BSP detection results demonstrated that methylation of miR-34a was decreased after MIAT siRNA transfection in PC9 cells. Solid circle, methylation; hollow circle, unmethylation. PCR products amplified from bisulfite-treated genomic DNA were cloned and sequenced to reveal the methylation status of individual CpG sites. Percentages of the methylated CpG sites (filled circles) among all scored sites are indicated. (G) Luciferase reporter analysis of luciferase activity in PC9 cells cotransfected with pGL3-miR-34a and MIAT siRNA lentivirus or an empty lentivirus. (H) Association of MIAT and Dnmts in PC9 cells. Cellular lysates from PC9 cells were used for RIP with antibody against Dnmt3a, Dnmt3b or Dnmt1. MIAT expression levels were detected using qRT-PCR. IgG was used as a negative control. (I) Western blot analysis for validated miR-34a target, c-Met after indicated treatment. GAPDH was used a loading control. ^∗P < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 4

MiR-34a inhibitor reverses the inhibitory effects of MIAT downregulation on PC9 cells. (A) CCK-8 assay was used to determine the cell viability after miR-34a inhibitor and MIAT siRNA treatment in PC9 cells. (B) Colony formation assay was used to determine the cell growth after miR-34a inhibitor and MIAT siRNA treatment in PC9 cells. (C) Flow cytometry was used to measured cell apoptosis after miR-34a inhibitor and MIAT siRNA treatment in PC9 cells. (D) Western blot analysis for p-EGFR, EGFR, p-PI3K, PI3K, p-AKT, AKT and c-Met after miR-34a inhibitor and MIAT siRNA treatment in PC9 cells. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 5

Knockdown of MIAT inhibits lung cancer growth in vivo. (A) the PC9/R cells transfected with MIAT siRNA, or together with miR-34a inhibitor were injected into nude mice. The cells transfected with empty plasmid were used as negative control. Mice were euthanized and tumors obtained from mice on day 28 after injection. (B) IHC was used to detect the expression of Ki67 in tumor section (upper), and quantification (lower). Scale bar, 100 μm. (C) TUNEL was performed to detect the apoptosis cells in tumor section (left), and the quantification of luciferase intensity (right). (D) qRT-PCR analysis for MIAT and miR-34a expression in xenografted tumor tissues. (E) Western blot analysis for c-Met and PI3K signaling proteins in xenografted tumor tissues. GAPDH was used a loading control. (F) qRT-PCR analysis for MIAT and miR-34a expression in human lung cancer tissues with high MIAT or low MIAT. ^∗P < 0.05.

FIGURE 6

Schematic diagram of mechanism of MIAT epigenetically regulating miR-34a. MIAT interacts with Dnmt3a and recruits it on miR-34a promotor to hyper-methylate miR-34a, increasing c-Met expression, and finally conferring chemoresistance, promoting proliferation and inhibiting apoptosis.