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. 2014 Aug 16:13:193.
doi: 10.1186/1476-4598-13-193.

miR-27a regulates cisplatin resistance and metastasis by targeting RKIP in human lung adenocarcinoma cells

Jipeng Li  1 , Yiping Wang, Yulan Song, Zhongming Fu, Wanjun Yu
Affiliations

miR-27a regulates cisplatin resistance and metastasis by targeting RKIP in human lung adenocarcinoma cells

Jipeng Li et al. Mol Cancer. .

Abstract

Background: MicroRNAs (miRNAs) have been identified as important posttranscriptional regulators involved in various biological and pathological processes of cells, but their association with tumor chemoresistance has not been fully understood.

Methods: We detected miR-27a expression in two lung adenocarcinoma cell lines, A549 and A549/CDDP, and then investigated the effects of miR-27a on the metastasis and the chemosensitivity of cancer cells, using both gain- and loss-of-function studies. The correlation between miR-27a level and chemoresistance was further investigated in clinical lung adenocarcinoma specimens.

Results: miR-27a was significantly up-regulated in cisplatin-resistant lung adenocarcinoma A549/CDDP cells compared with parental A549 cells. miR-27a regulates epithelial-mesenchymal transition (EMT) and cisplatin resistance in vitro and modulates response of lung adenocarcinoma cells to cisplatin in vivo. Further studies identified Raf Kinase Inhibitory Protein (RKIP) as a direct and functional target of miR-27a. Small interfering RNA-mediated RKIP knockdown revealed similar effects as that of ectopic miR-27a expression, while overexpression of RKIP attenuated the function of miR-27a in lung adenocarcinoma cells. Increased miR-27a expression was also detected in tumor tissues sampled from lung adenocarcinoma patients treated with cisplatin-based chemotherapy and was proved to be correlated with low expression of RKIP, decreased sensitivity to cisplatin, and poor prognosis.

Conclusion: Our results suggest that up-regulation of miR-27a could suppress RKIP expression and in turn contribute to chemoresistance of lung adenocarcinoma cells to cisplatin.

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Figures

Figure 1
Figure 1
Differences between A549/CDDP cells and parental A549 cells are shown. (A) A549/CDDP cells exhibiting fibroblastic morphology and the A549 cells showing epithelial-like appearance (original magnification, ×100). (B) MTT assay shows that A549/CDDP cells are much more resistant to cisplatin than A549 cells. (C) Western blotting illustrates reduced expression of E-cadherin and increased expression of vimentin in A549/CDDP cells. β-actin was used as an internal control. (D) Invasion assay reveals significant enhancement of invasion ability of A549/CDDP cells in vitro. (E) qRT-PCR indicates a significant up-regulation of miR-27a in A549/CDDP cells compared with A549 cells. miRNA abundance was normalized to U6 RNA. Data are means of three separated experiments ± SD; *P < 0.01.
Figure 2
Figure 2
miR-27a promotes EMT and cisplatin resistance in vitro . A549 cells were transfected with NC or miR-27a mimics, A549/CDDP cells were transfected with anti-NC or miR-27a inhibitors respectively. Western blotting was used to detect E-cadherin and vimentin expression, β-actin was used as an internal control (A). transwell invasion assay (B) and MTT assay (C and D) were used to measure invasion ability and cisplatin sensitivity. Data are means of three separated experiments ± SD; *P < 0.01.
Figure 3
Figure 3
miR-27a regulates response of lung adenocarcinoma cells to cisplatin in vivo. (A) A549 cells were performed after transduction by miR-27a-expressing or vector lentivirus. Representative images of nude mouse lungs (scale bars: 5 mm) and H&E stain of lungs (scale bars: 100 μm) are shown. (B) A549/CDDP cells were performed after transduction by miR-27a-inhibiting or antagomir-NC. Representative images of nude mouse lungs (scale bars: 5 mm) and H&E stain of lungs (scale bars: 100 μm) are shown. *P < 0.01.
Figure 4
Figure 4
RKIP is a direct target of miR-27a. (A) The predicted miR-27a binding site within RKIP 3′UTR and its mutated version by site mutagenesis are as shown. (B) Variable RKIP expression in A549 and A549/CDDP was obtained by western blot. (C) A549 cells were transfected with NC or miR-27a mimics, A549/CDDP cells were transfected with anti-NC or miR-27a inhibitors respectively. Western blotting was used to detect RKIP expression; β-actin was used as an internal control. (D) Luciferase assay was performed in A549 cells that were co-transfected with miRNA mimics and reporter vectors carrying RKIP 3′ UTR with wild type versus mutated miR-27a response element. Data are means of three separated experiments ± SD; *P < 0.01.
Figure 5
Figure 5
RKIP is involved in miR-27a-induced EMT and cisplatin resistance. (A) A549 cells were transfected with RKIP siRNAs, then RKIP, E-cadherin and vimentin protein levels were detected by western blot analysis. β-actin was used as an internal control. MTT assays were used to measure cisplatin sensitivity. (B) A549 cells were transfected with NC, miR-27a mimics or plasmid lacking 3′UTR along with miR-27a, RKIP, E-cadherin and vimentin protein levels were detected by western blot analysis. β-actin was used as an internal control. MTT assays were used to measure cisplatin sensitivity. Data are means of three separated experiments ± SD; *P < 0.01.
Figure 6
Figure 6
The inverse correlation between miR-27a and RKIP expression in lung adenocarcinoma tissue samples and the clinical significance of miR-27a are shown. Relative expression levels of (A) miR-27a and (B) RKIP mRNA were detected in cisplatin-sensitive (n =13) and insensitive (n = 17) lung adenocarcinoma tissues via qRT-PCR. Abundance of miRNA and RKIP mRNA was normalized to U6 RNA and GAPDH, respectively. (C) Expression levels of miR-27a and RKIP mRNA are inversely correlated among all the tissue samples (n = 30) as indicated by two-tailed Pearson’s correlation analysis, r = −0.691; p < 0.01. (D) Kaplan-Meier survival curve indicates that patients with high miR-27a expression have shorter overall survival than those with low miR-27a expression (log-rank test, P < 0.01).
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