MiRNA-195-5p Functions as a Tumor Suppressor and a Predictive of Poor Prognosis in Non-small Cell Lung Cancer by Directly Targeting CIAPIN1

Abstract

Accumulating evidence suggests that microRNAs (miRNAs) has been proven to be a critical regulator in the tumor progression, of which miR-195-5p was reported to function as tumor suppressor in prostate cancer and oral squamous cell carcinoma. However, studies on the clinical significance and biological function of miR-195-5p in non-small cell lung cancer (NSCLC) were still unavailable. Here, we reported that the expression of miR-195-5p was decreased in NSCLC tissues and cell lines. Downregulation of miR-195-5p was significantly associated with TNM stage, tumor size and lymph node metastasis. The Kaplan-Meier survival analysis demonstrated that the survival time of NSCLC patients with high expression of miR-195-5p was longer than those with low expression during the 5-year follow up period (p = 0.0410). COX regression analysis indicated that miR-195-5p expression was an independent prognostic indicator for the survival of NSCLC patients (HR = 2.45, 95% CI: 1.53-4.63; p = 0.007). Results of functional analyses revealed that overexpression of miR-195-5p in A549 cells inhibited cell proliferation, induced cell cycle G0/G1 phase arrest and apoptosis using MTT and flow cytometry analysis. Furthermore, bioinformatics and luciferase reporter assays demonstrated that cytokine-induced apoptosis inhibitor 1 (CIAPIN1), an anti-apoptotic molecule was a direct target of miR-195-5p in NSCLC cells. Meta-analysis based on Oncomine database showed CIAPIN1 was significantly up-regulated in human lung cancer tissues. Consistently, knockdown of CIAPIN1 phenocopied the inhibitory effects of miR-195-5p overexpression in NSCLC cell function. These findings suggest that miR-195-5p could be used as a potential prognostic predictor and tumor suppressor in NSCLC.

Keywords: CIAPIN1; Non-small cell lung cancer; Overall survival; Prognosis; miR-195-5p.

Fig. 1. Downregulation of miR-195-5p correlated with poor prognosis of NSCLC.

(a) Relative expression of miR-195-5p in NSCLC samples (n = 60) and adjacent non-cancerous tissues (n = 60) was measured by quantitative real-time PCR. (b) Kaplan-Meier method was evaluate the five-year survival rate of the patients with high (n = 22) and low (n = 38) expression of miR-195-5p

Fig. 2. Effects of miR-195-5p overexpression on cell proliferation, cell cycle progression and apoptosis in NSCLC cells.

a Expression of miR-195-5p in four NSCLC cell lines and normal bronchial epithelial cell BEAS-2B was determined by qRT-PCR. A549 cells were transfected with miR-195-5p or miR-NC for 48 h, respectively. b The expression of miR-195-5p was measured by quantitative real-time PCR. c Cell proliferation, (d) cell cycle distribution and (e) apoptosis were determined using MTT assay, flow cytometry with PI staining and flow cytometry with Annexin V/PI double staining assay, respectively in A549 cells. All values are represented as mean ± SD of three replicates. *p < 0.05, **p < 0.01, ***p < 0.001 compared with BEAS-2B cells, or miR-NC group

Fig. 3. CIAPIN1 was a direct target of miR-195-5p in NSCLC.

a Diagrams show the miR-195-5p putative binding sites and corresponding mutant sites of CIAPIN1. b miR-195-5p overexpression significantly suppressed the luciferase activity of CIAPIN1 containing a wild-type (WT) of 3′-UTR but not a mutant (MUT) 3′-UTR in A549 cells. All values are represented as mean ± SD of three replicates. ***p < 0.001 compared with miR-NC group; (c) miR-195-5p overexpression reduced the expression of CIAPIN1 protein in A549 cells

Fig. 4. CIAPIN1 mRNA expression in lung cancer tissues vs. normal lung tissues was analyzed by using Oncomine microarray database.

a Total seven microarray datasets regarding CIAPIN1 mRNA expression in lung cancer tissues vs. normal were included in our meta-analysis. Data are shown as the median rank of CIAPIN1 through each dataset analysis. P value for CIAPIN1 was presented using the median ranked analysis. b Three datasets, including Selamat Lung, Landi Lung and Okayama Lung were extracted for analyzing CIAPIN1 mRNA expression in NSCLC tissues vs. normal and data were presented as Log2 median-centered intensity

Fig. 5. Knockdown of CIAPIN1 phenocopied the inhibitory effects of miR-195-5p overexpression in NSCLC.

A549 cells were transfected with siCIAPIN1 or siNC for 48 h. a The expression of CIAPIN1 protein was determined by western blot analysis. b Cell proliferation was determined using MTT assay. c Flow cytomery was performed to detect cell cycle distribution and the percentage of cells at G0/G1, S and G2/M phase was calculated. d Flow cytometry was used to detect cell apoptosis. All values are represented as mean ± SD of three replicates. ***p < 0.01, ***p < 0.001 compared with siNC group