miRNA-503 inhibition exerts anticancer effects and reduces tumor growth in mesothelioma

Abstract

Background: Malignant mesothelioma (MM) is a rare and aggressive form of cancer that affects the mesothelial surfaces, associated with exposure to asbestos fibres. To date, no cure is available for MM and therapeutically approved treatments are based on the use of platinum compounds often used in combination with other drugs. We have previously analysed the efficacy of a cisplatin/piroxicam (CDDP/P) combined treatment showing that this treatment was able to reduce in vivo tumor growth. Several studies reported that platinum-drug sensitivity in cancer is connected to modulation of the expression of non-coding RNAs. In this study we analysed if the CDDP/P treatment was able to modulate miRNAs expression in MM.

Methods: miRNA sequencing performed on MSTO-211 H cells treated with CDDP with CDDP/P led us to identify miRNA-503 - downregulated by CDDP/P - as a novel miRNA that acts as an oncomiR in MM. The effect of miRNA-503 inhibition was evaluated in vitro in mesothelioma cells analysing apoptosis induction and reduction of cancer properties. Inhibition of miR-503 expression in vivo, was analysed in ectopic mouse model of MM by using LNP encapsulating anti-mir-503 and miR-503 expression was evaluated in human MM samples.

Results: In vitro and in vivo analysis confirmed miR-503 acts as oncogene in MM since its inhibition was able to reduce cell cancer properties and tumor growth in ectopic mouse model of MM. Its expression was found upregulated in human MM patients compared to normal pleura. Bioinformatic analysis indicated BTG1, CCNG1, EDG1, and TIMP2 as putative target genes of miRNA-503. These genes showed an opposite expression compared to miR-503 levels both in cells and in MM samples. Finally, microarray analysis indicated that miR-503 inhibition affected the expression of the well-known MM biomarkers: CXCL8, SERPINE1 and Osteopontin.

Conclusions: Our study is the first reporting an oncomiR role for miR-503 in MM and suggests that its inactivation could have a clinical value in MM patients. This study reveals that miRNA-503 acts as an oncomiR in MM suggesting that its inhibition, through LNP delivery, has the potential to be considered as a novel therapeutic strategy in MM.

Keywords: Apoptosis; LNPs; Mesothelioma; Tumor growth inhibition; miRNA-503 inhibition.

Fig. 1

miR-503 downregulation affects cancer properties in MM in vitro. A NGS expression values of DEG miRNAs selected after single CDDP and combined CDDP/P treatments and q-PCR validation in MM cells. B Effects on cell viability of miR-503 inhibition at different time. 48 h treatment resulted the best time and reduction was 46% MSTO, 66% NCI and 43% Mes2. A lower decrease was detected after 24 h (75% MSTO, 68% NCI and 84% Mes2) while after 72 h the effect was comparable to the 48 h (49% MSTO, 69% NCI and 50% Mes2). C FACS analysis confirms that cell viability decrease after 48 h treatments is due to apoptotic increase in all MM cells treated with anti-miR-503 (33% MSTO, 35% NCI and 16% Mes2) compared to nc-anti-miR-503 (6% MSTO, 4% NCI and 4% Mes2), used as control. Histograms report a data summary of the apoptotic index for both treatments. D Crystal violet colony assay in MM cells transfected for 48 h with anti-miR-503 or with negative control (nc-anti-miR-503). Cells were grown for 7 days before transfection and after additional 7 days the colonies were stained with 0.4% (w/v) crystal violet and counted using Image J. Reduction of colony numbers were: 50% MSTO, 50% NCI and 45% Mes2. Data are shown in the histogram. E Wound-healing assay shows that miR-503 inhibition after 48 h greatly reduced the migration capability of MM cells. The graph reports the residual gap in treated anti-miR-503 cells compared to control (80% MSTO, 80% NCI and 76% Mes2). Data are presented as the mean ± SD of at least three independent experiments (n = 3). **p < 0.01 ***p < 0.001 ****p < 0.0001 vs. control

Fig. 2

In vivo anticancer effects of miR-503 inhibition. A Experimental scheme showing design and details of the mesothelioma xenograft mouse model of MM. Treatments was performed inoculating i.v., after tumor growth, LNPs-anti-miR-503 or empty LNPs. B Tumor growth reduction evaluated by analysing tumor masses volume at the days of treatment. Data are expressed as tumor mass volume means ± SEM for each group and the linear regression (dashed line) is reported. Images of tumor masses from mice treated with LNPs-anti-miR-503 or with empty LNPs at the end of experiment are shown. C Effects of miR-503 inhibition on cell proliferation (Ki67) and apoptosis (TUNEL) in tumor masses compared to control (empty LNPs) (original magnification 20X). Bar plots indicates no changes for Ki67 and higher apoptotic index in tumors treated with LNPs-anti-miR-503 compared to empty LNPs, confirming the pro-apoptotic role of miR-503 inhibition in vivo. D Expression analysis of miR-503 putative target genes by q-PCR in MM cells after miR-503 inhibition E q-PCR analysis in human MM samples of the miR-503 putative target genes confirms their inverse expression and the concomitant up-regulation of miR-503. F Base pair alignment among miR-503 and the 3’UTR region of the putative target genes. Data are presented as the mean ± SD of at least three independent experiments (n = 3) for the in vitro experiments while for the in vivo analysis numbers of samples are reported in Methods. *p < 0.05, **p < 0.01 ***p < 0.001 ****p < 0.0001 vs. control

Fig. 3

Histological analysis of miR-503 target genes and transcriptomic analysis of miR-503 inhibition. A Evaluation of the expression of BTG1, CCNG1, EDG1 and TIMP2, in normal pleural (PL) and pleural mesothelioma tissues (EPM epithelial istotype; SPM, sacomatoid istotype). Yellow Scale bar = 100 μm. Data shown are mean ± S.D. of five independent experiments. B IHC analysis of the putative miR-503 target genes on mice tumors treated with LNPs-anti-miR-503 or empty LNPs (original magnification 20X). A and B confirm the in vitro results. C Transcriptome analysis in MSTO cells after miR-503 inhibition detected about 500 deregulated genes (red upregulated, green downregulated). D Expression analysis of MM biomarkers deregulated by inhibition of miR-503 in MM cells. E q-PCR analysis in human MM samples of the miR-503 MM biomarkers confirms the inverse expression only for CXCL8, SERPINE1 and SPP. F IHC analysis of the miR-503 deregulated MM biomarkers on mice tumors treated with LNPs-anti-miR-503 or empty LNPs (original magnification 20X). For each IHC panel bar plots report the score for the samples analysed. Data presented as the mean ± SD of at least three independent experiments (n = 3) for the in vitro experiments while for the in vivo analysis numbers of samples are reported in Methods. *p < 0.05, **p < 0.01 ***p < 0.001 ****p < 0.0001 vs. control