A Signature of Differentially Expressed Micrornas in Lymphoblastoid Cells from Shwachman-Diamond Syndrome Patients Indicates Possible Molecular Targets for Mirna Therapeutics

Abstract

In the Shwachman-Diamond syndrome (SDS), mutations of the gene encoding for the SBDS protein causes several hematological disorders, including neutropenia and myelodysplastic syndrome (MDS), with high risk of acute myeloid leukemia (AML).MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation. Low miRNA expression is associated with the accumulation of target mRNAs; high miRNA content causes low expression of target mRNAs. In almost all hematological disorders microRNAs were found dysregulated, and often these alterations of miRNAlevels are associated with the phenotype of the disease and tumor onset and progression. In order to verify possible involvement of microRNA in the SDS phenotype, we determined whether the microRNA pattern differentiate Lymphoblastoid Cell Cultures (LCC) derived from SDS patients from healthy subjects-derived LCC. To this aim Next Generation Sequencing (NGS) was performed using Illumina NextSeq500 platform and a signature of 11 up-regulated (hsa-miR-1260b, hsa-miR-1270, hsa-miR-1278, hsa-miR-1294, hsa-miR-296-5p, hsa-miR-34a-3p, hsa-miR-423-3p, hsa-miR-4513, hsa-miR-4521, hsa-miR-6732-3p, hsa-miR-744-3p) and 7 down-regulated (hsa-miR-148a-3p, hsa-miR-148a-5p, hsa-miR-181a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-30d-5p, hsa-miR-660-5p) miRNAs was found in SDS cell cultures compared with controls. The differential expression of the microRNAs identified as dysregulated was confirmed by droplet-digital RT-PCR (ddRT-PCR). In consideration of the possible involvement of microRNAs in the STAT3 hyperactivation and interleukin-6 (IL-6) dysregulation, the 7 miRNAs down-regulated in SDS-LCC were analyzed. Among this miRNA set, miR-148a-5p might be of interest, because it targets the 3'UTR of IL-6 mRNA (position 71-78 of IL-6 3'UTR). MicroRNA miR-148a-5p down-regulation can participate to the IL-6 upregulation in SDS-LCC, demonstrated in LCC supernatants by Bio-Plex analysis of the secretome. Moreover, in consideration of the fact that LCC from SDS patients are characterized by up-regulated mTOR phosphorylation (Bezzerri et al, Sci Rep 2016) possibly associated with down-regulation of PTEN (a validated inhibitor of mTOR phosphorylation), PTEN transcript and protein levels were analyzed in SDS-LCC by RT-PCR and Western Blotting analysis. The data obtained suggest a down regulation of PTEN in SDS-LCC. In order to investigate the possible correlation between PTEN down-regulation and microRNA content in lymphoblastoid cells of SDS patients, we verified whether some of the up-regulated microRNAs found in the NGS analysis were potential regulators of PTEN. When the 11 up-regulated miRNAs were compared with 81 validated PTEN regulating miRNAs (www. mirtarbase.mbc.nctu.edu.tw), two microRNAs were identified: miR-34a-3p and miR-744-3p. Of relevance, the relative expression of miR-34a-3p and miR-744-3p was found variable among the cells from the different SDS patients. When the PTEN expression was related with the miR-34a-3p and miR-744-3p levels, an inverse correlation was found between the miR-34a-3p and miR-744-3p levels and PTEN gene expression. This study allows proposing molecules mimicking miR-148a-5p function in order to reduction of IL-6 expression. On the other hand, miR-34a-3p and miR-744-3p might be considered as possible targets of antagomiRNA molecules, for increasing PTEN expression in SDS patients. Altogether, these data support the concept themicroRNAs might participate to the phenotype of lymphoblastoid cells of SDS patients, therefore being potential molecular targets for miRNA therapeutics of Shwachman-Diamond syndrome. Acknowledgements: This study was supported by a FIR and a FAR fund of Ferrara University to AF, and by a fund from the Italian Ministry of Health (MOH) to VB, MB and GB. Disclosures Bezzerri: Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome". Cipolli:Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome".