Regulatory Non-Coding RNAs as Cellular Rheostats: Modulating Biological Functions to Pathophysiology of Diseases

Abstract

The non-coding RNAs (ncRNAs) which are previously considered as ‘junk’ have garnered the attention of researchers because of their ability to perform diverse biological functions by modulating gene expression at various levels like transcription, RNA processing, and translation. The major group of ncRNAs encompassing small RNAs such as microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs) and long ncRNAs (lncRNAs) are highly tunable and are considered as molecular rheostat controlling gene expression. In this study, we deciphered various facets of ncRNA-mediated regulations performed by miRNA, piRNA and lncRNA and their impacts on normal biological processes as well as pathophysiology of diseases. We first studied the impact of single nucleotide polymorphisms (SNPs) associated with late-onset Alzheimer’s disease (LOAD) on miRNA binding by performing target prediction of differentially expressed miRNAs of LOAD against genes harboring SNPs in 3/UTRs followed by gene ontology, and interactome analysis. We reported three candidate MRE-SNPs (rs10876135, rs5848 & rs5786996) altering the binding sites of hsa-miR-197-5p, hsa-miR-185-5p and hsa-miR-34a-5p respectively, which are implicated in the progression of LOAD. Further, we extended our study to piRNAs, the newly discovered class of small ncRNAs whose functions were previously believed to be restricted to germ lines as ‘TE (Transposable elements) traps’, but are now reported to function in somatic cells including neurons and also abnormalities like cancer. Based on their evidences in neuronal functions such as synaptic plasticity in mouse, memory storage in Aplysia neurons, we conjectured the presence of these tiny RNAs in human brain and AD. We performed small RNA sequencing and identified a catalog of 564 and 451 piRNAs expressed in healthy human brain and AD-affected brain respectively. We characterized these piRNAs based on their origin, length, nucleotide biasness, conservation, presence in piRNA clusters, etc. We functionally characterised the deregulated piRNAs in AD by looking for their targets and involvement in AD associated pathways. Our study showed four piRNAs targeting and modulating four genes (CYCS, LIN7C, KPNA6, and RAB11A) that are enriched in five most significant AD-associated pathways. Based on increasing evidences of piRNAs in cancer, in the subsequent study, we investigated the piRNA transcriptome in human neuroblastoma (NB) cell lines, IMR-32 and SH-SY-5Y after confirming the expression of three PIWILs at both mRNAs and protein level by qRT-PCR and immunofluorescence respectively. We found a common pool of 525 piRNAs expressed in these NB cell lines. The genome-wide functional analysis revealed key piRNAs and their target genes enriched in cancer-related networks, pathways and biological processes indicating their crucial roles in oncogenesis of the malignancy. We then selected a repeat derived piRNA, piR-39980 abundantly expressed in NB and investigated its potential role in the oncogenic processes. Our study unveiled that piR-39980 act as an oncopiR and promotes tumor progression by inducing cell viability, cell proliferation, metastasis, and inhibiting cellular senescence. We also found JAK3 as the molecular target of the piRNA which was validated by inverse expression analysis through qRT-PCR as well as dual luciferase reporter assay. Additionally, piR-39980 was found to desensitize the effect of Doxorubicin, a commonly used chemotherapeutic drug against NB and inhibit cell death. This study revealed that piR-39980 could be used as a potential therapeutic target for this pediatric neoplasm. Finally, we studied the possible gene regulatory mechanism of an lncRNA, Khps1 in the transcriptional activation of a proto-oncogene, SPHK1-B. We observed that the lncRNA physically interact with E2F1 near a triplex formation region which might be responsible for the recruitment of TF to the transcriptional start site of the mRNA leading to its activation. This study revealed that Khps1 lncRNA acts as a molecular scaffold. To sum up, the present study identified and decrypted the molecular mechanisms through which three ncRNAs- miRNAs, piRNAs, and lncRNAs exert their gene regulatory functions on normal biological processes and pathophysiology of diseases, which might aid in developing ncRNA-based therapeutics of diseases.