MicroRNA-29b-3p degenerates terminally differentiated dopaminergic SH-SY5Y cells by perturbation of mitochondrial functions.

Mitochondrial dysfunction is the main cause of gradual deterioration of structure and function of neuronal cells, eventually resulting in neurodegeneration. Studies have revealed a complex interrelationship between neurotoxicant exposure, mitochondrial dysfunction, and neurodegenerative diseases. Alteration in the expression of microRNAs (miRNAs) has also been linked with disruption in mitochondrial homeostasis and bioenergetics. In our recent research (Cellular and Molecular Neurobiology (2023) https://doi.org/10.1007/s10571-023-01362-4), we have identified miR-29b-3p as one of the most significantly up-regulated miRNAs in the blood of Parkinson's patients. The findings of the present study revealed that neurotoxicants of two different natures, that is, arsenic or rotenone, dramatically increased miR-29b-3p expression (18.63-fold and 12.85-fold, respectively) in differentiated dopaminergic SH-SY5Y cells. This dysregulation of miR-29b-3p intricately modulated mitochondrial morphology, induced oxidative stress, and perturbed mitochondrial membrane potential, collectively contributing to the degeneration of dopaminergic cells. Additionally, using assays for mitochondrial bioenergetics in live and differentiated SH-SY5Y cells, a reduction in oxygen consumption rate (OCR), maximal respiration, basal respiration, and non-mitochondrial respiration was observed in cells transfected with mimics of miR-29b-3p. Inhibition of miR-29b-3p by transfecting inhibitor of miR-29b-3p prior to exposure to neurotoxicants significantly restored OCR and other respiration parameters. Furthermore, we observed that induction of miR-29b-3p activates neuronal apoptosis via sirtuin-1(SIRT-1)/YinYang-1(YY-1)/peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)-regulated Bcl-2 interacting protein 3-like-dependent mechanism. Collectively, our studies have shown the role of miR-29b-3p in dysregulation of mitochondrial bioenergetics during degeneration of dopaminergic neurons via regulating SIRT-1/YY-1/PGC-1α axis.

Ageing at Molecular Level: Role of MicroRNAs.

The progression of age triggers a vast number of diseases including cardiovascular, cancer, and neurodegenerative disorders. Regardless of our plentiful knowledge about age-related diseases, little is understood about molecular pathways that associate the ageing process with various diseases. Several cellular events like senescence, telomere dysfunction, alterations in protein processing, and regulation of gene expression are common between ageing and associated diseases. Accumulating information on the role of microRNAs (miRNAs) suggests targeting miRNAs can aid our understanding of the interplay between ageing and associated diseases. In the present chapter, we have attempted to explore the information available on the role of miRNAs in ageing of various tissues/organs and diseases and understand the molecular mechanism of ageing.

Transcriptomics and Proteomics Approach for the Identification of Altered Blood microRNAs and Plasma Proteins in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder caused by the selective destruction of dopaminergic neurons (DA-nergic). Clinically, PD is diagnosed based on developing signs and symptoms. A neurological and physical examination and sometimes medical and family history also help in the diagnosis of PD. However, most of these features are visible when more than 80% of the dopaminergic neurons have degenerated. An understanding of the selective degeneration process at the cellular and molecular level and the development of new biomarkers are required for effective PD management. Several studies have been carried out using a selected set of miRNAs/ mRNAs and proteins to develop biomarkers of PD; however, an unbiased and combined miRNA-protein profiling study was required to identify the markers of progressive and selected degeneration of dopaminergic neurons in PD patients. In the present study, we have carried out global protein profiling through LC-MS/MS and miRNA profiling by using a "brain-specific" miRNA array panel of 112 miRNAs in PD patients and healthy controls to find the unprejudiced group of proteins and miRNAs that are deregulating in PD. In the whole blood samples of PD patients compared to healthy controls, the expression of 23 miRNAs and 289 proteins was significantly increased, whereas the expression of 4 miRNAs and 132 proteins was considerably downregulated. Network analysis, functional enrichment, annotation, and analysis of miRNA-protein interactions were also performed as part of the bioinformatics investigation of the discovered miRNAs and proteins revealing several pathways that lead to PD development and pathogenesis. Based on the analysis of miRNA and protein profiling, we have identified four miRNAs (hsa-miR-186-5p, miR-29b, miR-139 & has-miR-150-5p) and four proteins (YWHAZ, PSMA4, HYOU1, & SERPINA1), which can be targeted for the development of new biomarkers of PD. In vitro studies have identified the role of miR-186-5p in regulating the levels of the YWHAZ/YWHAB & CALM2 gene, which has shown maximum downregulation in PD patients and is known for its role in neuroprotection from apoptotic cell death & calcium regulation. In conclusion, our research has identified a group of miRNA-proteins that can be developed as PD biomarkers; however, future studies on the release of these miRNAs and proteins in extracellular vesicles circulating in the blood of PD patients can further validate these as specific biomarkers of PD.

Identification of chemoresistance targets in doxorubicin-resistant lung adenocarcinoma cells using LC-MS/MS-based proteomics.

Acquired chemoresistance remains a significant challenge in the clinics as most of the treated cancers eventually emerge as hard-to-treat phenotypes. Therefore, identifying chemoresistance targets is highly warranted to manage the disease better. In this study, we employed a label-free LC-MS/MS-based quantitative proteomics analysis to identify potential targets and signaling pathways underlying acquired chemoresistance in a sub-cell line (A549DR) derived from the parental lung adenocarcinoma cells (A549) treated with gradually increasing doses of doxorubicin (DOX). Our proteomics analysis identified 146 upregulated and 129 downregulated targets in A549DR cells. The KEGG pathway and Gene ontology (GO) analysis of differentially expressed upregulated and downregulated proteins showed that most abundant upregulated pathways were related to metabolic pathways, cellular senescence, cell cycle, and p53 signaling. Meanwhile, the downregulated pathways were related to spliceosome, nucleotide metabolism, DNA replication, nucleotide excision repair, and nuclear-cytoplasmic transport. Further, STRING analysis of upregulated biological processes showed a protein-protein interaction (PPI) between CDK1, AKT2, SRC, STAT1, HDAC1, FDXR, FDX1, NPC1, ALDH2, GPx1, CDK4, and B2M, proteins. The identified proteins in this study might be the potential therapeutic targets for mitigating DOX resistance.

A protein-miRNA biomic analysis approach to explore neuroprotective potential of nobiletin in human neural progenitor cells (hNPCs).

Chemical-induced neurotoxicity is increasingly recognized to accelerate the development of neurodegenerative disorders (NDs), which pose an increasing health burden to society. Attempts are being made to develop drugs that can cross the blood-brain barrier and have minimal or no side effects. Nobiletin (NOB), a polymethoxylated flavonoid with anti-oxidative and anti-inflammatory effects, has been demonstrated to be a promising compound to treat a variety of NDs. Here, we investigated the potential role of NOB in sodium arsenate (NA)-induced deregulated miRNAs and target proteins in human neural progenitor cells (hNPCs). The proteomics and microRNA (miRNA) profiling was done for different groups, namely, unexposed control, NA-exposed, NA + NOB, and NOB groups. Following the correlation analysis between deregulated miRNAs and target proteins, RT-PCR analysis was used to validate the selected genes. The proteomic analysis showed that significantly deregulated proteins were associated with neurodegeneration pathways, response to oxidative stress, RNA processing, DNA repair, and apoptotic process following exposure to NA. The OpenArray analysis confirmed that NA exposure significantly altered miRNAs that regulate P53 signaling, Wnt signaling, cell death, and cell cycle pathways. The RT-PCR validation studies concur with proteomic data as marker genes associated with autophagy and apoptosis (HO-1, SQSTM1, LC-3, Cas3, Apaf1, HSP70, and SNCA1) were altered following NA exposure. It was observed that the treatment of NOB significantly restored the deregulated miRNAs and proteins to their basal levels. Hence, it may be considered one of its neuroprotective mechanisms. Together, the findings are promising to demonstrate the potential applicability of NOB as a neuroprotectant against chemical-induced neurotoxicity.

Crosstalk Between miRNA and Protein Expression Profiles in Nitrate-Exposed Brain Cells.

Growing evidence reported a strong association between the nitrate ingestion and adverse health consequences in humans, including its detrimental impact on the developing brain. The present study identified miRNAs and proteins in SH-SY5Y human neuroblastoma cells and HMC3 human microglial cells using high-throughput techniques in response to nitrate level most prevalent in the environment (India) as X dose and an exceptionally high nitrate level as 5X dose that can be reached in the near future. Cells were exposed to mixtures of nitrates for 72 h at doses of X and 5X, 320 mg/L and 1600 mg/L, respectively. OpenArray and LCMS analysis revealed maximum deregulation in miRNAs and proteins in cells exposed to 5X dose. Top deregulated miRNAs include miR-34b, miR-34c, miR-155, miR-143, and miR-145. The proteomic profiles of both cell types include proteins that are potential targets of deregulated miRNAs. These miRNAs and their targeted proteins involve in multiple functions, including metabolic processes, mitochondrial functions, autophagy, necroptosis, apoptosis, neuronal disorders, brain development, and homeostasis. Furthermore, measuring mitochondrial bioenergetics in cells exposed to nitrate revealed that a 5X dose causes a significant reduction in oxygen consumption rate (OCR) and other bioenergetic parameters in both cell types. In summary, our studies have demonstrated that a 5X dose of nitrate significantly alters cellular physiology and functions by deregulating several miRNAs and proteins. However, X dose of nitrate has not caused any adverse effects on any cell type.

Identification of Altered Blood MicroRNAs and Plasma Proteins in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is the age-related neurological disorder characterized by the degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). PD is based on motor deficits which start to appear when up to 80% of the DA neurons of SNpc have been lost. Effective management of PD requires the development of novel biomarkers. Therefore, the present study aimed to characterize biomarkers of PD using miRNomics, proteomics, and bioinformatics approaches. Rats exposed to rotenone (2.5 mg/kg b.wt) for 2 months were used as an animal model to identify the unbiased set of miRNAs and proteins deregulated in blood samples. OpenArray, a real-time PCR-based array, is used for high-throughput profiling of miRNAs, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to carry out the global protein profiling. Systematic bioinformatics analysis of miRNAs and proteins was also performed, including annotation, functional classification and functional enrichment, network analysis, and miRNA-protein interaction analysis. Expression of 19 miRNAs and 96 proteins was significantly upregulated in the blood, while 22 proteins were significantly downregulated in blood samples of rotenone-exposed rats. In silico pathway analysis of deregulated proteins and miRNAs in rotenone-exposed rats has identified multiple pathways leading to PD. In summary, we have identified a set of miRNAs (miR-144, miR-96, and miR-29a) and proteins (PLP1, TUBB4A, and TUBA1C), which can be used as a potential biomarker of PD, while further validation required large human population studies.

Studies on Regulation of Global Protein Profile and Cellular Bioenergetics of Differentiating SH-SY5Y Cells.

The SH-SY5Y cells differentiated by sequential exposure of retinoic acid (RA) and brain-derived neurotrophic growth factor (BDNF) are a well-employed cellular model for studying the mechanistic aspects of neural development and neurodegeneration. Earlier studies from our lab have identified dramatic upregulation (77 miRNAs) and downregulation (17 miRNAs) of miRNAs in SH-SY5Y cells differentiated with successive exposure of RA + BDNF and demonstrated the essential role of increased levels of P53 proteins in coping with the differentiation-induced changes in protein levels. In continuation to our earlier studies, we have performed unbiased LC-MS/MS global protein profiling of naïve and differentiated SH-SY5Y cells and analyzed the identified proteins in reference to miRNAs identified in our earlier studies to identify the cellular events regulated by both identified miRNAs and proteins. Analysis of LC-MS/MS data has shown a significant increase and decrease in levels of 215 and 163 proteins, respectively, in differentiated SH-SY5Y cells. Integrative analysis of miRNA identified in our previous studies and protein identified in the present study is carried out to discover novel miRNA-protein regulatory modules to elucidate miRNA-protein regulatory relationships of differentiating neurons. In silico network analysis of miRNAs and proteins deregulated upon SH-SY5Y differentiation identified cell cycle, synapse formation, axonogenesis, differentiation, neuron projection, and neurotransmission, as the topmost involved pathways. Further, measuring mitochondrial dynamics and cellular bioenergetics using qPCR and Seahorse XFp Flux Analyzer, respectively, showed that differentiated cells possess increased mitochondrial dynamics and OCR relative to undifferentiated cells. In summary, our studies have identified a novel set of proteins deregulated during neuronal differentiation and establish the role of miRNAs identified in earlier studies in the regulation of proteins identified by LC-MS/MS-based global profiling of differentiating neurons, which will help in future studies related to neural development and neurodegeneration.

Advances and Implications in Nanotechnology for Lung Cancer Management.

BACKGROUND: Lung cancer is one of the most fatal chronic diseases in the field of respiratory medicine. The purpose of this paper is to address the side effects of conventional treatment strategies and to report the findings of till date drug nanocarriers researches performed for lung cancer therapy. This review also highlights the outstanding results of several researches employing pulmonary delivery system of nano-based drug formulations suitable for lung cancer. OBJECTIVE: Summarizing the advances made in the field of nanotechnology-based lung cancer management. METHODS: We systematically searched for research literature using a well-framed review question and presented data in the tabular forms for readers' convenience. RESULTS: Sixty-four papers were included in the review, the majority of which represent latest researches in the field of nanoparticle-based drug delivery for lung cancer therapy. Conventional treatment strategies for lung cancer lack specificity and are limited by undesirable toxicities in normal cells, as well as a high rate of recurrence. Intervention of nanotechnology has revolutionized the therapy of lung cancer upto a great extent by overcoming the current constraints in conventional therapies. Pulmonary delivery of nano-based drug formulations has resulted in potentially more effective and advanced lung cancer therapy. CONCLUSION: Several nanoscale drug delivery systems for lung cancer treatment are at present in clinical trials and some of them already exist in commercially available forms in the marketplace. However, although nanoscale drug carriers for lung cancer treatment have demonstrated stupendous therapeutic potential at both preclinical and clinical trials, but there are still many limitations to be solved.