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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
miR-101-3p overexpression suppresses NSCLC progression through Immune-Related gene CALCRL regulation and lncRNA NEAT1
Gulnaz Tabassum1, Shweta Arora1, Prithvi Singh2
1Department of Biotechnology, Faculty of Life Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
Introduction:
Non-Small Cell Lung Cancer (NSCLC), a predominant subtype of lung cancer, remains a major cause of cancer-related mortality worldwide and continues to present significant clinical challenges. While microRNAs (miRNAs) are established regulators of cellular pathways, their specific involvement in modulating immunometabolic processes in NSCLC remains underexplored. This study aimed to uncover novel regulatory mechanisms in lung adenocarcinoma (LUAD) through an integrative approach combining computational and experimental strategies, with particular emphasis on miRNA, mRNA, and long non-coding RNA (lncRNA) interactions.
Methods:
By analyzing RNA-Seq datasets, we identified differentially expressed miRNAs, genes, and immune-associated transcripts specific to LUAD. Subsequent enrichment and network analyses highlighted key molecular players involved in immune dysregulation and cancer progression. A competing endogenous RNA (ceRNA) network involving miR-101-3p, the gene CALCRL, and lncRNA NEAT1 were constructed to elucidate regulatory hierarchies. Experimental validation included qRT-PCR, Western blot, and luciferase reporter assays to confirm miRNA-mRNA-lncRNA interactions. Functional assays were conducted to assess cancer cell proliferation, oxidative stress, mitochondrial integrity, and apoptosis.
Results:
Analysis revealed significant downregulation of miR-101-3p and concurrent upregulation of CALCRL and NEAT1 in LUAD cell lines. Experimental assays confirmed that miR-101-3p directly targets and negatively regulates CALCRL, while NEAT1 enhances CALCRL expression by competitively binding miR-101-3p. Restoration by doing overexpression of miR-101-3p led to reduced cancer cell growth, increased oxidative stress, disrupted mitochondrial function, decreased ATP levels, and increased apoptotic cell death.
Conclusion:
The study identifies the miR-101-3p/NEAT1/CALCRL regulatory axis as a key mediator of immunometabolic remodeling in LUAD. These findings underscore the potential of targeting this axis for miRNA-guided therapeutic interventions, presenting a novel strategy to counteract tumor progression in NSCLC.
Insights
This study reveals the miR-101-3p/NEAT1/CALCRL axis regulates immunometabolic processes in lung adenocarcinoma. Targeting this axis offers a novel therapeutic strategy for non-small cell lung cancer.
Area of Science:
- Oncology
- Molecular Biology
- Immunometabolism
Background:
- Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality, with lung adenocarcinoma (LUAD) presenting significant challenges.
- The role of microRNAs (miRNAs) in regulating immunometabolic pathways within LUAD is not well understood.
- Investigating novel regulatory interactions, including miRNA, mRNA, and long non-coding RNA (lncRNA), is crucial for understanding LUAD progression.
Purpose of the Study:
- To uncover novel regulatory mechanisms in lung adenocarcinoma (LUAD) by integrating computational and experimental approaches.
- To identify key miRNA, mRNA, and lncRNA interactions involved in immunometabolic dysregulation in LUAD.
- To elucidate the specific roles of miR-101-3p, CALCRL, and NEAT1 in LUAD pathogenesis.
Main Methods:
- RNA-sequencing (RNA-Seq) analysis to identify differentially expressed transcripts in LUAD.
- Construction of a competing endogenous RNA (ceRNA) network involving miR-101-3p, CALCRL, and NEAT1.
- Experimental validation using qRT-PCR, Western blot, luciferase assays, and functional assays to assess proliferation, oxidative stress, mitochondrial function, and apoptosis.
Main Results:
- Downregulation of miR-101-3p and upregulation of CALCRL and NEAT1 were observed in LUAD cell lines.
- miR-101-3p was confirmed to directly target and downregulate CALCRL, while NEAT1 competitively bound miR-101-3p, enhancing CALCRL expression.
- Overexpression of miR-101-3p reduced cancer cell growth, increased oxidative stress, disrupted mitochondrial function, decreased ATP levels, and promoted apoptosis.
Conclusions:
- The miR-101-3p/NEAT1/CALCRL regulatory axis plays a critical role in immunometabolic remodeling in LUAD.
- This axis represents a potential therapeutic target for miRNA-guided interventions in NSCLC.
- Targeting this axis offers a novel strategy to combat tumor progression in non-small cell lung cancer.
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