Related Experiment Video
Updated: Sep 25, 2025
An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
Pan-cancer analysis of microRNA expression profiles highlights microRNAs enriched in normal body cells as effective
Sharif Moradi1, Aryan Kamal2, Hamidreza Aboulkheyr Es1,3
1Cell Science Research Center, Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Background:
MicroRNAs (miRNAs) are frequently deregulated in various types of cancer. While antisense oligonucleotides are used to block oncomiRs, delivery of tumour-suppressive miRNAs holds great potential as a potent anti-cancer strategy. Here, we aim to determine, and functionally analyse, miRNAs that are lowly expressed in various types of tumour but abundantly expressed in multiple normal tissues.
Methods:
The miRNA sequencing data of 14 cancer types were downloaded from the TCGA dataset. Significant differences in miRNA expression between tumor and normal samples were calculated using limma package (R programming). An adjusted p value < 0.05 was used to compare normal versus tumor miRNA expression profiles. The predicted gene targets were obtained using TargetScan, miRanda, and miRDB and then subjected to gene ontology analysis using Enrichr. Only GO terms with an adjusted p < 0.05 were considered statistically significant. All data from wet-lab experiments (cell viability assays and flow cytometry) were expressed as means ± SEM, and their differences were analyzed using GraphPad Prism software (Student's t test, p < 0.05).
Results:
By compiling all publicly available miRNA profiling data from The Cancer Genome Atlas (TCGA) Pan-Cancer Project, we reveal a small set of tumour-suppressing miRNAs (which we designate as 'normomiRs') that are highly expressed in 14 types of normal tissues but poorly expressed in corresponding tumour tissues. Interestingly, muscle-enriched miRNAs (e.g. miR-133a/b and miR-206) and miRNAs from DLK1-DIO3 locus (e.g. miR-381 and miR-411) constitute a large fraction of the normomiRs. Moreover, we define that the CCCGU motif is absent in the oncomiRs' seed sequences but present in a fraction of tumour-suppressive miRNAs. Finally, the gain of function of candidate normomiRs across several cancer cell types indicates that miR-206 and miR-381 exert the most potent inhibition on multiple cancer types in vitro.
Conclusion:
Our results reveal a pan-cancer set of tumour-suppressing miRNAs and highlight the potential of miRNA-replacement therapies for targeting multiple types of tumour.
Insights
Tumor-suppressing microRNAs (miRNAs) are downregulated in many cancers. This study identifies specific miRNAs, like miR-206 and miR-381, that can inhibit cancer growth, offering potential for miRNA-replacement therapy.
Area of Science:
- Oncology
- Molecular Biology
- Bioinformatics
Background:
- MicroRNAs (miRNAs) are crucial regulators of gene expression and are frequently dysregulated in cancer.
- While blocking oncogenic miRNAs (oncomiRs) is a strategy, delivering tumor-suppressive miRNAs presents a promising anti-cancer approach.
- Identifying miRNAs with high expression in normal tissues but low expression in tumors is key for therapeutic development.
Purpose of the Study:
- To identify and functionally analyze tumor-suppressive miRNAs (normomiRs) that are downregulated in multiple cancer types.
- To investigate the potential of these normomiRs as therapeutic agents for cancer treatment.
- To explore sequence motifs differentiating tumor-suppressive miRNAs from oncomiRs.
Main Methods:
- Utilized miRNA sequencing data from The Cancer Genome Atlas (TCGA) Pan-Cancer Project for 14 cancer types.
- Employed bioinformatics tools (limma, TargetScan, miRanda, miRDB, Enrichr) to identify differentially expressed miRNAs and predict gene targets.
- Performed in vitro experiments, including cell viability assays and flow cytometry, to assess the functional impact of candidate normomiRs.
Main Results:
- Identified a set of 'normomiRs' highly expressed in normal tissues but poorly expressed in 14 cancer types.
- Muscle-enriched miRNAs (e.g., miR-133a/b, miR-206) and those from the DLK1-DIO3 locus (e.g., miR-381, miR-411) were prominent normomiRs.
- Demonstrated that miR-206 and miR-381 significantly inhibit the growth of multiple cancer cell types in vitro, with a distinct CCCGU motif found in some tumor-suppressive miRNAs.
Conclusions:
- Discovered a pan-cancer set of tumor-suppressing miRNAs with therapeutic potential.
- Highlights the promise of miRNA-replacement therapies for targeting diverse cancer types.
- miR-206 and miR-381 show potent anti-cancer activity, warranting further investigation for clinical application.
Related Concept Videos
01:22MicroRNAs
02:39lncRNA - Long Non-coding RNAs
01:05Cancer-Critical Genes II: Tumor Suppressor Genes
When the function of certain critical genes, especially those involved in cell cycle regulation and cell growth signaling cascades, gets disrupted, it upsets the cell cycle progression. Such cells with unchecked cell cycles start proliferating uncontrollably and eventually develop into tumors.
Such genes that act...
01:12Loss of Tumor Suppressor Gene Functions
When the tumor suppressor genes develop mutations or are lost, cells start growing out of control, leading to cancer. However, a single functional copy of the tumor suppressor gene is enough for the cells to maintain their normal functions and cell...