MiR-361-5p As a Promising QRT-PCR Internal Control for Tumor and Normal Breast Tissues

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2021 Jun 8

PMID 34101749

Citations 5

Authors

Sogol Ghanbari

Adel Salimi

Saeid Rahmani

Nahid Nafissi

Ali Sharifi-Zarchi

Seyed Javad Mowla

Affiliations

Soon will be listed here.

Abstract

Background: One of the most widely used evaluation methods in miRNA experiments is qRT-PCR. However, selecting suitable internal controls (IC) is crucial for qRT-PCR experiments. Currently, there is no consensus on the ICs for miRNA qRT-PCR experiments in breast cancer. To this end, we tried to identify the most stable (the least expression alteration) and promising miRNAs in normal and tumor breast tissues by employing TCGA miRNA-Seq data and then experimentally validated them on fresh clinical samples.

Methods: A multi-component scoring system was used which takes into account multiple expression stability criteria as well as correlation with clinical characteristics. Furthermore, we extended the scoring system for more than two biological sub-groups. TCGA BRCA samples were analyzed based on two grouping criteria: Tumor & Normal samples and Tumor subtypes. The top 10 most stable miRNAs were further investigated by differential expression and survival analysis. Then, we examined the expression level of the top scored miRNA (hsa-miR-361-5p) along with two commonly used ICs hsa-miR-16-5p and U48 on 34 pairs of Primary breast tumor and their adjacent normal tissues using qRT-PCR.

Results: According to our multi-component scoring system, hsa-miR-361-5p had the highest stability score in both grouping criteria and hsa-miR-16-5p showed significantly lower scores. Based on our qRT-PCR assay, while U48 was the most abundant IC, hsa-miR-361-5p had lower standard deviation and also was the only IC capable of detecting a significant up-regulation of hsa-miR-21-5p in breast tumor tissue.

Conclusions: miRNA-Seq data is a great source to discover stable ICs. Our results demonstrated that hsa-miR-361-5p is a highly stable miRNA in tumor and non-tumor breast tissue and we recommend it as a suitable reference gene for miRNA expression studies in breast cancer. Additionally, although hsa-miR-16-5p is a commonly used IC, it's not a suitable one for breast cancer studies.

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References

Bianchi N, Zuccato C, Finotti A, Lampronti I, Borgatti M, Gambari R . Involvement of miRNA in erythroid differentiation. Epigenomics. 2012; 4(1):51-65. DOI: 10.2217/epi.11.104. View

Liu J, Yang J, Yu L, Rao C, Wang Q, Sun C . miR-361-5p inhibits glioma migration and invasion by targeting SND1. Onco Targets Ther. 2018; 11:5239-5252. PMC: 6118279. DOI: 10.2147/OTT.S171539. View

Barra G, Rita T, Mesquita P, Jacomo R, Nery L . Analytical Sensitivity and Specificity of Two RT-qPCR Protocols for SARS-CoV-2 Detection Performed in an Automated Workflow. Genes (Basel). 2020; 11(10). PMC: 7599812. DOI: 10.3390/genes11101183. View

Cao Z, Huang Y, Yao L, Liu Y, Hu X, Hou Y . Positive expression of miR-361-5p indicates better prognosis for breast cancer patients. J Thorac Dis. 2016; 8(7):1772-9. PMC: 4958835. DOI: 10.21037/jtd.2016.06.29. View

Chapman J, Waldenstrom J . With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies. PLoS One. 2015; 10(11):e0141853. PMC: 4640531. DOI: 10.1371/journal.pone.0141853. View

Yang Z, Liu Z . The Emerging Role of MicroRNAs in Breast Cancer. J Oncol. 2020; 2020:9160905. PMC: 7354667. DOI: 10.1155/2020/9160905. View

Gharbi S, Shamsara M, Khateri S, Soroush M, Ghorbanmehr N, Tavallaei M . Identification of Reliable Reference Genes for Quantification of MicroRNAs in Serum Samples of Sulfur Mustard-Exposed Veterans. Cell J. 2015; 17(3):494-501. PMC: 4601870. DOI: 10.22074/cellj.2015.9. View

Han J, Yu J, Dai Y, Li J, Guo M, Song J . Overexpression of miR-361-5p in triple-negative breast cancer (TNBC) inhibits migration and invasion by targeting RQCD1 and inhibiting the EGFR/PI3K/Akt pathway. Bosn J Basic Med Sci. 2018; 19(1):52-59. PMC: 6387672. DOI: 10.17305/bjbms.2018.3399. View

Dai Y, Cao Y, Kohler J, Lu A, Xu S, Wang H . Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes. BMC Genomics. 2021; 22(1):27. PMC: 7789813. DOI: 10.1186/s12864-020-07318-y. View

10.

Chugh P, Dittmer D . Potential pitfalls in microRNA profiling. Wiley Interdiscip Rev RNA. 2012; 3(5):601-16. PMC: 3597218. DOI: 10.1002/wrna.1120. View

11.

Kontomanolis E, Koutras A, Syllaios A, Schizas D, Mastoraki A, Garmpis N . Role of Oncogenes and Tumor-suppressor Genes in Carcinogenesis: A Review. Anticancer Res. 2020; 40(11):6009-6015. DOI: 10.21873/anticanres.14622. View

12.

Liu T, Xu Z, Ou D, Liu J, Zhang J . The miR-15a/16 gene cluster in human cancer: A systematic review. J Cell Physiol. 2018; 234(5):5496-5506. DOI: 10.1002/jcp.27342. View

13.

Xu T, Su N, Liu L, Zhang J, Wang H, Zhang W . miRBaseConverter: an R/Bioconductor package for converting and retrieving miRNA name, accession, sequence and family information in different versions of miRBase. BMC Bioinformatics. 2019; 19(Suppl 19):514. PMC: 6311916. DOI: 10.1186/s12859-018-2531-5. View

14.

Tilli T, Castro C, Tuszynski J, Carels N . A strategy to identify housekeeping genes suitable for analysis in breast cancer diseases. BMC Genomics. 2016; 17(1):639. PMC: 4986254. DOI: 10.1186/s12864-016-2946-1. View

15.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

16.

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

17.

Davoren P, McNeill R, Lowery A, Kerin M, Miller N . Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer. BMC Mol Biol. 2008; 9:76. PMC: 2533012. DOI: 10.1186/1471-2199-9-76. View

18.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

19.

Svec D, Tichopad A, Novosadova V, Pfaffl M, Kubista M . How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments. Biomol Detect Quantif. 2016; 3:9-16. PMC: 4822216. DOI: 10.1016/j.bdq.2015.01.005. View

20.

Heneghan H, Miller N, Lowery A, Sweeney K, Newell J, Kerin M . Circulating microRNAs as novel minimally invasive biomarkers for breast cancer. Ann Surg. 2010; 251(3):499-505. DOI: 10.1097/SLA.0b013e3181cc939f. View