Modulation of microRNAs and claudin-7 in Caco-2 cell line treated with <i>Blastocystis</i> sp., subtype 3 soluble total antigen

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Abstract

Background

Blastocystis sp., is a eukaryote of the large intestine, which is reported from almost all countries. The pathogenesis of this protist is not clear. The current study aimed to analyze the effects of Blastocystis sp., ST3 soluble total antigen (B3STA) on the microRNAs (miRNAs) involved in the gut permeability and also pro-inflammatory cytokines, occludin, and claudin-7.

Methods

Blastocystis sp., ST3 isolated from stool sample was purified, and its soluble total antigen was extracted using freeze and thawing. The Caco-2 cell line was treated with B3STA for 24 h and the expression levels of mir-16, mir-21, mir-29a, mir-223, and mir-874 were analyzed. In addition, the expression levels of il -8, il -15, occludin, and claudin-7 genes were assessed.

Results

B3STA significantly upregulated the expression of mir-223, and mir-874, and downregulated mir-29a. The expression of mir-16 and mir-21 was not significant. In addition, the expression of il -8 and il -15 was not significant. B3STA significantly decreased the expression level of claudin-7 ( P-value < 0.0001), but the expression of occludin was not significant. Our results showed significant correlation between all studied miRNAs, except mir-29a, with downregulation of claudin-7.

Conclusions

This is the first study investigating the effects of Blastocystis sp., ST3 isolated from symptomatic subjects on the expression levels of miRNAs involved in the gut permeability. Our results demonstrated that B3STA may change miRNA expression, which are involved in the gut barrier integrity, and downregulates claudin -7, which is known as sealing factor.

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MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in regulating gene expression. The majority of miRNAs are transcribed from DNA sequences into primary miRNAs and processed into precursor miRNAs, and finally mature miRNAs. In most cases, miRNAs interact with the 3′ untranslated region (3′ UTR) of target mRNAs to induce mRNA degradation and translational repression. However, interaction of miRNAs with other regions, including the 5′ UTR, coding sequence, and gene promoters, have also been reported. Under certain conditions, miRNAs can also activate translation or regulate transcription. The interaction of miRNAs with their target genes is dynamic and dependent on many factors, such as subcellular location of miRNAs, the abundancy of miRNAs and target mRNAs, and the affinity of miRNA-mRNA interactions. miRNAs can be secreted into extracellular fluids and transported to target cells via vesicles, such as exosomes, or by binding to proteins, including Argonautes. Extracellular miRNAs function as chemical messengers to mediate cell-cell communication. In this review, we provide an update on canonical and non-canonical miRNA biogenesis pathways and various mechanisms underlying miRNA-mediated gene regulations. We also summarize the current knowledge of the dynamics of miRNA action and of the secretion, transfer, and uptake of extracellular miRNAs.

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Real-time quantification of microRNAs by stem–loop RT–PCR

Caifu Chen, Dana A Ridzon, Adam J. Broomer … (2005)

A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

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MicroRNA: Biogenesis, Function and Role in Cancer

Leigh Ann MacFarlane, Paul Murphy (2010)

MicroRNAs are small, highly conserved non-coding RNA molecules involved in the regulation of gene expression. MicroRNAs are transcribed by RNA polymerases II and III, generating precursors that undergo a series of cleavage events to form mature microRNA. The conventional biogenesis pathway consists of two cleavage events, one nuclear and one cytoplasmic. However, alternative biogenesis pathways exist that differ in the number of cleavage events and enzymes responsible. How microRNA precursors are sorted to the different pathways is unclear but appears to be determined by the site of origin of the microRNA, its sequence and thermodynamic stability. The regulatory functions of microRNAs are accomplished through the RNA-induced silencing complex (RISC). MicroRNA assembles into RISC, activating the complex to target messenger RNA (mRNA) specified by the microRNA. Various RISC assembly models have been proposed and research continues to explore the mechanism(s) of RISC loading and activation. The degree and nature of the complementarity between the microRNA and target determine the gene silencing mechanism, slicer-dependent mRNA degradation or slicer-independent translation inhibition. Recent evidence indicates that P-bodies are essential for microRNA-mediated gene silencing and that RISC assembly and silencing occurs primarily within P-bodies. The P-body model outlines microRNA sorting and shuttling between specialized P-body compartments that house enzymes required for slicer –dependent and –independent silencing, addressing the reversibility of these silencing mechanisms. Detailed knowledge of the microRNA pathways is essential for understanding their physiological role and the implications associated with dysfunction and dysregulation.

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Author and article information

Contributors

Hamed Mirjalali: hamedmirjalali@sbmu.ac.ir , hamed_mirjalali@hotmail.com

Journal

Journal ID (nlm-ta): BMC Microbiol

Journal ID (iso-abbrev): BMC Microbiol

Title: BMC Microbiology

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2180

Publication date (Electronic): 22 April 2022

Publication date PMC-release: 22 April 2022

Publication date Collection: 2022

Volume: 22

Electronic Location Identifier: 111

Affiliations

[1 ]GRID grid.411600.2, Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, , Shahid Beheshti University of Medical Sciences, ; Tehran, Iran

[2 ]GRID grid.411600.2, Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver Diseases, , Shahid Beheshti University of Medical Sciences, ; Tehran, Iran

[3 ]GRID grid.411600.2, Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, , Shahid Beheshti University of Medical Sciences, ; Tehran, Iran

Article

Publisher ID: 2528

DOI: 10.1186/s12866-022-02528-8

PMC ID: 9027909

PubMed ID: 35459091

SO-VID: 4aa497e3-c759-4037-8180-542b8c9f4e81

License:

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 9 January 2022

Date accepted : 13 April 2022

Funding

Funded by: Sahahid Beheshti University of Medical Sciences

Award ID: RIGLD-1072

Custom metadata

ScienceOpen disciplines: Microbiology & Virology

Keywords: blastocystis sp., st3,microrna,intestinal permeability,claudin-7,inflammation

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ScienceOpen disciplines: Microbiology & Virology

Keywords: blastocystis sp., st3, microrna, intestinal permeability, claudin-7, inflammation

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Modulation of microRNAs and claudin-7 in Caco-2 cell line treated with Blastocystis sp., subtype 3 soluble total antigen

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Abstract

Background

Methods

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Microbiology Independent Research Journal (MIR Journal)

Most cited references 99

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MicroRNA: Biogenesis, Function and Role in Cancer

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