Skip to main content

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

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-15occludin, 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.

Peer Review reports

Background

Blastocystis sp., is a single cell protist which colonizes the large intestine of humans and a wide range of animals [1, 2]. This protist is transmitted via fecal-oral rout, contaminated food and water, and close contact to animals [3, 4]. This protist is one of the most prevalent eukaryotes, which its prevalence rate reaches up to even 100% [5].

Based on the phylogenetic and molecular analyses of a signature region through the small subunit ribosomal RNA (SSU rRNA) gene of Blastocystis sp., at least 22 distinct subtypes have been reported from humans and animals [6]. From these subtypes, subtype (ST) 3 thought to be the most prevalent subtypes with wide distribution all over the world [4]. Most of cases that carry Blastocystis sp., in their intestine do not complain from specific symptoms [7,8,9]; however, available evidence has linked Blastocystis sp., colonization with some clinical manifestations. Accordingly, most of studies have associated the presence of ST1 and ST3 with some clinical manifestations such as gastrointestinal disorders and urticarial [10,11,12].

It was documented that Blastocystis sp., may affect its hosts via manipulation of either the gut microbiome or the host’s immune system [13,14,15]. Although it is controversially [13, 16,17,18], Blastocystis sp., is suggested to increase the gut microbiota diversity and to be a healthy gut indicator [19,20,21,22]. Nevertheless, the release of proteases from Blastocysis sp., supposes to disrupt tight junctions (TJ) or destroy the secretory immunoglobulin A (sIgA) [23,24,25,26]. A recent study evaluated protease activity and the effects of Blastocystis sp., subtypes 1–3 and 6 isolated from symptomatic and asymptomatic subjects in HT-29 cell line, and claimed higher protease activity of Blastocystis sp. isolated from symptomatic carriers [27]. They showed a significant higher protease activity of Blastocystis sp., ST3 isolated from symptomatic subjects compared to those isolated from asymptomatic individuals [27]. However, a lot of aspects remain unclear such as how Blastocystis sp., communicate with its hosts.

MicroRNAs (miRNAs) are 17–25 nucleotides, non-coding RNA fragment, which was firstly discovered in Caenorhabditis elegans. From 1993, which the first miRNA, lin-4, was described, new miRNAs are still being characterized. MiRNAs mostly interact with 3′ UTR of target messenger RNAs (mRNAs) to regulate their expression [28, 29]. Although there is evidence of communication between parasites and their hosts via miRNAs, there is no study describing the role of Blastocystis sp., on the expression of miRNAs involved in the gastrointestinal homeostasis. The current study aimed to investigate the effects of soluble total antigen (STA) of Blastocystis sp., ST3 on the expression changes of miRNAs: mir-16, mir-21, mir-29a, mir-223, and mir-874, which play roles in the integrity of the intestine, in the Caco2 cell line.

Methods

Ethical approval

No human or animal tissues were analyzed in this study. The study was approved by the ethics committee/institutional review board of the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

All experimental protocols were approved by the Research Institute for Gastroenterology and Liver Diseases and all procedures of this study were in accordance with the ethical standards (IR.SBMU.RIGLD.REC.1398.048) released by the Ethical Review Committee of the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran. In addition, all methods were carried out in accordance with relevant guidelines and regulations.

Blastocystis isolate

In the current study, Blastocystis sp., ST3 was from stool sample of a symptomatic subject from our previous study [7, 27]. Briefly, Blastocystis sp., was isolated from stool samples, which were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, MA, USA) containing penicillin-streptomycin (Sigma, USA), (1000-unit penicillin and 4 mg/mL streptomycin) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, USA), and were incubated in an anaerobic condition at 37 °C. The studied Blastocystis sp., ST3 was purified using Ficoll gradient (Ficoll-Paque™ PREMIUM) and several consecutive subculture in combination with antibiotic cocktail for about six months [27]. To characterize the subtype of isolated Blastocystis sp., the barcoding fragment was amplified using specific primers and sequenced [30]. To confirm that Blastocystis sp., is purified, consecutive cultivation of the protist was performed accompanied with a mixture of active antibiotics (4000 mg/ml of ampicillin, 1000 mg/ml of streptomycin, and 1000 units of penicillin together with amphotericin B (50 mg/mL) to eliminate yeasts or filamentous fungi), which were determined using antibiogram. Details of purification are mentioned elsewhere [27].

Blastocystis sp. ST3 soluble total antigen (B3STA)

To prepare soluble total antigen from purified Blastocystis sp., 1 × 105 parasites/mL of the parasite was washed three times in PBS at 300×g for 5 min at 4 °C and counted with Neubauer’s improved cell counting chamber (perci color HBG; Germany). In order to prepare the B3STA, three freeze-thaw cycles in liquid nitrogen and a 37 °C water bath were employed and the resultant subject was filtered using polyethersulfone (PES) filters with 0.22 μm pore size to eliminate probable remained bacteria [27].

Cell culture

The human colorectal adenocarcinoma cell line (Caco-2; ATCC HTB-37) was cultivated in a 25-cm2 culture flask (Cell culture Flask, SPL, Korea) supplemented with 5 mL of high-glucose DMEM medium (DMEM High Glucose, Biosera), 5% (v/v) heat-inactivated FBS, 2 mM L-glutamine, and 1% antibiotic-antimycotic agents (penicillin: 100 U/ml, streptomycin: 100 mg/ml Sigma-Aldrich, USA). Cultivated Caco-2 cells were incubated in 5% CO2 and 100% humidity at 37 °C. Upon 70–80% confluency, the cells were washed with sterile PBS (pH = 7), detached using 0.25% trypsin-EDTA (Gibco, USA), and the number of alive cells were counted by 0.025% (w/v) trypan blue solution (Gibco, USA) and Neubauer’s improved cell counting chamber.

Co-incubation of B3STA with Caco-2 cell line

For this purpose, 1 × 105 Caco-2 cells were counted and seeded in each well of a six-well plate. The plate was incubated in 5% CO2 at 37 °C overnight. After 70–80% confluency, the B3STA prepared from 105 of Blastocystis sp. ST3 was added to the sample well. In addition, 20 ng/mL LPS (Santa Cruz Biotechnology Cat No. sc-3535) were used to compare the induction pattern to the B3STA. A well full of Caco-2 cell without any treatment either by B3STA or LPS, was considered as control group. All groups were in duplicate and evaluated 24 h after exposure.

MicroRNA selection and primer designing

In order to evaluate the expression level of miRNAs: mir-16, mir-21, mir-29a, mir-223, and mir-874, mature sequences of human miRNAs were selected from the miRBase database (https://www.mirbase.org/) according following accession numbers: MIMAT0000069, MIMAT0000076, MIMAT0000086, MIMAT0000280, and MIMAT0004911, respectively. The primer designing was performed based on stem-loop and regarding the protocol, which was previously explained [31] (Table 1). The stem-loop reverse transcriptase (RT) and real-time PCR primers provide higher specificity and efficacy.

Table 1 Stem-loop RT primers designed for each studied miRNA and its forward real-time PCR primer

RNA extraction, cDNA synthesis, and quantitative real-time PCR

Total RNA was extracted using total RNA purification mini kit (YTA, Tehran, Iran). In order to adjust the RNA concentration before complementary DNA (cDNA) synthesis, the concentration of extracted RNAs was determined by a NanoDrop (NanoDrop Technologies, USA) apparatus. The cDNA synthesis specific for each miRNA was constructed using cDNA synthesis kit (YTA, Tehran, Iran) as explained previously [31]. During cDNA synthesis, stem–loop RT primers are used instead of conventional RT primers, and bind to the 3′ end of miRNA to increase the length of target miRNA [31].

To amplify and quantify targeted miRNA using real-time PCR, miRNA-specific forward primer and a universal reverse primer are used. Forward primers for real-time PCR are designed to bind to the 5′ end of miRNA, which was constructed using stem-loop RT primers, as tailed forward primer, and to increase the melting temperature (Tm) of target miRNA sequence [31].

To analyze the effects of B3STA on the inflammatory biomarkers and TJ, the expression levels of IL-8, IL-15, occludin, and claudin-7 were evaluated (Table 2). Relative expression of the miRNAs in treated and untreated cells were determined by quantitative (q) real-time PCR using Rotor-Gene Q (Qiagen, Germany) in a 20 μL reaction mixture containing 10 μL SYBR Green qPCR master mix 2X (Ampliqon, Denmark), 5 ρM of each primer, and 2 μL of constructed cDNA as template. The amplification conditions for miRNAs were adjusted with previously released protocol [31]. Real-time PCR for inflammatory markers and TJ was performed using Rotor-Gene Q (Qiagen, Germany) thermocycler in a 20 μL reaction mixture containing 10 μl SYBR Green qPCR Master Mix 2X (Ampliqon, Denmark), 5 μM of primers, and 2 µL of cDNA under conditions: initial denaturation 95 °C for 10 min, followed by denaturation at 95 °C for 20 s, annealing at 58–63 °C for 30 s, and extension at 72 °C for 20 s.

Table 2 Employed primers to study the expression of cytokines and tight junction’s genes.

To avoid from non-specific amplification, melting curve analysis was employed for each run. Subsequently, the relative quantification (RQ) of each miRNA relative to U6 snRNA [37] and inflammatory markers and TJ relative to beta-actin (BACT) was calculated using 2-∆∆CT incorporated in relative expression software tool (REST). All tests were performed in duplicate.

Statistical analysis

Student’s t-test was applied to analyze the real-time PCR data. P value < 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism software version 8.3.0.538.

Results

Relative expression of miRNAs

The B3STA did not significantly changes mir-16 3 (.041 folds; P-value = 0.0754), but LPS significantly downregulated the levels of mir-16 for 2.328 folds in the Caco-2 cell line (P-value < 0.0001). The comparison of the expression levels of mir-16 between the B3STA and LPS showed significant difference (P-value = 0.0014) (Fig. 1A).

Fig. 1
figure 1

The expression levels of (A) mir-16, (B) mir-21, (C) mir-29a, (D) mir-223, and (E) mir-874 in the Caco-2 cell line co-incubated with B3STA, isolated from symptomatic carrier, 24 h after exposure. Accordingly, the B3STA significantly downregulated mir-29a, and upregulated mir-223 and mir-874. In addition, LPS significantly downregulated mir-16, while upregulated mir-29a, mir-223, and mir-874. Comparison between LPS and B3STA was statistically significant only in mir-16, mir-29a, and mir-874. * P value < 0.05; ** P value < 0.01; *** P value < 0.001; **** P value < 0.0001. Comparisons were carried out using the Student’s t-test. Mir: microRNA; Caco-2: human colon carcinoma; B3STA: Blastocystis sp., ST3 soluble total antigen; LPS: lipopolysaccharide; NS: not significant

The expression level of mir-21 was not significantly changed with the B3STA (1.387 folds; P-value = 0.567), and LPS (2.716 folds; P-value = 0.174) (Fig. 1B). The B3STA significantly downregulated the expression levels of mir-29 (2.497 folds; P-value < 0.0001), while LPS significantly upregulated mir-29 (1.224 folds; P-value < 0.0001). Indeed, the comparison of the expression of mir-29 between the B3STA and LPS was statistically significant (P-value < 0.0001) (Fig. 1C). Our result showed that the B3STA and LPS significantly increased the expression levels of mir-223 (3.463 folds; P-value = 0.0128) and (2.425 folds; P-value = 0.0011), respectively (Fig. 1D). Finally, the expression of mir-874 was evaluated in Caco-2 cell line sensed by the B3STA that the results showed statistically significant overexpression in the cells, which were treated with the B3STA (3.186 folds; P-value = 0.0004) and LPS (1.37 folds; P-value = 0.0391). The comparison of the expression of mir-874 between the B3STA and LPS was statistically significant (P-value = 0.0018) (Fig. 1E).

Relative expression of inflammatory and TJ markers

The results of relative expression showed no statistically significant changes in il-8 (1.148 folds; P-value = 0.0584) and il-15 genes (2.017 folds; P-value = 0.7737) (Fig. 2). The B3STA significantly downregulated claudin-7 (1.582 folds; P-value < 0.0001), but did not induce significant changes in occludin (Fig. 3).

Fig. 2
figure 2

The expression levels of (A) il-8 and (B) il-15 genes in the Caco-2 cell line co-incubated with B3STA, 24 h after exposure. Our analysis indicated that changes in both cytokines were not statistically significant. Comparisons were carried out using the Student’s t-test. IL: interleukin; Caco-2: human colon carcinoma; B3STA: Blastocystis sp., ST3 soluble total antigen

Fig. 3
figure 3

The expression levels of (A) occludin and (B) claudin-7 genes in the Caco-2 cell line co-incubated with B3STA, 24 h after exposure. Statistically significant downregulation was seen in claudin-7. Claudin-7 is known as sealing factor and plays important role in reducing the gut permeability. **** P value < 0.0001. Caco-2: human colon carcinoma; B3STA: Blastocystis sp., ST3 soluble total antigen. Comparisons were carried out using the Student’s t-test

Correlation between miRNAs and expression of inflammatory and TJ markers

The correlation between the expression of il-8 and il-15 genes with investigated miRNAs was assessed and showed that only il-8 had a significant correlation with mir-29a (P-value = 0.007) and mir-874 (P-value = 0.011), while the correlation between il-8 and il-15 and all other miRNAs was not statistically significant. The correlation between the expression of occludin and miRNAs was not statistically significant. In addition, except mir-29a, all studied miRNAs were significantly correlated with elevated level of claudin-7 (Table 3).

Table 3 The statistical correlation of each miRNA with studied cytokines and tight junctions

Discussion

In the current study, we employed B3STA, which was obtained from a symptomatic subjects from our previous study [27]. The main reason for choosing this isolate was to investigate the effects of a clinically isolated Blastocystis sp., because it is documented that continuous cultivation of eukaryotes in axenic conditions may affect their physiological and pathogenic features [38, 39].

Focuses on the interaction between parasites and their hosts have pointed out the critical role of host or parasite originated miRNAs in orchestrating the immune responses and pathogenesis of parasites [40,41,42]. Blastocystis sp., is a prevalent protist, which its pathogenicity is still unclear. Nevertheless, the number of studies, which are describing pathogenic role for Blastocystis sp., are increasing. It was documented that Blastocystis sp., is not only able to dysregulate immune responses during colonization in the intestine [26, 43], but also affects the permeability of the gut [24, 44], both using its proteases. For example, Puthia et al., (2006) [45] observed that B. ratti induced apoptosis and increased the permeability of the gut epithelium. Additionally, Mirza et al., (2012) [24] demonstrated that Blastocystis sp., is able to manipulate cell permeability, transepithelial resistance, and phosphorylation of myosin light chain via a rho kinase (ROCK)-dependent manner. Another study by the same team, which was performed in Caco-2 cell line, suggested that ST7 changed the permeability and tight junction localization, which that led to disruption of the intestinal barrier [46].

The intestinal barrier plays crucial role in keeping homeostasis of the gut and protecting from translocation of the gut contents into the lower layers, such as mucus and the circulation system [47]. Therefore, an intestinal barrier with impaired functions has been linked to a broad spectrum of immunological disorders in gastrointestinal system [47, 48]. Tight junction proteins keep the integrity of intestinal barriers, and disruption of these proteins increases paracellular and transcellular permeability [47]. Claudins, occludin, and zonula occludens are the most important TJ proteins that are involved in maintenance of the intestinal barriers [48,49,50]. In the current study the effect of B3STA on occludin was almost without change, while it significantly decreased the expression level of claudin-7claudin-7. Claudin-7 in humans is observed in the large intestine [51], where is colonized by Blastocystis sp. Although controversially [49], claudin-7 is categorized among pore-sealing groups of claudins and are responsible for sealing junctions and reducing permeability [52].

The gut barrier integrity and permeability are also suggested to be regulated by miRNAs [52, 53] (Table 4). As results, B3STA significantly upregulated mir-223 and mir-874, and downregulated mir-29a. It was documented that mir-16 may be involved in the intestinal barrier dysfunction [78]. Although it was claimed that the level of mir-16 was downregulated in irritable bowel syndrome (IBS) diarrhea predominant patients [78], it was shown that in IBD patients the expression level of mir-16 was elevated in inflammatory bowel diseases (IBD) patients [79], and therefore, inhibiting of mir-16 could be an alternative therapeutic strategy [80]. Similar to mir-16, mir-21 seems to be associated with the impaired functions of intestinal barrier. Zhang et al., (2015) [61] assessed mir-21 in Caco-2 cell line and showed a significantly increased expression of this miRNA in the intestinal TJ barrier defect model, which was associated with overexpression of IL-8. In this line, it was claimed that mir-21 is correlated with ischemia reperfusion [81], flare up in IBD [82,83,84], and proliferation and invasion of colon adenocarcinoma [85], which all are related to the intestinal barrier dysfunction. As a result, B3STA downregulated mir-29. The mir-29 family thought to be involved in development of fibrosis, particularly in IBD [86,87,88]. On the other hand, in a clinical study in IBS patients, it was proposed that overexpression of mir-29a could be related to the glutamine synthesis and gut permeability [89]. Actually, mir-29a increases the gut permeability via controlling glutamine synthesis [89]. In this line, an experimental study performed in intestinal epithelial cell line (IPEC-1) suggested that inhibiting mir-29a was related to improvement of the monolayer integrity [87]. Our results also showed that B3STA increased the expression levels of mir-223 and mir-874. It was documented that mir-223 is associated with inflammation through the intestine tissue [90,91,92]. In addition, Li et al., (2020) suggested that mir-223-enriched mast cell-derived exosomes inhibited TJ proteins and destroyed intestinal barrier functions [92]. Notable, it was demonstrated that mir-874 induced paracellular permeability and intestinal barrier dysfunction via changes in expression of aquaporin 3 (AQP3) protein [73, 93]. Many studies reported a low prevalence rate of Blastocystis sp., colonization in IBD patients [13, 94,95,96], and a high prevalence of the protist in IBS patients [97,98,99,100]. However, it is though that the colonization of Blastocystis sp., probably is an indicator for healthy gut [19, 21, 22, 94].

Table 4 Targets and biological functions of studied miRNAs in intestinal permeability

Collectively, the significant expression changes of mir-29a, mir-223, and mir-874 in Caco-2 cell line treated with B3STA together with the significant correlation between overexpression of mir-223, and mir-874 with claudin-7 suggest a cross-talk between Blastocystis sp., and its host that should be scrutinized not only using in vitro models, but also by in vivo studies.

The main limitation of the current study was investigation of the effects of Blastocystis sp., in cell culture. Although in vitro investigations are important part of molecular biology studies, inferring the role of Blastocystis sp., in manipulation of the gut permeability in humans needs more experimental and clinical surveys.

Conclusion

Our results also showed that B3STA isolated from symptomatic carrier, is able to change miRNA expression of mir-29a, mir-223, and mir-874, which are involved in the gut barrier integrity. In addition, Blastocystis sp., can downregulates claudin-7, which is known as sealing factor. This study provides a clue about the role of miRNAs on pathogenesis of Blastocystis sp., but further studies, in vitro and in vivo, are needed to clear correlation between Blastocystis sp., and expression changes of host’s miRNAs.

Availability of data and materials

Generated data including figures and tables were not submitted elsewhere and are included in the article. In this study, DNA or RNA sequences were not generated to be submitted in relevant databases.

References

  1. Popruk S, Adao DEV, Rivera WL. Epidemiology and subtype distribution of Blastocystis in humans: a review. Infect Genet Evol. 2021;95:105085.

    CAS  PubMed  Article  Google Scholar 

  2. Hublin JSY, Maloney JG, Santin M. Blastocystis in domesticated and wild mammals and birds. Res Vet Sci. 2021;135:260–82.

    CAS  PubMed  Article  Google Scholar 

  3. Javanmard E, et al. Molecular analysis of Blastocystis sp. and its subtypes from treated wastewater routinely used for irrigation of vegetable farmlands in Iran. J Water Health. 2019;17(5):837–44.

    PubMed  Article  Google Scholar 

  4. Nemati S, et al. Molecular prevalence and subtype distribution of Blastocystis sp. in Asia and in Australia. J Water Health. 2021;19(5):687–704.

    PubMed  Article  Google Scholar 

  5. El Safadi D, et al. Children of Senegal River basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infect Dis. 2014;14:164.

    PubMed  PubMed Central  Article  Google Scholar 

  6. Stensvold CR, Clark CG. Pre-empting pandora's box: Blastocystis subtypes revisited. Trends Parasitol. 2020;36(3):229–32.

    PubMed  Article  Google Scholar 

  7. Rezaei Riabi T, et al. Genetic diversity analysis of Blastocystis subtypes from both symptomatic and asymptomatic subjects using a barcoding region from the 18S rRNA gene. Infect Genet Evol. 2018;61:119–26.

    CAS  PubMed  Article  Google Scholar 

  8. Jalallou N, et al. Subtypes distribution and frequency of Blastocystis sp. isolated from diarrheic and non-diarrheic patients. Iran J Parasitol. 2017;12(1):63–8.

    PubMed  PubMed Central  Google Scholar 

  9. Mardani Kataki M, Tavalla M, Beiromvand M. Higher prevalence of Blastocystis hominis in healthy individuals than patients with gastrointestinal symptoms from Ahvaz, southwestern Iran. Comp Immunol Microbiol Infect Dis. 2019;65:160–4.

    PubMed  Article  Google Scholar 

  10. El Safadi D, et al. Molecular epidemiology of Blastocystis in Lebanon and correlation between subtype 1 and gastrointestinal symptoms. Am J Trop Med Hyg. 2013;88(6):1203–6.

    PubMed  PubMed Central  Article  Google Scholar 

  11. Rajic B, et al. Eradication of Blastocystis hominis prevents the development of symptomatic Hashimoto's thyroiditis: a case report. J Infect Dev Ctries. 2015;9(7):788–91.

    CAS  PubMed  Article  Google Scholar 

  12. Verma R, Delfanian K. Blastocystis hominis associated acute urticaria. Am J Med Sci. 2013;346(1):80–1.

    PubMed  Article  Google Scholar 

  13. Azimirad M, et al. Blastocystis and Clostridioides difficile: evidence for a synergistic role in colonization among IBD patients with emphasis on ulcerative colitis. Turk J Gastroenterol. 2021;32(6):500–7.

    PubMed  PubMed Central  Article  Google Scholar 

  14. Deng L, et al. New insights into the interactions between Blastocystis, the gut microbiota, and host immunity. PLoS Pathog. 2021;17(2):e1009253.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Tito RY, et al. Population-level analysis of Blastocystis subtype prevalence and variation in the human gut microbiota. Gut. 2018;68(7):1180–9.

    PubMed  Article  CAS  Google Scholar 

  16. Nourrisson C, et al. Blastocystis is associated with decrease of fecal microbiota protective bacteria: comparative analysis between patients with irritable bowel syndrome and control subjects. PLoS One. 2014;9(11):e111868.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  17. Defaye M, et al. Fecal dysbiosis associated with colonic hypersensitivity and behavioral alterations in chronically Blastocystis-infected rats. Sci Rep. 2020;10(1):9146.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Nagel R, et al. Comparison of faecal microbiota in Blastocystis-positive and Blastocystis-negative irritable bowel syndrome patients. Microbiome. 2016;4(1):47.

    PubMed  PubMed Central  Article  Google Scholar 

  19. Stensvold CR, et al. Stool microbiota diversity analysis of Blastocystis-positive and Blastocystis-negative individuals. Microorganisms. 2022;10(2):326.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Billy V, et al. Blastocystis colonization alters the gut microbiome and, in some cases, promotes faster recovery from induced colitis. Front Microbiol. 2021;12:641483.

    PubMed  PubMed Central  Article  Google Scholar 

  21. Kim MJ, et al. Gut microbiome profiles in colonizations with the enteric protozoa Blastocystis in Korean populations. Microorganisms. 2021;10(1):34.

    PubMed  PubMed Central  Article  Google Scholar 

  22. Asnicar F, et al. Microbiome connections with host metabolism and habitual diet from 1,098 deeply phenotyped individuals. Nat Med. 2021;27(2):321–32.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Nourrisson C, et al. On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers. Parasitology. 2016;143(13):1713–22.

    CAS  PubMed  Article  Google Scholar 

  24. Mirza H, et al. Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced by Blastocystis cysteine proteases. Cell Microbiol. 2012;14(9):1474–84.

    CAS  PubMed  Article  Google Scholar 

  25. Abdel-Hameed DM, Hassanin OM. Proteaese activity of Blastocystis hominis subtype3 in symptomatic and asymptomatic patients. Parasitol Res. 2011;109(2):321–7.

    PubMed  Article  Google Scholar 

  26. Puthia MK, et al. Degradation of human secretory immunoglobulin a by Blastocystis. Parasitol Res. 2005;97(5):386–9.

    PubMed  Article  Google Scholar 

  27. Karamati SA, et al. Association of Blastocystis ST6 with higher protease activity among symptomatic subjects. BMC Microbiol. 2021;21(1):285.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. O'Brien J, et al. Overview of microRNA biogenesis, mechanisms of actions, and circulation. Front Endocrinol (Lausanne). 2018;9:402.

    CAS  Article  Google Scholar 

  29. Macfarlane LA, Murphy PR. MicroRNA: biogenesis, function and role in cancer. Curr Genomics. 2010;11(7):537–61.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Scicluna SM, Tawari B, Clark CG. DNA barcoding of Blastocystis. Protist. 2006;157(1):77–85.

    CAS  PubMed  Article  Google Scholar 

  31. Chen C, et al. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucl Acid Res. 2005;33(20):e179.

    Article  CAS  Google Scholar 

  32. Saegusa S, et al. Cytokine responses of intestinal epithelial-like Caco-2 cells to non-pathogenic and opportunistic pathogenic yeasts in the presence of butyric acid. Biosci Biotechnol Biochem. 2007;71(10):2428–34.

    CAS  PubMed  Article  Google Scholar 

  33. Nemeth ZH, et al. Crohn's disease and ulcerative colitis show unique cytokine profiles. Cureus. 2017;9(4):e1177.

    PubMed  PubMed Central  Google Scholar 

  34. Yu MH, et al. Up-regulated CKS2 promotes tumor progression and predicts a poor prognosis in human colorectal cancer. Am J Cancer Res. 2015;5(9):2708–18.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Chung CY, et al. Progressive proximal-to-distal reduction in expression of the tight junction complex in colonic epithelium of virally-suppressed HIV+ individuals. PLoS Pathog. 2014;10(6):e1004198.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  36. Fu J, et al. Identification of genes for normalization of quantitative real-time PCR data in ovarian tissues. Acta Biochim Biophys Sin Shanghai. 2010;42(8):568–74.

    CAS  PubMed  Article  Google Scholar 

  37. Galiveti CR, et al. Application of housekeeping npcRNAs for quantitative expression analysis of human transcriptome by real-time PCR. RNA. 2010;16(2):450–61.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Padilla-Vaca F, et al. Down regulation of Entamoeba histolytica virulence by monoxenic cultivation with Escherichia coli O55 is related to a decrease in expression of the light (35-kilodalton) subunit of the gal/GalNAc lectin. Infect Immun. 1999;67(5):2096–102.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Costa AO, et al. Pathogenicity of Entamoeba dispar under xenic and monoxenic cultivation compared to a virulent E. histolytica. Rev Inst Med Trop Sao Paulo. 2006;48(5):245–50.

    PubMed  Article  Google Scholar 

  40. Alizadeh Z, et al. MicroRNAs in helminth parasites: a systematic review. Curr Mol Med. 2021.

  41. Varikuti S, et al. MicroRNA-21 deficiency promotes the early Th1 immune response and resistance toward visceral leishmaniasis. J Immunol. 2021;207(5):1322–32.

    CAS  PubMed  Article  Google Scholar 

  42. Paul S, et al. Human microRNAs in host-parasite interaction: a review. 3. Biotech. 2020;10(12):510.

    Google Scholar 

  43. Puthia MK, Lu J, Tan KS. Blastocystis ratti contains cysteine proteases that mediate interleukin-8 response from human intestinal epithelial cells in an NF-kappaB-dependent manner. Eukaryot Cell. 2008;7(3):435–43.

    CAS  PubMed  Article  Google Scholar 

  44. Ajjampur SS, Tan KS. Pathogenic mechanisms in Blastocystis spp. - interpreting results from in vitro and in vivo studies. Parasitol Int. 2016;65(6 Pt B):772–9.

    CAS  PubMed  Article  Google Scholar 

  45. Puthia MK, et al. Blastocystis ratti induces contact-independent apoptosis, F-actin rearrangement, and barrier function disruption in IEC-6 cells. Infect Immun. 2006;74(7):4114–23.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Wu Z, et al. Strain-dependent induction of human enterocyte apoptosis by Blastocystis disrupts epithelial barrier and ZO-1 organization in a caspase 3- and 9-dependent manner. Biomed Res Int. 2014;2014:209163.

    PubMed  PubMed Central  Google Scholar 

  47. Schoultz I, V. Keita Å. The intestinal barrier and current techniques for the assessment of gut permeability. Cells. 2020;9(8):1909.

    CAS  PubMed Central  Article  Google Scholar 

  48. Lee SH. Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases. Intest Res. 2015;13(1):11–8.

    PubMed  PubMed Central  Article  Google Scholar 

  49. Garcia-Hernandez V, Quiros M, Nusrat A. Intestinal epithelial claudins: expression and regulation in homeostasis and inflammation. Ann N Y Acad Sci. 2017;1397(1):66–79.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. Slifer ZM, Blikslager AT. The integral role of tight junction proteins in the repair of injured intestinal epithelium. Int J Mol Sci. 2020;21(3):972.

    CAS  PubMed Central  Article  Google Scholar 

  51. Lameris AL, et al. Expression profiling of claudins in the human gastrointestinal tract in health and during inflammatory bowel disease. Scand J Gastroenterol. 2013;48(1):58–69.

    CAS  PubMed  Article  Google Scholar 

  52. Kozieł MJ, Ziaja M, Piastowska-Ciesielska AW. Intestinal barrier, claudins and mycotoxins. Toxins (Basel). 2021;13(11):758.

    Article  CAS  Google Scholar 

  53. Zhao X, et al. Tight junctions and their regulation by non-coding RNAs. Int J Biol Sci. 2021;17(3):712–27.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Wu F, et al. MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage inflammatory peptide-2 alpha. Gastroenterology. 2008;135(5):1624–1635.e24.

    CAS  PubMed  Article  Google Scholar 

  55. Wu F, et al. Identification of microRNAs associated with ileal and colonic Crohn's disease. Inflamm Bowel Dis. 2010;16(10):1729–38.

    PubMed  Article  Google Scholar 

  56. Paraskevi A, et al. Circulating microRNA in inflammatory bowel disease. J Crohns Colitis. 2012;6(9):900–4.

    PubMed  Article  Google Scholar 

  57. Konstantinidis A, et al. Colonic mucosal and serum expression of microRNAs in canine large intestinal inflammatory bowel disease. BMC Vet Res. 2020;16(1):69.

    CAS  Article  Google Scholar 

  58. James JP, et al. MicroRNA biomarkers in IBD-differential diagnosis and prediction of colitis-associated cancer. Int J Mol Sci. 2020;21(21):7893.

    CAS  PubMed Central  Article  Google Scholar 

  59. Stiegeler S, et al. The impact of microRNAs during inflammatory bowel disease: effects on the mucus layer and intercellular junctions for gut permeability. Cells. 2021;10(12):3358.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  60. Diamantopoulos MA, et al. Upregulated miR-16 expression is an independent indicator of relapse and poor overall survival of colorectal adenocarcinoma patients. Clin Chem Lab Med. 2017;55(5):737–47.

    CAS  PubMed  Article  Google Scholar 

  61. Zhang L, et al. MicroRNA-21 regulates intestinal epithelial tight junction permeability. Cell Biochem Funct. 2015;33(4):235–40.

    PubMed  Article  CAS  Google Scholar 

  62. Schaefer JS, et al. MicroRNA signatures differentiate Crohn's disease from ulcerative colitis. BMC Immunol. 2015;16:5.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  63. Liu Z, et al. MicroRNA-21 increases the expression level of occludin through regulating ROCK1 in prevention of intestinal barrier dysfunction. J Cell Biochem. 2019;120(3):4545–54.

    CAS  PubMed  Article  Google Scholar 

  64. Nakata K, et al. Commensal microbiota-induced microRNA modulates intestinal epithelial permeability through the small GTPase ARF4. J Biol Chem. 2017;292(37):15426–33.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. Tokumaru Y, et al. Low expression of miR-29a is associated with aggressive biology and worse survival in gastric cancer. Sci Rep. 2021;11(1):14134.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. Chen L, et al. miR-29a suppresses growth and invasion of gastric cancer cells in vitro by targeting VEGF-a. BMB Rep. 2014;47(1):39–44.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  67. He B, et al. hTERT mediates gastric cancer metastasis partially through the indirect targeting of ITGB1 by microRNA-29a. Sci Rep. 2016;6:21955.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  68. Liu X, et al. MicroRNA-29a inhibits cell migration and invasion via targeting roundabout homolog 1 in gastric cancer cells. Mol Med Rep. 2015;12(3):3944–50.

    CAS  PubMed  Article  Google Scholar 

  69. Zhao Z, et al. Reduced miR-29a-3p expression is linked to the cell proliferation and cell migration in gastric cancer. World J Surg Oncol. 2015;13:101.

    PubMed  PubMed Central  Article  Google Scholar 

  70. Zhang H, et al. Cell-derived microvesicles mediate the delivery of miR-29a/c to suppress angiogenesis in gastric carcinoma. Cancer Lett. 2016;375(2):331–9.

    CAS  PubMed  Article  Google Scholar 

  71. Fasseu M, et al. Identification of restricted subsets of mature microRNA abnormally expressed in inactive colonic mucosa of patients with inflammatory bowel disease. PLoS One. 2010;5(10):e13160.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  72. Sun D, et al. C/EBP-β-activated microRNA-223 promotes tumour growth through targeting RASA1 in human colorectal cancer. Br J Cancer. 2015;112(9):1491–500.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. Zhi X, et al. MiR-874 promotes intestinal barrier dysfunction through targeting AQP3 following intestinal ischemic injury. FEBS Lett. 2014;588(5):757–63.

    CAS  PubMed  Article  Google Scholar 

  74. Yuan RB, et al. MiR-874-3p is an independent prognostic factor and functions as an anti-oncomir in esophageal squamous cell carcinoma via targeting STAT3. Eur Rev Med Pharmacol Sci. 2018;22(21):7265–73.

    PubMed  Google Scholar 

  75. Duan L, et al. Molecular mechanisms and clinical implications of miRNAs in drug resistance of colorectal cancer. Ther Adv Med Oncol. 2020;12:1758835920947342.

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Que K, et al. Downregulation of miR-874-3p promotes chemotherapeutic resistance in colorectal cancer via inactivation of the hippo signaling pathway. Oncol Rep. 2017;38(6):3376–86.

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Han J, et al. MicroRNA-874 inhibits growth, induces apoptosis and reverses chemoresistance in colorectal cancer by targeting X-linked inhibitor of apoptosis protein. Oncol Rep. 2016;36(1):542–50.

    CAS  PubMed  Article  Google Scholar 

  78. Martínez C, et al. miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea. Gut. 2017;66(9):1537–8.

    PubMed  Article  CAS  Google Scholar 

  79. Zhou R, et al. Identification of microRNA-16-5p and microRNA-21-5p in feces as potential noninvasive biomarkers for inflammatory bowel disease. Aging (Albany NY). 2021;13(3):4634–46.

    CAS  Article  Google Scholar 

  80. Chen Y, et al. Inhibition of miR-16 ameliorates inflammatory bowel disease by modulating bcl-2 in mouse models. J Surg Res. 2020;253:185–92.

    CAS  PubMed  Article  Google Scholar 

  81. Zhang L, et al. MicroRNA-21 is upregulated during intestinal barrier dysfunction induced by ischemia reperfusion. Kaohsiung J Med Sci. 2018;34(10):556–63.

    CAS  PubMed  Article  Google Scholar 

  82. Ando Y, et al. Downregulation of microRNA-21 in colonic CD3+ T cells in uc remission. Inflamm Bowel Dis. 2016;22(12):2788–93.

    PubMed  Article  Google Scholar 

  83. Yang Y, et al. Overexpression of miR-21 in patients with ulcerative colitis impairs intestinal epithelial barrier function through targeting the rho GTPase RhoB. Biochem Biophys Res Commun. 2013;434(4):746–52.

    CAS  PubMed  Article  Google Scholar 

  84. Shi C, et al. MicroRNA-21 knockout improve the survival rate in DSS induced fatal colitis through protecting against inflammation and tissue injury. PLoS One. 2013;8(6):e66814.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  85. Yu W, et al. Overexpression of miR-21-5p promotes proliferation and invasion of colon adenocarcinoma cells through targeting CHL1. Mol Med. 2018;24(1):36.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  86. Giuffrida P, et al. Biomarkers of intestinal fibrosis - one step towards clinical trials for stricturing inflammatory bowel disease. United European Gastroenterol J. 2016;4(4):523–30.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  87. Zhu Y, et al. MicroRNA-29a mediates the impairment of intestinal epithelial integrity induced by intrauterine growth restriction in pig. Am J Physiol Gastrointest Liver Physiol. 2017;312(5):G434–g442.

    PubMed  Article  Google Scholar 

  88. Nijhuis A, et al. In Crohn's disease fibrosis-reduced expression of the miR-29 family enhances collagen expression in intestinal fibroblasts. Clin Sci (Lond). 2014;127(5):341–50.

    CAS  Article  Google Scholar 

  89. Zhou Q, et al. MicroRNA-29a regulates intestinal membrane permeability in patients with irritable bowel syndrome. Gut. 2010;59(6):775–84.

    CAS  PubMed  Article  Google Scholar 

  90. Fassan M, et al. Early miR-223 upregulation in gastroesophageal carcinogenesis. Am J Clin Pathol. 2017;147(3):301–8.

    CAS  PubMed  Article  Google Scholar 

  91. Neudecker V, et al. Myeloid-derived miR-223 regulates intestinal inflammation via repression of the NLRP3 inflammasome. J Exp Med. 2017;214(6):1737–52.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  92. Li M, et al. Mast cells-derived MiR-223 destroys intestinal barrier function by inhibition of CLDN8 expression in intestinal epithelial cells. Biol Res. 2020;53(1):12.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  93. Su Z, et al. LncRNA H19 functions as a competing endogenous RNA to regulate AQP3 expression by sponging miR-874 in the intestinal barrier. FEBS Lett. 2016;590(9):1354–64.

    CAS  PubMed  Article  Google Scholar 

  94. Mirjalali H, et al. Distribution and phylogenetic analysis of Blastocystis sp. subtypes isolated from IBD patients and healthy individuals in Iran. Eur J Clin Microbiol Infect Dis. 2017;36(12):2335–42.

    CAS  PubMed  Article  Google Scholar 

  95. Navarro-Llavat M, et al. Prospective, observational, cross-sectional study of intestinal infections among acutely active inflammatory bowel disease patients. Digestion. 2009;80(1):25–9.

    PubMed  Article  Google Scholar 

  96. Cekin AH, et al. Blastocystosis in patients with gastrointestinal symptoms: a case-control study. BMC Gastroenterol. 2012;12:122.

    PubMed  PubMed Central  Article  Google Scholar 

  97. Cifre S, et al. Blastocystis subtypes and their association with irritable bowel syndrome. Med Hypotheses. 2018;116:4–9.

    PubMed  Article  Google Scholar 

  98. Das R, et al. Molecular characterization and subtyping of Blastocystis species in irritable bowel syndrome patients from North India. PLoS One. 2016;11(1):e0147055.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  99. Jimenez-Gonzalez DE, et al. Blastocystis infection is associated with irritable bowel syndrome in a Mexican patient population. Parasitol Res. 2012;110(3):1269–75.

    PubMed  Article  Google Scholar 

  100. Khademvatan S, et al. Blastocystis and irritable bowel syndrome: frequency and subtypes from Iranian patients. Parasitol Int. 2017;66(2):142–5.

    PubMed  Article  Google Scholar 

Download references

Acknowledgments

The authors thank all members of the Foodborne and Waterborne Diseases Research Center for their collaborations.

Funding

This study was financially supported by the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences with grant number: RIGLD-1072.

Author information

Affiliations

Authors

Contributions

HM designed the study. HMR contributed in performing the experiments. HMR AY contributed in analyzing data. HAA MRZ contributed in providing reagents/facilities/instruments. HM HMR contributed in writing the manuscript. All authors read and confirmed the manuscript. The author(s) read and approved the final manuscript.

Corresponding author

Correspondence to Hamed Mirjalali.

Ethics declarations

Ethics approval and consent to participate

No human or animal tissues were analyzed in this study. The study was approved by the ethics committee/institutional review board of the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

All experimental protocols were approved by the Research Institute for Gastroenterology and Liver Diseases and all procedures of this study were in accordance with the ethical standards (IR.SBMU.RIGLD.REC.1398.048) released by the Ethical Review Committee of the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran. In addition, all methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

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, visithttp://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.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mohammad Rahimi, H., Yadegar, A., Asadzadeh Aghdaei, H. et al. Modulation of microRNAs and claudin-7 in Caco-2 cell line treated with Blastocystis sp., subtype 3 soluble total antigen. BMC Microbiol 22, 111 (2022). https://doi.org/10.1186/s12866-022-02528-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12866-022-02528-8

Keywords

  • Blastocystis sp., ST3
  • MicroRNA
  • Intestinal permeability
  • Claudin-7
  • Inflammation