Entamoeba histolytica Up-Regulates MicroRNA-643 to Promote Apoptosis by Targeting XIAP in Human Epithelial Colon Cells

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that function as negative regulators of gene expression. Recent evidences suggested that host cells miRNAs are involved in the progression of infectious diseases, but its role in amoebiasis remains largely unknown. Here, we reported an unexplored role for miRNAs of human epithelial colon cells during the apoptosis induced by Entamoeba histolytica. We demonstrated for the first time that SW-480 colon cells change their miRNAs profile in response to parasite exposure. Our data showed that virulent E. histolytica trophozoites induced apoptosis of SW-480 colon cells after 45 min interaction, which was associated to caspases-3 and -9 activation. Comprehensive profiling of 667 miRNAs using Taqman Low-Density Arrays showed that 6 and 15 miRNAs were significantly (FC > 1.5; p < 0.05) modulated in SW-480 cells after 45 and 75 min interaction with parasites, respectively. Remarkably, no significant regulation of the 6-miRNAs signature (miR-526b-5p, miR-150, miR-643, miR-615-5p, miR-525, and miR-409-3p) was found when SW-480 cells were exposed to non-virulent Entamoeba dispar. Moreover, we confirmed that miR-150, miR-643, miR-615-5p, and miR-525 exhibited similar regulation in SW-480 and Caco2 colon cells after 45 min interaction with trophozoites. Exhaustive bioinformatic analysis of the six-miRNAs signature revealed intricate miRNAs-mRNAs co-regulation networks in which the anti-apoptotic XIAP, API5, BCL2, and AKT1 genes were the major targets of the set of six-miRNAs. Of these, we focused in the study of functional relationships between miR-643, upregulated at 45 min interaction, and its predicted target X-linked inhibitor of apoptosis protein (XIAP). Interestingly, interplay of amoeba with SW-480 cells resulted in downregulation of XIAP consistent with apoptosis activation. More importantly, loss of function studies using antagomiRs showed that forced inhibition of miR-643 leads to restoration of XIAP levels and suppression of both apoptosis and caspases-3 and -9 activation. Congruently, mechanistic studies using luciferase reporter assays confirmed that miR-643 exerts a postranscripcional negative regulation of XIAP by targeting its 3'-UTR indicating that it's a downstream effector. In summary, we provide novel lines of evidence suggesting that early-branched eukaryote E. histolytica may promote apoptosis of human colon cells by modulating, in part, the host microRNome which highlight an unexpected role for miRNA-643/XIAP axis in the host cellular response to parasites infection.

Keywords: Entamoeba histolytica; SW480; XIAP; apoptosis; microRNAs.

Figure 1

E. histolytica induces apoptosis in SW-480 cells. (A) Representative transmission electron microscopy ultrastructure image of confluent SW-480 cells showing a well-spread cell with irregular microvilli, mitochondria distributed through the cytoplasm and enlarged nuclei. Scale bar 5 μM. (B) Image shows transmission electron microscopy ultrastructure images of trophozoites in contact with colon cells after 15, (C) 30 and (D) 45 min of co-culture. (D) SW-480 cells showing typical early changes associated with apoptosis including a general cell-shrinkage, loss of microvilli and nuclear chromatin condensation denoted by arrows (close up in D). Scale bar 10 μM. (E) Apoptosis assays. SW-480 cells after interaction with E. histolytica trophozoites for 0, 15, 30, 45, and 75 min were analyzed by flow cytometry using annexin V. SW-480 cells without exposure to trophozoites and cells treated with cisplatin (50 μM) for 24 h were used as controls. Data are representative of three independent experiments, performed in triplicate. Bars represent the mean of three independent experiments ± S.D. **p < 0.01. ***p < 0.001. (F) Western blot assays showing cropped images for immunodetection of caspase-9 and−3. Whole protein extracts were obtained from SW-480 cells after interaction with trophozoites for 0, 15, 30, and 45 min and submitted to Western blot assays using caspase-3 and caspase-9 antibodies. Actin antibodies were used as loading control. Immunodetected bands intensity was quantified by densitometry and normalized against actin. Numbers between panels represent the fold change values relative to control (time 0).

Figure 2

Validation of differential expression of cellular miRNAs after interaction of colon cells with trophozoites. (A) Expression levels of the 6-miRNAs signature during the interplay of SW-480 cells with E. histolytica for 45 min using qRT-PCR assay (black bars) in comparison with data obtained using spent E. histolytica media, during interaction of Entamoeba dispar parasites with SW-480 cells and relative to TLDAs profiling (white bars). qRT-PCR assays of Caco2 cells interacting with trophozoites were carried out as an additional control (gray bars). Cell lines, treatments and experiments performed for the 6-miRNAs profiling are denoted in the box inset. (B) qRT-PCR assays of the 6-miRNAs in SW-480 and Caco2 colon cells treated with cisplatin as apoptosis control for 24 h. Data is representative of three independent experiments that were performed in duplicate, error bars represent S.D.

Figure 3

Co-regulation networks of modulated miRNAs and predicted target mRNAs. Image depicts the interaction networks of miRNAs modulated at (A) 45 min and (B) 75 min after interaction of SW-480 cells with E. histolytica. Target mRNAs were predicted by at least two of the three programs used (DIANA-microT, TargetScan, and miRanda). Genes related to apoptosis were also included even if they were targeted by only one miRNA.

Figure 4

XIAP 3′UTR is a target of miR-643. (A) Western blot assays for XIAP expression. SW-480 cells were incubated with trophozoites for 0, 15, 30, and 45 min. Total protein were extracted and XIAP expression was determined by Western blot assays using XIAP antibody (Cell signaling). Actin antibodies (Santa Cruz) were used as control. Numbers represents the fold change in XIAP expression quantified by densitometry and normalized against actin. Values are expressed relative to the control group (time 0 h). (B) Schematic representation of pGL3-XIAP construct containing the 3′UTR of XIAP gene cloned at the XbaI site downstream of firefly luciferase gene (Luc+) of pGL3 Luciferase Reporter Vector. The miR-643 seed sequence is indicated in the colored box. (C) qRT-PCR assays for expression analysis of miR643. SW-480 cells were incubated with trophozoites for 45 min and transfected with miR-643 inhibitor at 30 nM and 50 nM or with scramble sequence (50 nM). (D) Luciferase reporter gene assays. SW-480 cells transfected with the pGL3-XIAP were incubated with trophozoites for 45 min and co-transfected with anti-miR-643 (30 and 50 nm) or scramble (50 nM). Luciferase activity was measured after 24 h. SW-480 cells transfected with pGL3-XIAP but not exposed to parasites were used as control. (E) Western blot assays showing cropped images for XIAP expression. SW-480 cells transfected with anti-miR-643, scramble or not transfected were incubated with trophozoites for 45 min and then submitted to immunoblotting with XIAP and actin antibodies. Bands intensity was quantified by densitometry and normalized to actin. In all cases, data are representative of three independent experiments by duplicate. Error bars represent S.D. The unpaired Student t-test was used to compare each condition with control cells that were not exposed to E. histolytica. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Inhibition of miR-643 impairs apoptosis of SW-480 cells interacting with trophozoites. (A) SW-480 cells were or not transfected with miR-643 inhibitor or scramble and interacting with trophozoites for 45 min. Then apoptosis of SW-480 cells was quantified using annexin V assays. SW-480 cells without exposure to E. histolytica and cells treated with cisplatin (50 μM, during 12 h) were used as controls. (B) Protein extracts from miR-643 deficient SW-480 cells interacting with E. histolytica trophozoites for 45 min and controls were submitted to Western blot assays using caspase-3 and caspase-9 antibodies. GADPH antibodies were used as loading control. (C) Immunodetected bands intensity in panel B were quantified by densitometry and normalized to GAPDH. Values are expressed relative to the control group (0 h, interaction). **p < 0.01; ***p < 0.001.

Figure 6

Working model that describes the possible participation of miR-643 in apoptosis of SW-480 cells driven by E. histolytica. Upregulation of miR-643 occurs after contact of SW-480 cells with parasites. Then miR-643 may target the XIAP 3′UTR inducing the downregulation of XIAP protein levels, which release the inhibition of caspase-3 and -9. In consequence the executioner caspase-3 triggers degradation of cellular proteins inducing the apoptosis of SW-480 cells.