Evaluation of MIR-21 and MIR-145 expression in cryptosporidium parvum infected HCT-8 and HT-29 cell lines

Abstract

Cryptosporidium parvum is a widespread pathogenic parasite which causes cryptosporidiosis in human. The host cell-C. parvum interaction causes alteration of expression of a series of microRNAs or miRNAs within the host cells due to activation of defense mechanism. In immunocompromised host cells, miRNAs play a key role in post-transcriptional gene regulation. The regulation of miRNAs in the infected cells may be identified as possible biomarkers in cancer onset and progression. Upregulation of oncomicroRNAs in the host epithelial cells due to the C. parvum infection may lead to colorectal cancer initiation and progression in human. HCT-8 and HT-29 were grown in different L-glutamine concentration and pH to observe the relative growth. The study was aimed to delineate the host epithelial colorectal cancer cells-C. parvum interaction and its associated miRNAs involvement in the immunocompromised host cells in vitro. The differential expression level of oncomiRNA, miR-21 and tumor suppressor miRNA, miR-145 in host cells upon C. parvum infection was observed in order to establish a relationship between C. parvum infection and colorectal cancer in humans. As Cryptosporidium mainly infects the epithelial cells of the colorectal region, the host cells of interest in this study are HCT-8 and HT-29 cell lines. The direct immunofluorescent staining method has been applied using fluorescein conjugated Vicia Villosa Lection (VVL) to observe the C. parvum infection sites on HCT-8 and HT-29 cell lines after 2.5 hours of infection period. In addition, scanning electron microscopy has been used to observe the surface micrographs of different life stages of C. parvum adhered to the cell lines. The real time quantitative PCR (R T-qPCR) was performed to observe the relative expression ofmiR-21 and miR-145 in infected HCT- 8 and HT-29 cell lines. Data normalization of the miRNA expression was carried out using reference gene RNU44. The result showed that, the cell growth was optimum for cell lines in higher L-glutamine concentration and higher pH. In the immunofluorescent staining assay, the micrographs show multiple sites of C. parvum infection on both HCT-8 and HT-29 cell lines. Meanwhile, scanning electron micrographs demonstrated the adherence of C. parvum oocysts in both HCT-8 and HT-29 cell lines after 24 hours of inoculation. Along with the surface meront I, several numbers of early trophozoites in their developmental stages were confined to the apical surfaces of HCT-8 and HT- 29 cell lines. Besides~ a few sporozoites have been observed to be attached on the HCT- 8 and HT-29 cell lines. The normalized expression quantification data obtained from RT-qPCR has shown upregulation of miR-21 expression and significant downregulation of miR-145 expression in HCT-8 and HT-29 cell lines upon C. parvum infection. The rniR-21 expression showed significantly higher upregulation in infected HCT-8 than in infected HT-29. On the other hand, miR-145 downregulation was significantly lower in HT-29 cells than in HCT-8 cells upon C. parvum infection. In this study, immunofluorescent micrographs and scanning electron micrographs indicated that C. parvum effective infects human colorectal cancer cell lines. The upregulation of oncomiRNA and downregulation of tumor suppressor miRNA in infected HCT-8 and HT-29 cells possibly indicates the influence of C. parvum in cancer progression. However, the findings are less sufficient to confirm the C. parvum as a pathogenic parasite for cancer progression. Future studies should be aimed to observe more targeted genomic and transcriptomic alterations, relative expression of sets of targeted miRNAs to absolutely verify the progression of colorectal cancer by C. parvum infection.