Analysis Of The Micrornas Expression Profiles In HCT-8 Cells Infected With Cryptosporidium Parvum Subtype Ⅱd

Cryptosporidium is an importantly obligate intracellular protozoan parasite that causes gastroenteritis in both humans and a variety of vertebrate hosts.Up to now,27 species are recognised as valid and of these,Cryptosporidium parvum and Cryptsporidium hominis are responsible for the majority of infections in humans.C.parvum is the most widely studied species in the world,and subtype Ⅱa in this species is dominant abroad,while subtype Ⅱd is the most popular and unique in China.Currently,there is no effective drug or vaccine against cryptosporidiosis,and pathogenesis of the disease is barely understood.So the study of Cryptosporidium parvum subtype Ⅱd has important public health implications.MicroRNAs,an important non-coding small molecules,act as endogenous regulate factors in cells,regulating gene expression at the posttranscriptional level.They play an important roles in development,cell proliferation,differentiation,apoptosis and metabolism.It was found that mircoRNAs expression has been shown to be highly conserved,temporally regulated as well as tissue-specific in many species.So far there is rarely research on microRNAs of host infected with Cryptosporidium parvum.More efforts on this aspect will make contribution to study the relationship between Cryptosporidium parvum and hosts.In this research,purifing methods of Cryptosporidium parvum oocysts subtype Ⅱd oocysts were established,the life cycle stages of Cryptosporidium parvum subtype Ⅱd were visualized by inoculating HCT-8 cells in vitro model,gene chip technology was applied to detect microRNAs expression profile in HCT-8 cells infected with Cryptosporidium parvum subtype Ⅱd.1.Establishment of purifing methods of Cryptosporidium parvum oocystsIn order to obtain a large number of Cryptosporidium parvum oocysts from feces,the separation and purification of method for Cryptosporidium parvum oocysts was established in the present study.Cryptosporidium parvum ocysts were propagated by infecting a Holstein male neonatal calf(not fed freshing colostrm),oocysts excreting and clinical manifestations were monitored.The results showed that the excrete peak of oocysts was the 5-7th days post infection,the patent period was 17 days and oocysts harvested were billions.The lipids in calf will affect acquisition of Cryptosporidium parvum oocysts,the treatment method of fecal fat was optimized.Different volume ratio of water-ether precipitation and water-ethyl acetate precipitation was applied to degrease feces separately.The number of oocysts obtained from the two methods was no significantly different.However,oocysts collected from the water-ether precipitation were purer and with few impurities under microscopic observation.And the fat layer is stable by water-ether precipitation.The results manifested that the degreasing effect of water-ether precipitation is better than water-ethyl acetate precipitation.The number of oocysts when the water and ether volume ratio was 1:1 had no insignificantly difference with the number when volume ratio was 1:2(P>0.05).Therefore,the former ratio was applied in the following experiments.Then the oocysts were further purified with sucrose and CsCl gradient centrifugation and the recovery rate was 71.57%and 89.69%respectively.The purified oocysts were pure in large quantity,with 77.78%average activity,which can be used in vitro cultivation,antibodies preparation and genome research,etc.2.Study on the Cryptosporidium parvum subtype Ⅱd endogenous developmental history in HCT-8 cellsIn order to understand the Cryptosporidium parvum subtype Ⅱd endogenous developmental history in HCT-8 cells,the purified oocysts from neonatal calf were inoculated in HCT-8 cell s with the cell to oocysts ratio 1:2 to observe the development and propagation of parasites by Giemsa staining and PCR amplifiation based on gp60 gene in this study.The results told us that the developmental stages of Cryptosporidium parvum subtype Ⅱd containing sporozoite,trophozoite,meront,macrogamont,microgamont,zygote and oocyst were identified from the cells.Cryptosporidium parvum subtype Ⅱd was successfully determined by PCR amplifiation at each time point.Our research provided theoretical basis for the endogenous developmental history,in-vitro culture of other Cryptosporidium ssp.and the exploitation of new antiprotozoal drug.3.Analysis of the microRNAs expression profiles in HCT-8 cells infected with Cryptosporidium parvum subtype ⅡdIn order to probe microRNAs differential expression in HCT-8 cells infected with Cryptosporidium parvum subtype Ⅱd and provide a basis for Cryptosporidium parvum invasion mechanism and screening of drug targets,total RNA of HCT-8 cells infected with Cryptosporidium parvum subtype Ⅱd at 4 h and 12h were determined using μParaflo microfluidic chip containing probe sets for 2555 human mature microRNAs in the present study.The results showed that the expression of hsa-miR-122-5p was significantly up-regulated and the expression of hsa-miR-3591-3p,hsa-miR-6074,hsa-miR-454-5p,hsa-miR-34b-5p,hsa-miR-4685-3p and hsa-miR-1908-3p were remarkably down-regulated at | log2(OI/OC)|>2 and p-value<0.05 at 4 h post-inoculation,the expression of hsa-miR-942-5p,hsa-miR-5580-3p and hsa-miR-6763-5p were significantly up-regulated,expression of hsa-miR-181d-3p,hsa-miR-18b-3p,hsa-miR-4689,hsa-miR-1256,hsa-miR-3976,hsa-let-7b-3p,hsa-miR-6721-5p,hsa-miR-3118 and hsa-miR-3121-5p were markedly down-regulated at | log2(OI/OC)|>2 and p-value<0.05 at 12 h post-inoculation.The three bioinformatics algorithms TargetScan,miRanda and PicTar were utilized to predict the target sites of several differentially expressed microRNAs.46 target genes,including MAPRE1,METTL9,CCDC6,ALDOA,CNOT6,ATP2A2 and LGR4 were obtained.Some of them may play an important role in Cryptosporidium parvum invasion procedure.Further investigations are needed to elucidate the pathogenesis of cryptosporidiosis and screen drug targets.