| Weaning piglet’s diarrhea and edema disease which can result in the death of piglets are important infectious diseases, and inflict economic damage on pig industry. E. coli F18 is the main pathogenic bacteria causing these two diseases. Due to the unclear molecular mechanism of Chinese local pig breeds resistance to E. coli F18, this study aimed at the regulation of microRNA and target genes. Firstly, based on microRNAs sequencing of E. coli F18 resistant and sensitive Sutai piglets with blood relationship of Taihu pig and Duroc pig, important miRNA, miR-192, was achieved. Then, A series of gene function verification, such as target genes prediction, qPCR, dual luciferase validation, bacterial infection on cell level, knockout by TALEN, and SNP detection of miRNA precursor, were done to analysis the function of miR-192 and target genes, aiming at revealing the regulatory mechanism of microRNAs in weaned piglets resistance to E. coli F18. The main results as below.(1) An efficient and accurate method testing Escherichia coli adhesion to intestinal epithelial cells will contribute to the research of bacteria pathogenic mechanism and function of receptor gene related with the adhesion. This study applied the method of real-time fluorescent quantitative PCR method. The quantitative PCR primer was designed from PILIN gene of E.coli F18ab, F18ac and K88ac and pig β-ACTIN gene, and the total DNA of Escherichia coli and intestinal epithelial cells was used as tamplate for relative quantitative PCR. The formula of 2-AACt was used to calculate the relative number of bacteria in different culture areas. Results showed that the relative number of F18ab, F18ac and K88ac adhering to intestinal epithelial cell was not significant different among the 6,12, and 24 well culture plates. That indicated that there was no relationship between the relative adhesion number of Escherichia coli and the area of cells, so that the method of real-time quantitative PCR could accurately test the relative number of Escherichia coli. This provided a convenient and reliable testing method for experiments of Escherichia coli adhesion in this study.(2) Combining the bioinformatic analysis of miR-192 target genes with the previous results of expression profile chip, five important target genes were obtained, including DLG5, ALCAM, FRMD4B, MIPOL1 and ZFHX3. By reason of the gene biological function and the results of literature mining, ALCAM and DLG5 were selected as the key target genes and studied for related function role in maintaining the integrity of plasma membrane of epithelial cell, so we studied the function of ALCAM and DLG5 as the key target genes. The recombinant vectors for luciferase report of key target gene UTR were constructed and cotransfected with miRNA mimics into 293 cell, and the targeting of miR-192 was analyzed. The result of luciferase activity showed that miR-192 mimics could inhibit ALCAM and DLG5 (P<0.05).(3) The expression profile of key target genes showed that, in 35 days old of Sutai resistant and sensitive pigs, the level of DLG5 were relatively higher in jejunum, duodenum, liver, lung and kidney tissues. The relative expression in resistance individuals was higher than sensitive individuals, the difference was significant in heart and lymph (P<0.05), and the fold change of all the tissues was from 1.049 to 6.464. The expression of ALCAM in Sutai pigs was relatively higher in liver, lung and kidney, the expression in stomach, lymphatic, duodenum and jejunum was moderate, and low in other tissues, and the expression of ALCAM in tissues had no difference between resistance and sensitive individuals. The results of different days,8,18,28, and 35 days, showed that the expression of DLG5 in muscle, lung and immune tissues, such as thymus, lymph and spleen, were decreasing with the growing of pigs, the expression of jejunum and duodenum reached lowest at the age of 28 days, while began rising at the age of 35 days. The expression of ALCAM in kidney was significantly higher at the age of 28 days than 8 days, and the expression in duodenum was significantly lower at the age of 28 and 35 days than 18 days which was on a downward trend.(4) The influence of the invasion of E. coli F18ab, F18ac, and K88ac on the expression of miR-192 in IPEC-J2 cell was analyzed, and the stimulation of E. coli F18ab, F18ac, and K88ac could not cause the significant change of miR-192 level, and neither could culture supernatants of bacteria. Also, the trend had no change with stimulus time. The expression level of DLG5 and ALCAM was significant down-regulated by bacteria stimulation (P<0.05). With the extension of dispose time, the level of DLG5 and ALCAM was reduced, and they were significant down-regulated (P<0.05).(5) The TALEN method was used to the sequence deletion of mature miR-192, and IPEC-J2 cells without miR-192 mature sequence were acquired. The expression analysis of key target genes showed that the expression of DLG5, ALCAM, FRMD4B in miR-192 knockout cell had no difference compared with the control cell, while the expression of MIPOL1 and ZFHX3 were significant lower (P<0.05). The result of E. coli adhesion level showed the adhesion level of E. coli F18ab, F18ac and K88ac were on the significant rise in knockout cell.In view of the genetic background differences between alien and Chinese indigenous pig breeds, the miRNAs research could not perfectly represent and reveal the regulatory mechanism of E. coli F18 resistance for Chinese indigenous pig breed. Moreover, Chinese indigenous breed, Meishan piglets, were chosen as the object of study. The E. coli F18 resistant and sensitive Meishan piglets were obtained by the methods of E. coli infection experiment, a series of phenotype detection, such as bacteria detection, bacteria adhesion of small intestinal epithelial cells in vitro and pathological examination. Differential miRNAs were selected from sensitive and resistance Meishan pigs, using Selex sequencing. Combined with the result of transcriptome sequencing, target prediction and interaction network were done to screen the key target genes and regulatory pathway. Then, using the RNAi technology and bacteria infection, the regulatory function of the key target and pathway were verified on the cell level, aiming at investigating their function and regulatory mechanism related with the E. coli F18 resistance of Chinese local weaned piglets.The main results as below.(1) miR-192, which played important regulative role in Sutai piglets, was functionally analyzed in Meishan pig, and the expression profile showed that the expression level in duodenum and jejunum was high, moderate in liver and kidney, low in heart, spleen, lung, stomach, thymus and lymph. Referencing the E. coli infection experiment of Meishan weaned piglets, the E. coli infection did not cause the miR-192 expression level significant changes. So, miR-192 might not play important function during the process of E. coli infecting Meishan weaned piglets.(2) Full sibs of E. coli F18 resistant and sensitive Meishan piglets confirmed by E. coli infection experiment and a series of verification, were analyzed by high throughput sequencing, and 24 differential miRNAs was screened in the duodenum, including 15 miRNAs upregulated and 9 miRNAs downregulated. The result of change fold by qRCR had coherence with the result of high throughput screening. Combining previous achievements of the transcriptome sequencing in the full sibs of E. coli F18 resistant and sensitive Meishan pig, with target predicting and network interacting, miR-7136-5p and its target, MyD88 gene, were chosen as the key. In consideration of the MyD88 played important role as an adaptor protein in the TLR4 signal pathway, the pathway was also selected as the key pathway, and its function and mechanism were analyzed during the E. coli infection.(3) The level of MyD88 reduced significantly in IPEC-J2 cell disposed by F18ac and K88ac, while TNFα, which is same in the TLR4 pathway, was significantly up-regulated (P<0.05). The level of MyD88 decreased sharp after 8 hours disposed by culture supernatant of F18ac and K88ac (P<0.05), and the TNFα was significantly up-regulated same as dealing with bacteria (P<0.05). Same as in IPEC-J2, MyD88 was decreased significantly in PK15 cell disposed by F18ac and K88ac (P<0.05). The F18ac could increase the expression of CD14 (P<0.01), but not K88ac. Neither F18ac nor K88ac could make the expression of IFNα significant exchange. IL-1β and TLR4 was slightly up-regulated. TNFα was increased significantly same as in IPEC-J2. Disposed by culture supernatant of F18ac and K88ac, the PK15 cell, in which MyD88, CD14, IFNα were all down-regulated. The supernatant of F18ac could extremely significantly increase the expression of TNFα(P<0.01), but not the K88ac.(4) Using RNAi technology, a vector expressing shRNA was obtained which targeted efficiently MyD88 gene. It was packed into lentivirus vector to construct pig cell lines with stably silent MyD88 gene. Eventually, IPEC-J2 and PK15 cells were harvested, whose MyD88 gene interference efficiency were 69.3% and 61.0%, respectively. The silence of MyD88 gene led to the significant down-regulation of TLR4 and IL-1β gene (P<0.05), while CD14, IFNα and TNFa had no significant change. The silence of MyD88 gene did not have influence on the immune factors of IL-1β and TNF-α in the cell culture supernatant.(5) The expression of MyD88 gene in RNAi PK15 cell was still reduced by the invasion of E. coli F18ac and K88ac. After disposed by E. coli, the variation trend of TLR4 and IL-1β in RNAi cell was larger than that of control cell. The variation trend of CD14 gene in RNAi cell was lower than that of control cell, and the variation trend of TNF-α gene in RNAi cell was similar to the control cell. IFNα in RNAi cell disposed by E. coli presented a significant down-regulation (P<0.05). Stimulated by the culture supernatant of E. coli F18ac and K88ac, the variation trend of TLR4, CD14, MyD88, IL-1β and IFN-α gene of RNAi PK15 cell was similar to the control, while the the variation trend of TNF-α gene in RNAi PK15 cell was lower than that of the control. The IL-1β and TNF-α factor level results showed that, stimulated by E. coli F18ac and K88ac, the level of IL-1β in the cell supernatant was increased significantly (P<0.01), and the up trend of IL-1β in RNAi group was higher than the control. The culture supernatant of E. coli K88ac could induce the IL-1β level, and the variation trend in two groups was similar. Neither the stimulation of E. coli F18ac and K88ac nor their supernatant could significantly change the level of TNF-a in the cell supernatant.Conclusion:The results of this study indicated that, microRNA played important regulatory function during the process of E. coli infecting Sutai and Meishan weaned piglets, and reminded that difference indeed exist in the genetic basis and regulatory mechanism between Meishan pig, Chinese indigenous pig breeds, and Sutai pig, cultivated breed containing foreign pig pedigree. Targeting DLG5 and ALCAM, miR-192 and its targets played important regulatory function in replying the invasion of E. coli, maintaining stability of intestine and regulating immunization in Sutai weaned piglets. Targeting MyD88, miR-7136-5p could influence TLR4 pathway, and play important roles in replying the invasion of E. coli and immune response in Sutai weaned piglets. |
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