Research paperEffects of mutations in porcine miRNA-215 precursor sequences on miRNA-215 regulatory function
Introduction
MicroRNAs (miRNAs) are a family of small noncoding RNAs that participate in post-transcriptional regulation of gene expression in animal and plant cells. MiRNAs can lead to mRNA degradation or translational repression by complete or incomplete complementary pairing with 3′-untranslational regions (3’UTRs) of target mRNAs, facilitating regulation of post-transcriptional expression of target genes and participation in various biological processes (Ambros, 2004). Recent studies demonstrated that miRNAs play an important regulatory role in porcine muscle development, fat metabolism, and a variety of viral and bacterial diseases (Xie et al., 2017; Lee et al., 2013; Loveday et al., 2012; Xia et al., 2013; Zhu et al., 2017). Diarrhea in weaned piglets is a kind of common and infectious disease caused by complicated factors in large-scale pig production, which have caused enormous economic losses to the pig industry. Thereinto, Escherichia coli F18 is one of the main pathogens responsible for diarrhea in weaned piglets, and it has two important serotypes (F18ab and F18ac). Our research group previously screened two miRNAs (miR-192 and miR-215) related to Escherichia coli F18 resistance in weaned piglets via high-throughput sequence analysis (Ye et al., 2012), and identified discs large homolog 5 (DLG5) and activated leukocyte cell adhesion molecule (ALCAM) as potential key target genes of miR-192/−215 (Wu et al., 2015). MiR-192 and miR-215 are homologous microRNAs sharing the same ‘seed’ region (Lagos-Quintana et al., 2003), and both are associated with cell proliferation and differentiation, tumour growth, cancer development, bacterial invasion, and many other physiological and pathological processes (Chiang et al., 2012; Khella et al., 2013). Jin et al. (2011) revealed that miR-215 and -192 were significantly upregulated in gastric cancer tissues to inhibit the expression of ALCAM, and they promoted cell growth and migration of gastric cancer, indicating their potential as prognostic markers of this disease. Kim et al. (2017) found that downregulation of miR-215 and -192 leads to upregulation of ALCAM and N-cadherin to improve migration and adhesion of mesenchymal cells, and they participate in carcinogenesis. Meanwhile, DLG5 plays a critical role in regulating cell growth, maintaining the structural integrity of epithelia cells, signal transduction, and embryonic development (Stoll et al., 2004; Reilly et al., 2015). In addition, DLG5 also influences the occurrence of cancer and inflammatory bowel disease (Li et al., 2016). ALCAM mediates cell adhesion, and is correlated with several physiological and pathological processes such as haematopoietic cell growth, intestinal stem cell homeostasis, blood brain barrier homeostasis, inflammation, and tumour invasion (Ohneda et al., 2001; Smith et al., 2017; Tan et al., 2012). In summary, we surmised that miR-215/-192 may play an important regulatory role in resistance to E. coli in weaned piglets by targeting DLG5 and ALCAM.
Previous research showed that miRNA expression and function can be influenced by genetic variation (Ryan et al., 2010). The specific conformation of miRNA precursors (pre-miRNAs) is processed by endonucleases Drosha and Dicer to generate mature miRNAs (Lee et al., 2003; Chekulaeva and Filipowicz, 2009). Thus, mutations in the miRNA precursor sequence may alter its conformation, mediating upregulation or downregulation by changing miRNA maturation, and ultimately affecting the regulation of target genes. If mutations occur in the seed sequence, microRNAs may lose their original target genes or engender new target genes, altering the corresponding biological traits, potentially causing diseases (Ryan et al., 2010). To date, research on polymorphisms of miRNA precursors has mainly focused on human diseases (Dikeakos et al., 2014; Bensen et al., 2013; Li et al., 2014), and few reports on porcine disease susceptibility have been published. Therefore, based on our previous findings, we used PCR-SSCP to detect genetic variation in miRNA-192 and -215 precursor sequences. In addition, wild-type (WT) and mutant miR-215 precursor expression vectors were constructed to study the effects of sequence mutations on expression of miR-215 and its target genes DLG5 and ALCAM, cytokine levels and adhesion of E. coli cells. The results will provide insight into the regulatory mechanisms of miR-215 in porcine resistance to E. coli, and provide a basis for screening and identifying effective markers for disease resistance breeding.
Section snippets
Ethics statement
The animal study proposal was approved by the Institutional Animal Care and Use Committee (IACUC) of the Yangzhou University Animal Experiments Ethics Committee (permit number: SYXK (Su) IACUC 2012-0029). All experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People's Republic of China.
Experimental materials
Meishan pigs (n = 59) and Sutai pigs (n = 75) were collected from Meishan pig
PCR-SSCP detection and sequencing analysis
PCR products of miR-192/215 were checked using SSCP analysis, and the results showed that amplified products of the miR-192 precursor were of a single band type, indicating no polymorphism (Supplementary Fig. 3A). PCR sequencing further confirmed that the miR-192 precursor sequence did not include single nucleotide polymorphism (SNP) sites. By contrast, three band types were observed in amplification products of the miR-215 precursor (Supplementary Fig. 3B). PCR sequencing revealed an insertion
Discussion
Recent studies on human and animal diseases has revealed that genetic variation in pre-miRNAs and miRNAs can alter disease susceptibility by affecting the expression of mature miRNAs, indicating their potential as molecular markers for disease diagnosis (Tian et al., 2009; Oner et al., 2015). In addition, genetic variations are correlated with the economics of livestock farming (Lei et al., 2011; Wang et al., 2015; Hong et al., 2012). Sequence mutations in miRNA precursors can influence the
Acknowledgements
This work was supported by the Postgraduate Research & Practice Innovation Program of Jiangsu Province [grant number KYCX17_1889] and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institution.
Author contributions
WB and SW conceived and designed the experiments. ZG, LS and KD performed the experiments. ZG and LS analysed the data. YD contributed reagents/materials/analysis tools. LS and WB contributed to the writing of the manuscript.
Conflicts of interest
The authors declare that they have no competing interests.
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2022, Digestive and Liver DiseaseCitation Excerpt :miRNAs can lead to mRNA degradation or translational repression by complete or incomplete complementary pairing with 3′-UTRs of target mRNAs, facilitating regulation of post-transcriptional expression of target genes and participation in various biological processes [31]. Multiple miRNAs have been identified to target DLG5 by binding to its 3′-UTR, and share a very significant negative correlation with the DLG5, such as miR-215 and miR-218-3p [32,33]. In our study, DLG5 was revealed to be a functional target of miR-454-3p by the TargetScan website (http://www.targetscan.org/vert_72/) in combination with the dual-luciferase reporter gene assay, and also, DLG5 expression was negatively regulated by miR-454-3p.
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Zhongcheng Gao and Li Sun contributed equally to this study.