Effects of mutations in porcine miRNA-215 precursor sequences on miRNA-215 regulatory function

Highlights

• The miR-192 precursor sequence was not mutated, but the miR-215 precursor included two mutations.

• Wild-type and mutant miR-215 precursor expression vectors were constructed.

• The sequence variation in the miRNA-215 precursor may affect the miRNA-215 regulatory network.

• The variation can alter the stability of intestinal epithelial cells and resistance to E. coli.

Abstract

MicroRNAs (miRNAs) play an important role in animal growth and disease development, and sequence variation in microRNAs can alter their functions. Herein, we explored the effects of mutations in the miRNA-215 precursor sequence on the miRNA-215 regulatory network and resistance to Escherichia coli (E. coli). Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to detect sequence variations in Sutai and Meishan pigs. The miR-192 precursor sequence was not mutated, but the miR-215 precursor included an AT insertion mutation at position 6 (start from the first base of the miR-215 precursor) and a C/T mutation at position 43. Wild-type (WT) and mutant miR-215 precursor expression vectors were constructed to investigate the effects of sequence variation on expression of miR-215 and its target genes DLG5 and ALCAM, cytokine levels and E. coli adhesion. Compared with the WT control group, cells harbouring the C/T mutant vector displayed reduced miR-215 expression, increased target gene expression, elevated cytokine levels and rising E. coli adhesion, whereas cells harbouring the AT insertion mutant vector were not significantly changed. The sequence variation in the miRNA-215 precursor may affect the miRNA-215 regulatory network, and alter the stability of intestinal epithelial cells (IPEC-J2 cells) and resistance to E. coli. Our findings provide guidance for future research on the regulatory mechanisms of miR-215 in porcine resistance to E. coli F18, and identifying effective genetic markers against this organism.

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.