| Micro RNAs(mi RNAs)are a class of single-stranded non-coding RNAs that are widely found in animals,plants,and microorganisms.They are about 21–24 nucleotides long,and control gene expression at the post-transcriptional level by cutting the m RNA of the target gene and inhibiting its translation.Thus,mi RNAs participate in the regulation of many biological processes,including growth,development,apoptosis,reproduction,and signal transduction.mi RNAs play regulatory roles not only inside the cell,but also outside.The mi RNAs that enter the circulatory system are known as “circulating mi RNAs”.Many studies have shown that circulating mi RNAs are present in human serum,saliva,milk,and urine.Circulating mi RNAs can enter the circulatory system mainly through secretion via exosomes,in conjunction with the RNA-binding protein and leakage from broken cells.As intercellular communication signaling molecules,circulating mi RNAs are loaded into exosomes and transported from biosynthesis sites to target organs or cells to regulate gene expression.Recent studies have shown that mi RNAs are able to regulate gene expression across different species.In 2012,Zhang et al.discovered that rice-derived mi R168 a crosses the mouse intestine wall into the bloodstream,where it is transported to the liver via microvesicles.In the liver,it regulates cholesterol levels by inhibiting the expression of LDLRAP1,the gene encoding lowdensity lipoprotein receptor adapter protein.Similarly,mi R162 a in pollen induces young bees to develop into worker bees by targeting down-regulation of the honeybee am TOR gene.In 2015,our research group used high-throughput sequencing technology to detect eight mulberryderived mi RNAs in silkworms.Based on those data,in this study,we used the silkworm Bombyx mori,an oligophagous insect that feeds only on mulberry leaves,to analyze the stability of mulberry-derived mi RNAs in the silkworm midgut lumen.Then,we selected mulberry mi RNAs that were resistant to digestive enzymes in the silkworm gut lumen.Silkworm genes targeted by mulberry-derived mi RNA were predicted by bioinformatics software.By conducting mi RNA mimic transfection of silkworm cell lines,double luciferase reporter experiments,silkworm organ culture in vitro,mi RNA mimic injection of silkworms,and feeding mimic studies,we preliminarily explored whether mulberry mi RNAs are able to regulate target genes in silkworm.The main results obtained in this research are as follows:1.Some mulberry mi RNAs are stable in the midgut lumen of silkworm.First,total RNAs were extracted from the mulberry leaves and incubated with silkworm digestive juice.The RNA status was evaluated by agarose gel electrophoresis and fluorescence quantitative PCR.Most of the RNAs were degraded,indicating that naked RNAs extracted from mulberry leaf are sensitive to enzymes in silkworm digestive juice.Secondly,mulberry leaf homogenates were incubated with silkworm digestive juice,and total RNAs in the supernatant were isolated.Most of the long fragment RNAs were degraded,compared with the control(without digestive juice).Compared with the control,the test group showed slightly lower degradation of mi RNAs,suggesting that some mi RNAs of mulberry leaves are protected against degradation by digestive enzymes in the silkworm midgut lumen.Unexpectedly,some mi RNAs(such as mi R166 b,mi R396b)were present at increased levels compared with the control.We speculated that the digestive enzymes of silkworm could accelerate the release of these mi RNAs from mulberry leaf cells,thereby increasing the mi RNA levels in the supernatant of homogenates.2.Mulberry-derived mi RNA are present in the exosomes of the silkworm hemolymph.The exosomes of silkworm hemolymph were isolated and the total mi RNAs were extracted from them.Using quantitative PCR,the indicated mulberry mi RNAs were detected in total mi RNAs from silkworm hemolymph exosomes.The results suggested that these mulberry mi RNAs are sorted into silkworm exosomes.The results of q PCR analyses showed that the detected mulberry mi RNAs displayed differential expression levels.Notably,mi R159 a was expressed at much higher levels than mi RNA396 b in mulberry leaves,but this pattern was completely reversed in silkworm hemolymph exosomes.These preliminary data suggested that silkworm might selectively transport mi RNAs from ingested mulberry leaves into the body cavity through exosomes to play a role in regulating gene expression.3.Putative target genes of mulberry mi RNAs in silkworm.Based on our previous results,the putative target genes of eight mulberry-derived mi RNAs(mi R156 c,mi R159 a,mi R162,mi R166 b,mi R166 c,mi R167 e,mi R396 b,and mi R398)that were detected in silkworm were subjected to further analyses using the software mi Randa and RNA22.In total,1757 and 1320 target genes were predicted by mi Randa and RNA22,respectively,and 247 target genes were commonly predicted by both software packages.To reduce the false discovery rate,much stricter parameters were then applied for mi RNA target prediction.This resulted in the identification of 58 putative targets.A GO functional analysis showed the target genes are involved in multiple physiological processes in silkworm.These data from target-prediction and bioinformatics analyses lay the foundation for subsequent screening and target gene verification.4.Mulberry-derived mi RNAs change expression levels of target genes in silkworm.First,the transcript levels of 14 potential target genes were determined using an organ culture system and silkworm larvae injection experiments.Based on these data,four target genes,APO,CCAP,Dna J,and RAD50,were selected for further verification.We used Bm N cells and Bm E cells for mi RNA target validation assays.The transcriptional level of these four genes(APO,CCAP,Dna J,and RAD50)putatively targeted by mi R156 c and mi R396 b were suppressed by mi R156 c mimic and mi R396 b mimic,as determined by quantitative PCR.Recombinant plasmids containing a luciferase reporter gene with its 3′end fused with the mi RNA-targeted fragment from silkworm Dna J or RAD50 genes and mi R396 b mimic were co-transfected into Bm N cells.The luciferase activity of cells transfected with mi RNA396 b mimic was significantly reduced,compared with those transfected with negative control mi RNAs.The reduced luciferase activity confirmed that mi R396 b is able to interact with Dna J or RAD50.Furthermore,when silkworm larvae were fed an artificial diet containing mi RNA396 b mimic,the mi RNA396 b mimic was detected in silkworm hemolymph by quantitative PCR.In addition,the transcript levels of Dna J and RAD50 were significantly reduced in silkworms fed with mi R396 b mimic,compared with the negative control.Taken together,these data proved that silkworm Dna J or RAD50 are target genes of mulberry mi R396 b. |
