Identification and characterization of miR-31 potential targets by RNA-seq

In our previous work, miR-31 displayed differential significant expression in Apostichopus japonicus sea cucumber with skin ulcer syndrome and modulated coelomocytes ROS production by targeting p105. To identify other promising targets ofmiR-31, 4 transcriptome libraries of coelomocytes, as well as 2 control libraries, were constructed frommiR-31 mimics (31 M) or AMO-miR-31 (31I) and injected into a sea cucumber at 12 and 24 h. A total of207,977 unigenes with an average length of 363 bp were assembled, in which17,204 distinct sequences (8.27% of the unigenes) were successfully matched with annotated protein sequences. Fragments per kilobase of transcript per million fragments mapped analysis indicated that 1325 unigenes displayed up-regulated expression profiles in the 31I-12 group and were depressed in the 31M - 12 group compared with the control group. A total of 1470 unigenes showed down-regulated expressions in 31I-12 and were induced in 31 M-12. Similarly, 2079 and 2098 unigenes were detected at 24 h post-injection. Among these unigenes, 36 unigenes (depressed expression in the 31 M group and induced in the 31I group) showed consistent expression patterns at 2 examined time points and were considered promising targets of miR-31. qPCR analysis confirmed that all 4 unigenes showed opposite expression profiles to miR-31 in cultured coelomocytes. Our present work provided a fast and feasible method of identifying miR-31 targets by transcriptome analysis. The results of this study would enhance our present understanding ofmiR-31 function insea cucumber immune regulation.