Targeting Relationship Between Let-7 Family And Cbx2 And Their Expression In The Gonads Of Japanese Flounder (Paralichthys Olivaceus)

Japanese flounder(Paralichthys olivaceus)is an important marine economic cultured fish.The female is larger than the male.It is a good animal model to study the gonadal differentiation and development of fish.For a long time,the study of gonadal differentiation and development has been the focus of fish genetics and developmental biology.Because the genomes of both sexes are basically the same,the differential expression of genes may contribute to the differentiation and development of gonads.Gene expression is mainly regulated by transcription level,different transcription factors or post transcription level.Micro RNAs,combined with target messenger RNA(m RNAs),can induce m RNA degradation or translation inhibition,and play a role in post transcriptional regulatory factors in gene expression.Our laboratory has previously completed high-throughput sequencing of micro RNAs in the testis and ovary of Japanese flounder,and screened a number of differentially expressed and highly expressed mi RNAs in male and female gonads.Let-7a,let-7b,Let-7c,let-7d,let-7e,let-7f,let-7g,let-7h,let-7i and let-7j were identified in Japanese flounder,and the expression of the mi RNA family during metamorphosis was detected.Let-7 family also plays an important role in the gonadal development of mammals and other animals.However,there are few reports on the target of let-7 family in gonads.It is important to find the target genes of let-7 family for explaining the function of let-7 family in vertebrate gonads.We also identified cbx2(chromobox homolog 2)and calb2(calretinin)genes in Japanese flounder.Therefore,in this study,we first predicted the targeting relationship between let-7family and candidate target genes calb2 and cbx2 by bioinformatics method(website RNAhybrid),and verified the relationship between let-7 family and cbx2 by dual luciferase reporter gene system.Bioinformatics prediction results showed that only one member of let-7 family(let-7c)was completely complementary to calb2 3’UTR,while five members of let-7 family were completely complementary to cbx2 3’UTR,including let-7b,let-7d,let-7e,let-7g and let-7j.The results of dual luciferase reporter gene technology showed that cbx2 was the target gene of let-7b,let-7d,let-7e,let-7g and let-7j.Among them,there were significant differences in the targeting relationship between let-7b and cbx2 3’UTR(P<0.05),while there were extreme significant differences in the targeting relationship between let-7d,let-7e,let-7g and let-7j(P<0.001).The target sites of four members(let-7d,let-7e,let-7g and let-7j)with extreme significant difference in targeting relationship were mutated in vitro,and then verified by dual luciferase reporter gene technology.The results showed that cbx2 3’UTR lacked binding sites with these four members due to site mutations and could not be regulated by let-7 family mi RNAs.On this basis,among the four let-7 family members that have been confirmed to have extreme significant targeting relationship with cbx2 3’UTR,let-7g,which has the highest expression level in the gonads of Japanese flounder,was selected.The expression of let-7g and its target gene cbx2 in the gonads of Japanese flounder was detected by real-time fluorescence quantitative method,and the localization of let-7g and its target gene cbx2 in the testis of Japanese flounder was analyzed by fluorescence in situ hybridization.Real time fluorescence quantitative analysis showed that let-7g was significantly higher expressed in testis than in ovary at different developmental stages,which was consistent with the results of high-throughput sequencing;Cbx2expression was significantly lower in testis than in ovary at different developmental stages,which was consistent with the theory that mi RNA negatively regulates target genes.During gonadal development,the expression of let-7g in testis and ovary increased first and then decreased,and reached the highest level in stage IV.the lowest level in testis was stage II,and the lowest level in ovary was stage Ⅴ.The expression level of cbx2 in ovary decreased first and then increased,and reached the lowest level in stage IV,while the expression level of cbx2 increased all the time during the development of testis.The results of fluorescence in situ hybridization showed that the expression pattern of cbx2 in testis was consistent with that of vasa.Strong positive hybridization signals of cbx2 could be detected in spermatogonia and spermatocytes around the seminiferous lobule,but weak cross signals of cbx2 were detected in spermatids inside the mature seminiferous lobule and mature spermatozoa;Let-7g was localized in germ cells of testis,mainly including spermatogonia and spermatocytes,but no signal was detected in germ cells of other developmental stages.In order to further explore the relationship between let-7g and cbx2 and the regulatory role of let-7g in the gonads of Japanese flounder,the expressions of let-7g,cbx2 and sf1 were detected by real-time fluorescence quantitative technique after the primary testicular cells were cultured in vitro by mi RNA overexpression test,inhibition test and m RNA interference test.The results showed that after the overexpression of let-7g,the expression of let-7g increased,but the expression of cbx2 and sf1 decreased;After the inhibition of let-7g,the expression of let-7g decreased and the expression of cbx2 and sf1 increased.After si RNA interference,the expression of cbx2 was significantly inhibited,and the expression of sf1 was also inhibited in the same direction.Based on the above results,we can find that let-7g can also negatively regulate cbx2 at the testicle primary cell level,and it is speculated that let-7g may regulate downstream gene sf1 by targeting cbx2,and then play a role in gonadal differentiation and development,which provides a meaningful basis for revealing the function of let-7g in the gonad of Japanese flounder.