Molecular Mechanism Of Let-7 MicroRNA Regulating The Development Of Silk Gland In The Silkworm,Bombyx Mori

Silkworm?Bombyx mori?has been domesticated from the wild progenitor Bombyx mandarina for over 5,000 years and was an important economic insect.By exploring the molecular mechanisms in the silk protein synthesis,the way of silk protein assembling,and the genetic basis of silk gland development,it is expected to make breakthroughs in improving silk yield,obtaining special properties silk fiber,and expanding the application of silk gland bioreactor.let-7 is an extremely conserved miRNA,which exists in the form of let-7 cluster?let-7C?in different species and play an important post-transcriptional regulatory role in timing development regulation,tumorigenesis,and cell pluripotency maintenance,but its function in the silk gland is unclear.In this study,let-7 and let-7C were knocked out in different divisions of the silk gland by CRISPR/Cas9 gene editing technology,and their effects on the growth of silk glands were investigated.let-7C miRNAs were separately overexpressed in the posterior silk gland?PSG?,so as to reversely verify the function of let-7C miRNAs in regulating the growth of silk glands.Then,transcriptome sequencing was used to explore the molecular mechanism of let-7 regulating silk gland growth.Target genes of let-7 were screened and identified,and their functions in the silk gland were explored too.The main results are shown as below.1.Explore the appropriate methods for the functional study of miRNAs in the silk gland of silkworm.Taking the silk gland-specific miR-274 as an example,here we aimed to apply the appropriate tools to effectively manipulate miRNAs expression in the silk gland cells.Firstly,miR-274 mimic and miR-274 antagomir were synthesised and modified in a conventional ways,and were then injected into silkworm larvae at the fifth instar,but they failed in changing the expression of miR-274 in the silk gland.Secondly,miR-274sponge were designed and cloned into the transgenic vector containing the A4 promoter.The transgenic silkworms which expressed miR-274 sponge were selected and the expression of miR-274 was down-regulated by about 20%in the silk gland.Thirdly,the sequence of a total of 279 bp,including 83 bp up-stream of pre-miR-274,and 101bp downstream of pre-miR-274,was PCR-amplified from the miR-274 locus of the silkworm genome,and this miR-274 overexpression sequence was cloned into different transgenic vectors with Ser1 or FibH promoter respectively.qPCR results showed that miR-274 was significantly overexpressed in both MSG and PSG.Finally,a pair of sgRNAs for the knockout of miR-274 were designed and successfully constructed into a sgRNA expression transgenic vector.The two-gRNA-expressed transgenic silkworms were selected and then crossed with the PSG-specific Cas9transgenic line?PSG-Cas9?to obtain the kncokout line?miR-274-PSG.Sanger sequencing results revealed highly polymorphic mutations in miR-274 locus,and q-PCR results showed that miR-274 expression decreased by about 54%in the PSG.Altogether,miRNA-transgenic overexpression is the applicable strategy to upregulate miRNA in silk gland while CRISPR/Cas9-based tissue-specific miRNA knockout strategy is more effective than miRNA sponge to down-regulate the expression of miRNA in the silk gland.2.Spatiotemporal expression and basic function analysis of let-7C miRNAsThe temporal and spatial expression profiles of the let-7C miRNAs?let-7-5p,let-7-3p,miR-100 and miR-2795?were detected by q-PCR.The results showed that these four miRNAs were highly expressed from the fourth molt to the adult stage,and expressed ubiquitously but weakly in the silk gland from fifth-instar day-3 larvae?D3IL5?.During the development of silk gland of fifth-instar larvae,let-7C miRNAs were expressed at a low level in the early fifth-instar,and then quickly increased to a peak level at D6 IL5,and finally declined at the end of cocoon spinning.This expression pattern suggests that let-7C miRNAs might be involved in silk gland cell endoreduplication.In order to explore the potential roles of let-7C miRNAs in the silkworm,the mimics and antagomirs of those four miRNAs were chemically synthesized and transfected into BmE and BmNs cell lines.In BmE cells,only the mimic of let-7-5p inhibited cell proliferation.In BmNs cells,the mimics of those four miRNAs inhibited cell proliferation,while their antagomirs promoted cell proliferation.These results suggest that let-7C miRNAs play a synergistic role in regulating cell proliferation of silkworm.3.Knockout of let-7 and let-7C promoted growth of the middle silk glandTo knock out let-7 and let-7C in the silk gland using CRISPR/Cas9 system,we constructed let-7-2gRNA and let-7C-2gRNA transgenic vectors.The surviving G0moths of let-7-2gRNA and let-7C-2gRNA were sib-mated or crossed to the individuals of MSG-specific Cas9 transgenic strain?MSG-Cas9?to generate G1 broods,namely the knockout strains?let-7-MSG and?let-7C-MSG,respectively.Sanger sequencing and q-PCR results showed that let-7 or let-7C was partially knocked out in the MSG cells.To verify the potential abnormal phenotypes in the MSG of?let-7-MSG and?let-7C-MSG strains,we dissected the larvae at different time points of the fifth instar and observed that the phenotypic difference between?let-7-MSG,?let-7C-MSG and the controls.The posterior part of middle silk gland?P-MSG?in?let-7-MSG was thicker and longer than that of controls,and was smooth in its outer surface.In?let-7C-MSG,the roughly bead-like surface was observed in both divisions of M-MSG and P-MSG.Together,these data indicate that knock out let-7 or let-7C promoted growth of the MSG,whereas some obvious phenotypic differences existed between?let-7C-MSG and?let-7-MSG.The rapid growth of the silk gland is achieved primarily via chromosome endoreduplication in silk gland cells at the early fifth instar larval stage.After let-7 or let-7C knockout,the DNA content increased by 170%and 120%in the P-MSG of?let-7-MSG and?let-7C-MSG,and the ATP level,which as a proxy for the amount of energy required for DNA replication,increased by 8-and 10-fold,respectively.These date suggest that enhanced endoreduplication happen in knockout strains.All sericin proteins stored in the silk gland lumen were used for silk production in wild type silkworms,but unexpectedly,some silk protein was left in the lumen of P-MSG of both?let-7-MSG and?let-7C-MSG after cocoon spinning.Consistently,the cocoon weight or silk yield was significantly decreased for both?let-7-MSG and?let-7C-MSG compared with the wild type silkworms.We consider that?let-7-MSG and?let-7C-MSG mutation individual could be used as advanced material to explore silk fiber assemble mechanism.4.Let-7 as a key negative regulator participates in regulation of posterior silk gland developmentIn order to verify whether knockout let-7 and let-7C effectively promote PSG growth,the surviving G0 moths of let-7-2gRNA and let-7C-2gRNA were then separately crossed to the individuals of PSG-Cas9.The positive let-7 and let-7C knockout individuals were selected by screening the green and red fluorescence markers in the eyes of late embryos and were named as?let-7-PSG and?let-7C-PSG.Then we dissected the larvae at different time points of the fifth instar larval stage to compare the silk glands.The PSGs of?let-7-PSG and?let-7C-PSG were significantly larger than those of the control groups from D3 IL5.The statistical results showed that the length of the PSG in the?let-7-PSG and?let-7C-PSG increased by about 100%and 60%,respectively.Furthermore,the weight of PSGs from both knockout lines increased by about 150%.We evaluated the silk yield at the pupal stage,and it was revealed that the average cocoon weight increased about 12%for both male and female silkworms of?let-7-PSG and?let-7C-PSG.Next,the PSGs were stained with Alexa Fluor488-labeled phalloidin and DAPI,and the cell volume of the PSG increased dramatically in both knockout lines.However,some PSG cells in?let-7C-PSG were atrophied,resulting in the serrated surface of the silk gland.After let-7 or let-7C knock out,the DNA content increased by 150%²00%in the PSG,indicating that the endoreduplication was enhanced in knockout strains.In order to reversely verify the function of let-7C miRNAs in regulating the growth of silk glands,we developed two transgenic silkworm strains which overexpressed let-7and miR-100₂795 in the PSG,named let-7-OE-PSG and miR-100₂795-OE-PSG,respectively.let-7 overexpression strongly inhibited the growth of PSGs in let-7-OE-PSG.The PSG cells in let-7-OE-PSG were atrophied and disordered;the DNA content of PSG in the let-7-OE-PSG decreased by about 75%and the cocoon shell weight is only 50%of the control.Those results show that endoreduplication in the PSG cells was severely hindered when let-7 was overexpressed.However,overexpression of miRNA-100 and miRNA-2795 exerted no significant effect on PSG development.PSG dysplasia affects the synthesis of fibroin.let-7-OE-PSG cocoon shell became thin and fragile,similar to the phenotype of the Nd-s mutant.Moreover,Statistical analysis found that silk fibroin of the let-7-OE-PSG only accounted for about 15%of the total silk weight,while the fibroin of miR-100₂795-OE-PSG accounted for about 60%of the total silk weight,suggesting that miR-100 and miR-2795 also contribute to the synthesis of fibroin.Together,let-7 acts as the main contributor in the let-7C to regulate PSG growth of silk gland and fibroin synthesis.5.Transcriptome analysis revealed the molecular mechanism involved by let-7 in the silk gland growth.To explore the molecular mechanism underlying the promoted enlargement of silk gland cells in response to the deletion of let-7,we performed comparative transcriptome analysis of?let-7-MSG and?let-7-PSG.A total of twelve samples at day-3 of fifth instar were analyzed by transcriptome sequencing.Totally,283 differentially expressed genes?DEGs?were identified in?let-7-MSG,including 184 up-regulated and 99down-regulated,and 548 DEGs were identified in?let-7-PSG,including 271 genes up-regulated and 277 down-regulated.Only 36 DEGs were common to?let-7-MSG and?let-7-PSG.Gene Ontology?GO?term analysis revealed that DEGs in the MSG of?let-7-MSG were most enriched in oxidation-reduction process and fatty acid biosynthetic process;DEGs in the PSG of?let-7-PSG mostly function in the metabolic process and carbohydrate transport.KEGG pathway analysis demonstrated that DEGs in?let-7-MSG were mainly enriched in metabolic pathways,followed by peroxisome,fatty acid metabolism and TCA cycle.However,DEGs in?let-7-PSG only enriched in folate-mediated one-carbon metabolism.These results collectively suggest that let-7target multiple gene or pathways to regulate DNA endoreduplication in different parts of silk gland.6.Target genes of let-7 involved in the silk gland growth regulation.The 3'UTR sequences of Bombyx mori genes were downloaded from NCBI.In all,504 genes were predicted to be the putative targets of let-7 by using the three algorithms,RNAhybrid,miRanda and TargetScan.Then we compared them with the 455up-regulated genes identified by RNA-seq analysis.This resulted in 22 overlapping genes,including 10 in?let-7-MSG and 12 in?let-7-PSG,and only 9 of them were negatively correlated with let-7 in the temporal expression,and were thus regarded as core candidate targets.The 3'UTR of 6 genes were amplified by 3'RACE and cloned into the psiCHECK^TM-2 dual luciferase reporter vector.We then co-transfected each recombinant vector with let-7 mimic into HEK-293FT cells.Dual Luciferase Reporter Assay results showed that only pyruvate carboxylase?PC?,ecdysteroid 22-kinase?EcK?and oocyte zinc finger protein XLCOF6?XLCOF6?were the authentic targets of let-7.PC and EcK were up-regulated in MSG while the XLCOF6 was up-regulated in PSG when let-7 was knockout.PC gene is a conserved target of let-7 in Drosophila melanogaster.Next,we predicted two binding sites of let-7 in PC 3'-UTR.Mutation of either sites only partially restored the activity of the reporter gene,but the activity of the reporter gene was completely restored when both sites were mutated simultaneously,indicating that both target sites are authentic.7.The function of PC in the silk gland.Pyruvate carboxylase is one rate-limiting enzyme in the gluconeogenesis,playing a crucial role in glucose metabolism.In order to reveal whether PC is an important target gene of let-7 regulating silk gland,we constructed two PC gene transgenic overexpression vectors in MSG and PSG,respectively.q-PCR results showed that PC was overexpressed in the PC-overexpressed strain,PC-OE-MSG.The MSGs of PC-OE-MSG were significantly longer than the controls.Further,the length and DNA content of MSGs increased by 25%and 77%at this stage,respectively.PC overexpression can promote MSG growth and endoreduplication in the PC-OE-MSG,so PC is one important target gene of let-7 regulating the growth of MSG.We constructed PC-2gRNA transgenic strain and obtained the PC knockout strains,?PC-MSG and?PC-PSG.Sanger sequencing results showed that base or fragment deletions occurred in the second exon of PC mRNA,ranging from 4 bp to 64 bp.However,deletion of PC gene in the MSG or PSG did not produced significant changes in the size or shape of silk glands.In summary,the results of the gain-of-function and lose-of-function showed that let-7 is a crucial negative regulate in silk gland growth.Especially,knockout of let-7 or let-7C resulted in silk gland enlargement and silk production increase.Furthermore,we screened and identified PC,EcK and XLCOF6 as let-7 targets.PC wss confirmed as one important target gene of let-7 involved in the growth of MSG.In this work,we obtained different gRNA expression strains,let-7 and let-7C knockout strains,as well as let-7overexpression strain,which provide abundant materials to further explore the molecular mechanisms in the silk gland growth and silk protein synthesis,and would contribute to improving silk quality and exploiting silk protein applications.