| Cetaceans(whales,dolphins and porpoises)are a group of highly specialized mammals adapted to aquatic lifestyles,whose ancestors returned from land to the sea about53 million years ago in what is known as a "secondary entry".During the evolutionary process of gradually adapting to the aquatic environment,their morphological and physiological have undergone a series of changes,among which one of the most obvious characteristics is the enlargement of body size.In general,while increasing body size would normally be associated with an increased risk of cancer,but large whales have been shown to have evolved a strong anti-cancer mechanism,consistent with Peto's paradox.However,the molecular mechanism of anti-cancer in cetaceans is poorly understood.The Hippo signaling pathway is evolutionarily conserved,involved in the regulation of organ size,and is closely associated with cancer.The key genes YAP1 and TEAD4 regulate cell proliferation,differentiation,migration and apoptosis,so as to control body size and cancer development.Therefore,this paper takes the representative species of whales as the research object,and the key genes of Hippo signaling pathway YAP1 and TEAD4 as candidate genes.Combined with the methods of bioinformatics analysis and functional experiments,the following scientific questions are explored: 1)Are there strong selection constraints on YAP1 and TEAD4 genes in whales,and cetacean specific mutations? 2)Do YAP1 and its specific amino acid locus play a role in the anti-cancer regulation of cetaceans?3)Do the different amino acid sites of TEAD4 in cetaceans of different body sizes have any influence on the formation of body size?Firstly,the orthologous coding sequences of YAP1 gene of 12 cetaceans and other mammals were obtained by downloading from databases(NCBI,Ortho Ma M)and executing the local BLAST program.The evolutionary pattern of YAP1 was analyzed by using the one ratio model in the Codeml program of PAML software.The results showed that the gene sequence was very conservative and subject to strong selection constraint,suggesting that the gene played an important role.In addition,the Fas Parser software was used to scan and analyze the sequence,and a specific amino acid site(Ser215)was found in whales.The Uniprot database and literature verified that the site had undergone radical changes in amino acid properties and was located near the important functional domain(WW)that interacts with other proteins.Previous studies have found that YAP1 gene is a key effector molecule in Hippo pathway.After phosphorylation,it plays a role in suppressing cancer and regulating the size of tissues and organs in the cytoplasm,while unphosphorylated YAP1 enters the nucleus to promote cancer.Thus,in order to verify whether this cetacean specific site mutation affects the biological function of YAP1,this study conducted nuclear localization detection in vitro.The result showed that the YAP1 protein was mainly localized in the cytoplasm of bottlenose dolphins(Tursiops truncatus)YAP1 and its specific mutations of cetacean,which was significantly different from the wild-type zebrafish Yap1.Combined with the biological characteristics of cancer occurrences,cell proliferation and migration were further detected in this study.Our results indicate that the mutation of the cetacean specific site does not cause significant changes in cell proliferation(P > 0.05),but can significantly inhibit cell migration(P < 0.05).In vitro,these results suggest that YAP1 specific mutation in cetaceans may cause differences in nuclear and cytoplasmic distribution and then affect the transcription of target genes and resulting in changes of cell migration ability.In order to further verifies the specific mutations influence which genes or path expression in the zebrafish model animals,after knocking down yap1 gene in the zebrafish(yap1-MO),transiently overexpress bottlenose dolphins YAP1(namely d YAP1),zebrafish yap1 with cetacean specific mutation(namely z YAP1-M),and zebrafish itself yap1(namely z YAP1)performed transcriptome sequencing together with the control groupwild-type zebrafish knock down the yap1 gene(MO + control).Transcriptome analysis found: 1)Compared with the control group(MO + control),a total of 507 differentially expressed genes were identified in the group overexpressing cetacean YAP1(MO +d YAP1),including 220 up-regulated genes and 287 down-regulated genes.Differentially expressed genes were mainly concentrated on the m TOR signaling pathway,and the expression levels of several proto-oncogenes were down-regulated,such as sgk1.2)Compared with the transient overexpression zebrafish yap1(MO + z YAP1),a total of 456 differentially expressed genes were identified in the group carrying cetacean-specific mutation(MO + z YAP1-M),including 242 up-regulated and 214 down-regulated genes.Differentially expressed genes were mainly concentrated in the ubiquitin-mediated proteolytic pathway and the expression of several hydrolases related to cell cycle progression was down-regulated.3)Compared with the control group(MO + control),the differentially expressed genes generated by the transient overexpression YAP1 gene of zebrafish(MO + z YAP1)were removed,the 70 differentially expressed genes shared by the MO + d YAP1 and MO + z YAP1-M groups are cetacean-specific differentially expressed genes,including 25 up-regulated and 45 down-regulated genes.Most of the differentially expressed genes were down-regulated proto-oncogenes(sgk1/2,jund,fosl1 a,etc.)and up-regulated tumor suppressor genes(fam98a,zpr1,cdk5rap3,etc.).Obviously,transcriptome sequencing results suggest that the specific mutation of YAP1 in cetacean may regulate tumor suppression through up-regulation of tumor suppressor genes and down-regulation of proto-oncogenes.Combined with the results of cell experiments and transcriptome analysis,it was speculated that dolphin YAP1 and its cetacean-specific mutation could play an important role in the regulation of cancer suppression.It is possible that cetaceans could prevent carcinogenesis by inhibiting cell migration as they grow in size.The key gene downstream of the Hippo pathway-TEAD4 gene is highly conserved in mammals,but near the important functional domain(TEA)that binds to DNA,we have identified a potential amino acid mutation specific to the large and small size cetaceans.that is,the site is alanine(Ala)in bowhead whales(Balaena mysticetus),gray whales(Eschrichtius robustus),sperm whales(Physieter catodon)and other large whales.In addition,the small body of whales such as finless porpoise(Neophocaena asiaeorientalis),porpoise(Phocoena phocoena)and bottlenose dolphin(Tursiops truncatus)is threonine(Thr).and this specific mutation has radically changed the nature of amino acids.Similarly,the locus shows similar differences among primate and rodent species of different body sizes.In order to verify the role of body size differential loci in TEAD4 on the formation of body size,transient overexpression of TEAD4 was used in this study to detect cell proliferation in vitro.The result showed that compared with the TEAD4 gene overexpressing small size mice or carrying small size specific mutations,overexpression of large size cetaceans or TEAD4 gene carrying large size specific mutation can significantly promote cell proliferation(P < 0.05).The following quantitative PCR assay showed that the expression levels of cyclins such as Ccnd1 and Ccne1 were significantly higher in cells with large body specific loci than with small body specific loci.In addition,the mouse Tead4 gene was overexpressed with large and small body-specific mutations,and it was found that the expression of Cdkn1 b gene in the group carrying the large-bodyspecific mutation was significantly lower than that of the group carrying the small-bodyspecific mutation(P < 0.05).Therefore,the results suggesting that the large and small body-specific mutations of TEAD4 gene affect cell proliferation by regulating the cell cycle process to achieve the difference in size and body shape.In summary,through the sequence analysis and functional experiments of YAP1 and TEAD4,the key genes of Hippo signaling pathway,we have initially revealed the molecular mechanism of cancer suppression and body size difference in cetaceans,and lay the foundation for a comprehensive understanding of the body evolutionary mechanism of mammals. |
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