| Cryptorchidism is the most common congenital defect in children’s genitourinary system.It is one of the risk factors for male infertility and testicular tumors.The occurrence of cryptorchidism is closely related to many factors,and the alteration of genes plays an important role in the occurrence of cryptorchidism.A large number of studies have confirmed that the formation of cryptorchidism is related to the differential expression of genes.Therefore,exploring new differential expression genes of cryptorchidism from the transcriptome level and studying the relationship between susceptibility genes and children with cryptorchidism can provide a new direction for studying the pathogenesis of cryptorchidism.Part Ⅰ:Preliminary study on transcriptomics in children with cryptorchidism based on high-throughput sequencingA transcriptome refers to the sum of all gene transcripts of a species at a particular developmental stage or in a sample under a certain physiological condition,including mRNA and non-coding RNA,and non-coding RNA,mainly miRNA.In the narrow sense,it refers to a collection of all mRNAs.Protein expression plays a major role in the function of cells.Proteome is the most direct description of cell function and state.Transcriptional composition is the main means of studying gene expression.Transcriptome is the inevitable link between genomic genetic information and biological function proteome.The regulation of transcriptional level is the focus of the study and the most important regulation of organisms.In this study,transcriptome sequencing was performed in children with cryptorchidism.Illumina HiSeqTM(high-throughput transcriptome sequencing platform RNA-Seq technology)was used for cryptorchidism in children with cryptorchidism,including mRNA and non-coding RNA.A new generation of high-throughput sequencing can be used to obtain almost all transcript sequence information of children with cryptorchidism in a comprehensive and rapid way.By means of revealing the differential genes and characteristics of children with cryptorchidism through human whole transcript sequencing,we will establish a platform for transcriptomics research in cryptorchidism.The main results of this part of the study are as follows:In order to check the difference between the genome-wide transcriptional profile of children with cryptorchidism and in normal control testis,we separately examined testicular tissue including the samples of 6 cases of children with cryptorchidism and 2 cases of testicular trauma children and 1 case of incarcerated inguinal hernias.The transcriptome sequencing of the 9 testicular tissues was performed by RNA-seq technique,and the RNA-seq data were compared by HISAT2 software.Mapping results showed that the transcription rate of the transcriptome was 0.957(0.956-0.959),which was consistent with the sequencing of human whole transcriptome.We analyzed the Mapping and genomic structure of the filtered Clean Reads.The results showed that the genome structure of 9 testis tissues was mainly distributed in the coding region(CDS,Coding Sequence)and exons(Exon,Expressed Region).In order to better study the phenotype-related gene expression,we explored the differential gene between the cryptorchidism group and the normal control group.We used LogFC>1 or<-1,P<0.05 as the differential screening criteria.The results indicated that there were 6145 significantly differentially expressed mRNA,with 3815 up-regulated and 2330 decreased.To detect the difference of differentially screened genes in different samples,we used differential genes to cluster different samples and cluster different genes to help us explore its biological significance.We used Fisher to perform significant functional annotation.Differential genes were analyzed by Gene Ontology(GO)analysis and then a gene regulatory network directed by GO-Term.The analysis revealed that significant pathways in the network ultimately pointed to Core Pathway,such as calcium signaling pathways,cell cycle signal transduction pathways,and cellular metabolic-related signaling pathways.In summary,our study revealed the differentially expressed genes between cryptorchid tissue in children with cryptorchidism and normal control testis tissue from the level of gene transcriptome.Further,we performed functional annotation,pathway analysis and interaction network regulation analysis of these differentially expressed genes.Part Ⅱ Screening for susceptibility genes for cryptorchidism based on multi-omics integration analysisBioinformatics is an interdisciplinary subject formed by the combination of life science and computer science.Bioinformatics has emerged along with high-throughput molecular techniques aimed at solving biological problems by using computer and statistical methods to analyze molecular biological data.Bioinformatics involves many fields of biological research,annotating and reorganizing massive raw data,and extracting useful and reliable results.TCGA and GEO are commonly used comprehensive databases.Data mining were based on GEO and TCGA database,we detected differential expression genes using big data,functional enrichment analysis and protein-protein interaction(PPI)network construction and module analysis,futher to seek out key genes(the susceptibility gene,hub gene).The key molecules were then obtained to verify the molecular phenotype.The main results of this section are as follows:We used the bioinformatics method to download the materials from the database GEO,and screened out the gene expression chip spectrum of children with cryptorchidism:GSE25518.The gene expression profile data of this group was derived from GPL570.The expression profile GSE25518 consisted of 19 cryptorchidism samples and 4 normal samples.We performed a comprehensive analysis of the sequencing results and the above GSE25518 microarray dataset.There were 6145 differentially expressed genes in the sequencing results,and 782 differentially expressed genes in the GSE25518 microarray.Finally,we found 108 common differentially expressed genes by Venn plot.Through the study of susceptibility gene screening,validation and functional analysis,we selected the DEGs one by one,and used the DAVID analysis tool for functional annotation.Then we further obtained five Hub genes(OFD1,NEK2,AHI1,ALMS1 and MZT2B)from the level of bioinformatics analysis through the STRING protein interaction database.The susceptibility genes were also compared in the 9 normal controls and cryptorchidism tissues we sequenced.It was found that the expression of MZT2B and NEK2 was down-regulated in cryptorchid tissues,while the expressions of AHI1,ALMS1 and OFD1 were up-regulated.Human protein map analysis showed that AHI1,ALMS1,MZT2B and NEK2 genes may be closely related to the occurrence of cryptorchidism.Finally,through gene website analysis and functional prediction,we identified NEK2 as a candidate gene and attempted to further confirm the possible expression and functional changes of NEK2 as a susceptibility gene for children with cryptorchidism.To sum up,we used bioinformatics analysis methods to generate gene expression data sets for cryptorchidism risk in children with cryptorchidism through large-scale keyword search and data mining on comprehensive databases.At the same time,combining the gene sequencing results of children with cryptorchidism,we finally obtained a susceptibility gene for cryptorchidism,by integrating the analysis of co-expressed differentially genes in all data sets.Part III Expression of cryptorchidism risk susceptibility gene NEK2 and its involvement in apoptosis in mouse cryptorchidism modelWe obtained five Hub genes,OFD1,NEK2,AHI1,ALMS1,and MZT2B from the level of bioinformatics analysis.We identified NEK2 as a candidate gene through paper review and gene website analysis.And we attempted to further demonstrate the possible expression and functional changes of NEK2 as a susceptibility gene for cryptorchidism.The purpose of this study was to observe NEK2 mRNA and protein expression of NEK2 in the cryptorchidism mice model,and to explore its role in apoptosis of testicular tissue.The results of the study were as follows:After the mouse cryptorchidism model was successfully constructed,the testicular volume did not change much in the first 6 days.On the 6th day,the testicular volume became significantly smaller and the volume gradually became smaller as time went on.On 15th day,the testis showed atrophy.In combination with morphological observation,HE staining results showed that the spermatogonia cells,primary spermatocytes and sperm cells in the seminiferous tubules were more severely damaged.By the 15th day,there were almost no normal sperm cells,and the damage of the muscle-like cells was more serious as time went on.On 15th day,the thin base film showed a thickening change.The results of Tunel showed that the number of apoptotic cells first increased and then decreased.The number of apoptotic cells in the first 3 days was small,from the 6th day apoptotic cells increased sharply,and decreased to the highest peak on the 9th day.The results of RT-PCR showed that NEK2 mRNA expression increased gradually on the 6th day after cryptorchidism modeling,and decreased to the lowest on the 15th day after the highest expression on the 6th day,and was significantly lower than the normal control group.Immunohistochemistry showed that NEK2 protein was expressed in the nucleus and cytoplasm in normal testis and cryptorchidism.In summary,NEK2 protein expression increased first and then decreased.NEK2 expression increased gradually in the first 6 days,peaked on the 6th day,and then decreased.In summary,we found that abnormal expression of NEK2 in the mouse cryptorchidism model might lead to infertility in cryptorchidism patients through apoptosis.Our study provided a new direction for clinical treatment of cryptorchidism,prevention of infertility and testicular tumors,and proposed that NEK2 might be used as a biomarker for diagnosing cryptorchidism and prognosis as well as a therapeutic target for male infertility and reproductive system diseases. |
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