The Study Of The Role And Mechanism Of TEX11 In Meiosis And Testicular Germ Cell Tumors

Spermatogenesis plays a key role in male reproductive health.Abnormal development of testicular germ cells will not only lead to arrest of spermatogenesis,but also the occurrence of testicular germ cell tumor（TGCT）.The process of spermatogenesis and the formation of TGCT is closely related,making it a central issue to study the development of testicular germ cells.1.The role and function of TEX11 in spermatogenesis.Purpose:To clarify the expression pattern and function of Testis-expressed gene 11（TEX11）during meiosis in spermatogenesis,to explore the role and its potential mechanism of TEX11 in the development of non-obstructive azoospermia（NOA）,in order to provide a new insight for the diagnosis and treatment of the infertile male patients.Methods:（1）To clarify the spatiotemporal expression of TEX11 in mice,using the method of spermatocyte chromosome spreading combined with immunofluorescence experiment,to clarify the dynamic expression pattern of TEX11 during meiosis;（2）Using CRISPR/Cas9 technology to construct a TEX11 knockout mice model,in order to explore the effect of TEX11 on male fertility and its role in meiosis;（3）Using transcriptome sequencing technology（RNA-seq）to screen the target molecules and the downstream regulatory networks of TEX11,thus preliminarily elucidate the underlying mechanism of TEX11 on spermatogenesis.Results:（1）TEX11 was specifically expressed in mouse testis,and distributed along the chromosomal synaptonemal complex during zygotene and early pachytene;（2）The TEX11 gene knockout resulted in a sterile phenotype in the male mice.The weight and size of the testis in TEX11^-/Y mice were significantly decreased,and the process of spermatogenesis was arrest at pachytene stage;（3）Compared with TEX11^+/Ymice,the spermatocytes of TEX11^-/Y mice exhibited asynapsis or aberrant synapsis,as well as reduced crossover formation,and the event of meiotic sex chromosome inactivation（MSCI）was affected as well;（4）The results of RNA-seq showed that compared with TEX11^+/Ymice,a total of 3584 genes were significantly down-regulated and 376 genes were significantly up-regulated in TEX11^-/Y mice,the KEGG analysis showed that the significantly down-regulated differential expressed genes were mainly enriched in glycolysis/gluconeogenesis,biosynthesis of amino acids,carbon metabolism and so on;（5）qRT-PCR validated that the TEX11 deletion led to significant down-regulation of Pgk2,Tssk6,Prm and Tnp;（6）The deletion of TEX11 gene cause abnormal splicing of a large number of genes,the main type of alternative splicing was exon skipping.The KEGG analysis showed that the differentially alternatively spliced genes were mainly enriched in cell adhesion and tight junction related pathways.Conclusions:（1）Loss of TEX11 leads to infertility in male mice;（2）TEX11regulated the process of spermatogenesis by promoting meiotic synapsis and crossover formation;（3）TEX11 may affect the expression and location of H2AX,affecting meiotic sex chromosome silencing and chromatin remodeling events;（4）TEX11 affects alternative splicing events in spermatogenesis,mainly through exon skipping affecting the intercellular tight junctions,thus promoting spermatogenesis.2.The role and function of TEX11 in testicular germ cell tumor.Purpose:To explore the role and potential function of TEX11 in testicular germ cell tumors（TGCT）,as well as to provide new molecular biomarkers and therapeutic targets for TGCT.Methods:（1）GEPIA database were used to analyze the expression of TEX11in TGCT,and its related pathological features;（2）The overexpression plasmid of TEX11 was constructed and transfected into Tcam-2 cell line and NCCIT cell line,using MTT assay,cell apoptosis assay,Transwell cell migration and invasion to explore the effects of TEX11 on the function of TGCT cells;（3）Use transcriptome sequencing technology to explore the downstream molecules and regulation network of TEX11,understanding its potential mechanism in the occurrence and development of TGCT.Results:（1）The GEPIA database showed that TEX11 was abnormally expressed in renal clear cell carcinoma,pancreatic cancer and TGCT.Besides,TEX11 was significantly decreased in TGCT and was correlated with the pathological stage of TGCT;（2）Overexpression of TEX11 could inhibited the proliferation of TGCT cells but not affected apoptosis,besides,overexpression of TEX11 could also inhibited the invasion and migration ability of TGCT cells;（3）After the transfection of TEX11 in the Tcam-2 cell line,the RNA-seq showed that 131 genes were differentially expressed,including 75 significantly up-regulated genes and 56significantly down-regulated genes.KEGG analysis showed that the significantly down-regulated differential genes were mainly enriched in cell adhesion-related pathways（CAMs）;（4）We focused on the differentially expressed gene ITGB2 in CAMs pathway,and considered ITGB2 as a target gene for the subsequent mechanism study.Using GEPIA2 database,it was found that ITGB2 was significantly highly expressed in various tumor which included TGCT,and was negatively correlated with the expression of TEX11;（5）After overexpression of TEX11 in TGCT cell,the protein expression of ITGB2,FAK and p-FAK were all were significantly down-regulated.Conclusion:（1）The expression of TEX11 is significantly decreased in TGCT and related with its pathological stage;（2）TEX11 is a co-regulator of spermatogenesis and testicular germ cell tumor,and may be a new candidate cancer-testis gene and involved in the development of TGCT;（3）TEX11 suppressed the proliferation,migration and invasion ability of TGCT cells;（4）TEX11 inhibited the invasion and migration ability of TGCT by suppressed ITGB2/FAK pathway-related molecules.