【作者】 周林；

【导师】 逄大欣；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2022， 博士

【摘要】 Cancer/testis lnc RNA是一类新定义的在正常睾丸和癌细胞中特异性高表达的长链非编码RNA。THOR是高度保守的睾丸特异性长链非编码RNA,代表了一类具有重要功能的Cancer/testis lnc RNA,其结构和功能都经历了正向进化的选择。已有研究揭示THOR参与生殖系统发育的调节,但是具体的机制尚未清晰,而且THOR在雄性动物的生殖系统发育中的作用及机制研究很少。本研究通过CRISPR/Cas9技术建立稳定遗传的THOR-KO基因编辑小鼠模型,研究THOR对雄性小鼠生育的影响。对THOR-KO小鼠的生存情况和雄性小鼠的生育能力进行检测和统计,包括血清雄性激素水平、体外受精能力、个体交配成功率、后代雌雄比例和生存曲线等。结果显示,THOR-KO小鼠的存活率降低,THOR-KO雄性小鼠生育能力降低,睾丸较小,精子活力减弱,睾丸生精细胞凋亡增加,后代雌雄比例失衡。进一步为了研究THOR通过何种途径参与雄性生殖系统发育,RT-q PCR检测THOR下游靶基因的m RNA表达水平。与WT小鼠相比,IGF2BP1、c-MYC、IGF1、IGF2的m RNA表达降低。Western Blot对IGF2下游MEK-ERK通路进行蛋白水平的验证,进一步发现,MEK-ERK信号通路表达降低。综上所述,THOR敲除后,调节靶基因IGF2的下游MEK-ERK信号通路,影响睾丸发育和精子发生。结直肠癌（Colorectal cancer,CRC）是常见的消化道恶性肿瘤之一,其患病率和死亡率逐年上升。关于THOR在CRC中的作用的探索目前仅在细胞水平进行。为了明确THOR在CRC发生和发展中的贡献,将APC^Min/+小鼠与THOR-KO交配繁殖,获得THOR基因敲除的APC^Min/+小鼠,并利用CRISPR/Cas9技术构建THOR敲除的人结直肠癌SW480细胞。结果表明,THOR基因敲除APC^Min/+小鼠的存活率比APC^Min/+小鼠高50%,个体比APC^Min/+小鼠大。THOR基因敲除APC^Min/+小鼠肠道炎性损伤得到缓解,结直肠腺瘤的发生降低。CCK-8结果发现,THOR基因敲除SW480的细胞增殖率降低大约50%。Transwell实验表明,THOR基因敲除SW480细胞的迁移能力降低。此外,为阐明THOR在CRC发生发展中的调控机制,通过RT-q PCR检测THOR下游靶基因的m RNA表达水平。结果表明,c-MYC、APC、SOX9、Wnt1、CDH1、Axin1、Axin2、LEF 1等的m RNA水平显着降低。Western Blot结果进一步证实Wnt/β-catenin信号通路的表达降低。综上所述,THOR基因敲除下调Wnt/β-catenin信号通路,调控下游转录因子TCF1和LEF1,影响c-MYC和Cyclin D1的表达,降低癌细胞的生长、增殖和迁移,从而参与CRC的发生发展。本研究成功构建了两种THOR基因不同片段长度敲除的小鼠,并通过交配繁殖获得THOR敲除的APC^Min/+小鼠,在哺乳动物水平探索THOR在雄性生殖系统发育和结直肠癌发生发展中的作用和调控机制。更多还原

【Abstract】 Cancer/testis lnc RNA is a newly defined class of long non-coding RNA that is specifically and highly expressed in cancer cells and normal testis.THOR is a highly conserved testis-specific long non-coding RNA,representing a class of Cancer/testis lnc RNA with important functions,whose structure and function have undergone forward evolutionary selection.Previous studies have revealed that THOR is involved in the regulation of the development of the reproductive system,but the specific mechanism is not yet clear,and the role and mechanism of THOR in the development of the reproductive system of male animals is rarely studied.In this study,a stable genetic THOR-KO transgenic mouse model was established by CRISPR/Cas9 technology,and the relationship between THOR and male fertility was investigated in adult male mice.The survival of THOR-KO mice and the fertility of male mice were detected and counted,including serum androgen level,in vitro fertilization ability,individual mating success rate,offspring male-female ratio and survival curve.The results showed that the survival rate of THOR-KO mice decreased,the fertility of THOR-KO male mice was reduced,the testes were smaller,the sperm motility was weakened,the apoptosis of testicular spermatogenic cells was increased,and the sex ratio of the offspring was unbalanced.In order to further investigate the pathway through which THOR is involved in the development of male reproductive system,the m RNA expression levels of targeted genes downstream of THOR were detected by RT-q PCR.Compared with WT mice,the m RNA expression of IGF2BP1,c-MYC,IGF1,and IGF2 decreased.Western Blot verified the protein level of MEK-ERK pathway downstream of IGF2,and further found that the expression of MEK-ERK signaling pathway decreased.In conclusion,THOR knockout regulates the downstream MEK-ERK signaling pathway of the target gene IGF2 and affects testicular development and spermatogenesis.Colorectal cancer（CRC）is one of the common gastrointestinal malignancies,and its prevalence and mortality are increasing year by year.The exploration of the role of THOR in CRC is currently only carried out at the cellular level.In order to clarify the contribution of THOR in the occurrence and development of CRC,APC^Min/+mice were mated with THOR-KO mice to obtain THOR gene knockout APC^Min/+mice,and THOR knockout human colorectal cancer SW480 cells were constructed using CRISPR/Cas9technology.THOR knockout APC^Min/+mice had a 50%higher survival rate than APC^Min/+mice,and the individuals were larger than that of APC^Min/+mice.THOR knockout APC^Min/+mice alleviated intestinal inflammatory damage and reduced the occurrence of colorectal adenomas.The CCK-8 results found that the cell proliferation rate of SW480 was reduced by approximately 50%after THOR gene knockout. Transwell experiment showed that THOR knockout SW480 cells had reduced migratory ability.In addition,in order to elucidate the regulatory mechanism of THOR in the occurrence and development of CRC,the m RNA levels of THOR downstream target genes were analyzed by RT-q PCR.The results showed that the m RNA levels of c-MYC,APC,Sox9,Wnt1,CDH1,AXIN1,AXIN2 and LEF1 decreased significantly.Western Blot further confirmed the decreased expression of Wnt/β-catenin signaling pathway on gene protein levels.Taken together,THOR knockout down regulated the Wnt/β-catenin signaling pathway in CRC,regulates the downstream transcription factors TCF1 and LEF1,affects the expression of c-MYC and Cyclin D1,and reduces the growth,proliferation and migration of cancer cells,and thus participates in the occurrence and development of CRC.In summary,this study successfully constructed two THOR gene knockout mice with different fragment lengths,and obtained THOR knockout APC^Min/+mice through mating and breeding,and explored the role of THOR in male reproductive system and the occurrence and development of colorectal cancer at the mammalian level.更多还原