Establishment Of LncRNA THOR Knockout Mouse Model And Its Application In The Study Of Related Mechanisms About Male Reproduction And The Occurrence And Development Of Colorectal Cancer

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.