Functional Studies Of TETs In Neural Stem Cells And Colorectal Cancer

TET(ten-eleven translocation)can convert 5mC to 5-hydroxymethylcytosine(5hmC),are key proteins for regulating DNA methylation levels.Technical protocols using small-molecule compounds(naloxone and vitamin C)to inhibit or promote TET protein activity have been established in our previous studies.The current study plans to use these small molecules to investigate the functions and mechanisms of TET in neural stem cells and colorectal cancer.Previous studies have shown that naloxone,can inhibit the DNA demethylation activity of TET 1 through a non-opioid receptor-dependent pathway in vitro,thereby promoting the proliferation and differentiation of NSCs,but whether naloxone regulates NSCs and their spatial memory in the hippocampus of mice by a similar pathway in vivo needs to be further explored.In the current study,naloxone was administered by micro-osmotic pressure pump to wild type(WT)and Tet1+/-mice.The results showed that naloxone treatment promoted the proliferation and differentiation of NSCs in the hippocampus of WT mice,while no such phenomenon was observed in the hippocampus of Tet1+/mice,which suggests that TET1 can mediate the proliferative and differentiationpromoting effects of naloxone on neural stem cells in the hippocampal in vivo.Meanwhile,the spatial learning memory ability of mice was tested by water maze experiment after naloxone administration to WT and Tet1+/-mice.The experimental results showed that naloxone could effectively promote the spatial learning memory of WT mice,while it had no effect on the spatial learning memory of Tet1+/-mice.This suggests that TET1 can mediate the facilitation effect of naloxone on spatial learning memory ability in mice.The role of TET2 in solid tumors,especially colorectal cancer,is unclear.Therefore,the current study focused on the roles and mechanisms of TET2 protein in colorectal cancer progression.After analyzing clinical data from a variety of datasets and 380 colorectal cancer samples,this study found that high expression and nuclear localization of TET2 was associated with good prognosis(longer survival)in colorectal cancer patients,while low expression and cytoplasmic localization of TET2 was the opposite.After human colorectal cancer cells SW620 were cultured in vitro and in vivo(CDX model)for long time culture,it was found that exogenous overexpression of TET2 localized to the cytoplasm at early stage and had no inhibitory effect on tumor growth.In contrast,it was mainly localized in the nucleus during the middle and late stages and inhibited tumor growth.Thereafter,the current study found that at the late stage during colorectal cancer progress,internal cells tend to undergo EMT and migrate to the outside due to excessive tumor(or cell mass)size and insufficient supply of oxygen and nutrients.In addition,the activation of the WNT signaling pathway normally accompanied during the EMT process,and βcatenin transfers TET2 from the cytoplasm to the nucleus and promotes its DNA demethylation activity to inhibit the proliferation of tumor cells.The above findings were further verified by analyzing the single cell transcriptome of SW620 cells cultured for a long period of time.Therefore,TET2 plays a braker role at the late stage during colorectal cancer progress.In addition,considering the low expression of TET2 protein in colorectal cancer tissues,this study used the WNT pathway agonist IM-12 to induce the transfer of TET2 from the cytoplasm to the nucleus,and also innovatively used vitamin C,a TET protein activator,to activate TET2 to enhance the inhibitory effect on tumor cell growth.In summary,the current study found that:1)TET1 mediates the receptorindependent abilities of naloxone to promote the proliferation and differentiation of NSCs and the contextual learning and memory ability in vivo.2)The DNA demethylation ability of TET2 inhibits the progress of colorectal cancer,while the nucleus entry of TET2 could be activated by EMT and WNT pathways.This study revealed the role of TETs proteins in two different biological processes and provided new insights to study the functions of TETs protein.