Mechanistic Study Of The Effect Of Ginkgo Biloba Extract On Colorectal Cancer Cells By Regulating TET2 Expression

Colorectal cancer(CRC)is the most common and deadly of all digestive system cancers.Despite the advances in the treatment of CRC,its prognosis is still not optimistic.Therefore,in-depth studies on the mechanisms of CRC development are needed to explore therapeutic targets for CRC and to reduce the pressure for patients and our healthcare system.A large number of studies have focused on the anti-cancer effects of natural products or herbal extracts.Ginkgo biloba is an ancient plant of Chinese origin,and ginkgo biloba extract(GBE)has rich pharmacological effects.A standardized GBE,EGB761,is currently available in the market,which can exert functions in regulating gene expression,modulating epigenetic modifications and antitumor properties.There is evidence that GBE can be used in the treatment of CRC,however,its specific mechanism has not been explored.Recent studies have found that the development of cancer is closely associated with abnormal epigenetic modifications.As an important epigenetic modification,DNA methylation is responsible for the regulation of gene expression,which mainly includes the establishment and erasure of methylation.In the process of DNA methylation erasure,the TETs protein family play an important role in catalyzing the cascade of 5-methylcytosine to 5-carboxycytosine.Aberrantly elevated overall methylation levels have been observed in CRC,and TETs protein family are in an aberrantly expressed state in a variety of cancers.In this study,CRC cells were first treated with GBE,and the effects of GBE on the proliferation,migration,invasion ability of CRC and the level of apoptosis were analyzed using CCK-8 assay,scratch healing assay,Transwell assay,and flow cytometry.The effect of GBE on the global DNA methylation level of CRC cells was analyzed using immunofluorescence staining.To investigate whether regulation of the TETs family might be the target of GBE action on CRC cells,the expression pattern of the TETs family in CRC was analyzed using bioinformatics,and the rescue effect of GBE on the TETs family was analyzed using q RT-PCR and WB.Subsequently,bioinformatics analysis and q RT-PCR and WB were used to predict and validate the micro RNA that mediates the regulation of TET2 by GBE.Then,overexpression vectors and si RNAs of TET2 were constructed to explore the effect of TET2 expression pattern on CRC cells.Finally,a CRC xenograft model was constructed using nude mice to explore the tumor suppressive effect of GBE-regulated TET2 expression in vivo.The results showed that:1.GBE could limit the cell proliferation ability of CRC and significantly reduce the invasive ability of CRC cells,but had no significant effect on the migration ability and apoptosis level of CRC cells.2.GBE could regulate the level of 5hm C in CRC cells and modulate the expression pattern of TETs protein family;among them,TET2 protein was in an abnormal state of reduced expression level in CRC,and GBE could promote the expression of TET2;the effect of GBE to increase the expression of TET2 was achieved through the inhibition of miR-29 a.3.The expression level of TET2 could regulate the proliferative ability,invasive ability and the level of 5hm C in CRC cells;consistent with the results observed in GBEtreated CRC,the expression level of TET2 had no significant effect on the migration ability and apoptosis level of CRC cells.4.GBE increased TET2 expression levels and inhibited CRC tumor growth in vivo;tumor growth was significantly inhibited in the xenograft model constructed using TET2 overexpressing CRC cell lines compared with the control group.In summary,this study demonstrated that GBE regulates TET2 expression through miR-29 a and thereby inhibits the proliferation and invasion of CRC cells and suppresses tumor growth in vivo,demonstrating that TET2 can be a potential therapeutic target for CRC,improving the theoretical basis of the mechanism related to CRC and bringing a new idea to the treatment of CRC.