The Role And Mechanism Of UGT2A3 In Regulating The Proliferation And Migration Of Colon Cancer

Objective:Colon cancer is a common malignancy and the second leading cause of cancer-related mortality in the world.Changes in the Extracellular matrix(ECM)play a key role in the proliferation and metastasis of tumors.Hyaluronic acid(HA),an important component of the Extracellular matrix,has been proved to be overexpressed in colon cancer,breast cancer,and other tumors,and can promote the survival,metastasis and even drug resistance of tumors.It HAS been reported that the production of HA is determined by the content of Hyaluronic acid synthesize(HAS)and the synthetic substrate of HA and the content of the substrate plays a dominant role.By studying the synthesis mechanism of HA,the applicant analyzed the synthesis substrate UDPGA of the UDP-glucuronosylhransferase(UGT)members of UGT2A3 metabolized HA and transferred its glucuronide group to some metabolites to increase its water solubility and then eliminate it from the cell,thereby reducing the amount of UDPGA,which has the potential to affect HA synthesis in tumors.The applicant’s previous work found a correlation between UGT2A3 and HA,and the expression of UGT2A3 in normal colon tissues and tumor tissues showed the greatest difference,which was related to tumor prognosis.Preliminary in vitro experiments verified that UGT2A3 was related to tumor function changes,and also affected the activation of PI3K/AKT pathway downstream of HA.Based on the above work,this project intends to carry out the following studies:(1)exploring the relationship between UGT2A3 and the progression of colon cancer;(2)Proving the relationship between UGT2A3 and HA;(3)Verifying that UGT2A3 worsens the progression of colon cancer by altering the synthesis of HA.Methods:1.TCGA database and bioinformatics analysis website GEPIA were used to analyze the expression changes of the UGT family in colon cancer and the prognostic relationship between screening family members and colon cancer.2.The accuracy of UGT2A3 bioassay was verified by gene level and protein expression level by using colon cancer and adjacent normal tissues and colon cancer cells combined with q-PCR and western blot,and cell lines were selected for subsequent tests.3.The SW480 plasmid interfering cell lines were constructed and transwell and CCK8 were used to investigate the effect of UGT2A3 on the proliferation and metastasis of colon cancer cells.SW480 and HCT116 cell lines were constructed with stable interference and overexpression groups,and their functions were verified by transwell,CCK8,and clonal formation.4.The changes of HA in SW480 and HCT116 stable strains were detected by the ELISA kit,and the effect of UGT2A3 on the downstream pathway of HA was verified.Meanwhile,exogenous HA addition was applied to verify the changes in the downstream pathway of HA,and western blot was used to verify the changes in the downstream pathway of HA.5.The functional changes of UGT2A3 in colon cancer cells were verified after exogenous HA was applied.Results:1.The expression of the UGT family as a whole is decreased in colon cancer.(1)According to the TCGA database,the overall UGT family in colon cancer cells was reduced,among which UGT2A3 and the remaining three UGT family members(UGT1A8,UGT1A10,and UGT2B15)showed the highest relative reduction.(2)According to the analysis of prognosis on the GEPIA website,only the UGT2A3 gene was significantly correlated with prognosis.2.UGT2A3 was consistent with bioinformatics analysis in colon cancer tissue validation.(1)UGT2A3 was consistent with bioassay in colon cancer tissue validation,and protein expression was consistent with gene expression.(2)Two of the nine groups of colon cancer cell lines(SW480 with the highest protein and gene expression,HCT116 with relatively low protein and gene expression)were compared.3.UGT2A3 inhibits the proliferation and metastasis of colon cancer.(1)Transwell results showed that UGT2A3 inhibited colon cancer metastasis.(2)The results of CCK8 and clone formation experiments showed that UGT2A3 could inhibit the proliferation and clone formation of colon cancer.4.UGT2A3 is related to HA and can regulate its downstream pathway PI3K/AKT(1)The content of HA in the medium of stable rotation expressing cell lines was relatively low,while the content of HA in the medium of stable rotation interfering cell lines was relatively high.(2)Overexpressed cell lines can significantly inhibit the activation of the PI3K/AKT pathway,while interfering cell lines can promote the activation of the PI3K/AKT pathway.5.The response experiments showed that the regulatory effect of UGT2A3 on tumor development was achieved by changing HA content.CCK8 and transwell results showed that the proliferation and metastasis of overexpressed cell lines with exogenous HA were recovered,and western blot results showed that PI3K/AKT activation was restored.Conclusion:1.The UGT family is downregulated in colon cancer,and UGT2A3 is associated with prognosis.2.Colon cancer tissue validation was consistent with bioinformatics analysis.3.UGT2A3 can inhibit the proliferation and metastasis of colon cancer.4.UGT2A3 is related to HA and can regulate its downstream pathway PI3K/AKT.5.UGT2A3 regulates colon cancer progression by regulating HA content.