| Background: Colorectal cancer(CRC)is the third most common cancer and the second most common cause of cancer death worldwide.The overall prognosis of patients is poor,which is mostly attributed to the recurrence and metastasis caused by chemotherapy resistance.5-fluorouracil(5-Fu)is the most classical and commonly used first-line chemotherapy drug.Our group has been devoted to the study of B7-H3.Previous studies have found that B7-H3 is highly expressed in CRC and regulates 5-Fu resistance in CRC.B7-H3 is a type Ⅰtransmembrane glycoprotein,belonging to the B7/CD28 immunoglobulin superfamily.It is considered a new tumor marker and a potential target for immunotherapy.CRC tissue microarray showed that high expression of B7-H3 and cytochrome CYP1B1 indicated a poor prognosis of CRC,and there was a positive correlation between them.CYP1B1 is a member of the cytochrome P450(CYP450)oxidase superfamily,which is involved in the metabolic reactions of many hormones,procarcinogens,and drugs.The expression of CYP1B1 in tumor tissues is much higher than that in normal tissues,and it is related to the chemosensitivity of paclitaxel,docetaxel,and other drugs.The association of B7-H3 and CYP1B1 with 5-Fu sensitivity in CRC has not been studied.In this study,we aimed to explore the effects of B7-H3 and CYP1B1 on the sensitivity of CRC to 5-Fu.Methods: First,Western blot was used to detect the expression of B7-H3 in CRC cells,and the cells were selected to construct cell lines with stable B7-H3 overexpression and knockdown.Western blot and q RT-PCR were used to verify the transfection efficiency.CCK8,Ed U,colony formation,flow cytometry,and animal experiments were used to verify the effect of B7-H3 on the sensitivity of CRC cells and tumor-bearing nude mice to 5-Fu.Secondly,bioinformatics analysis was used to identify the possible downstream target of B7-H3,CYP1B1,and the expression relationship between B7-H3 and CYP1B1 was detected by Western blot,,immunofluorescence,and CO-IP experiments.CCK8 assay was used to detect the effect of CYP1B1 knockdown on 5-Fu sensitivity.At the same time,the expression of B7-H3 and the effect of CYP1B1 knockdown on the sensitivity of the tumor-bearing model to5-Fu were investigated by intratumoral injection.Finally,Western blot was used to detect the relationship between the expression of AKT,p-AKT,and B7-H3 in the stable cell lines,and then the changes in the expression of B7-H3,CYP1B1,AKT,and p-AKT were detected after treatment with PI3K-AKT pathway inhibitor LY294002.Results: In the first part,B7-H3 overexpression cell SW480-B7-H3 and control cell SW480-NC were constructed by selecting the B7-H3 low expression cell SW480 and high expression cell Caco-2 according to the results of Western blot.B7-H3 knockdown expression cell Caco-2-sh B7-H3 and control cell Caco-2-sh NC.After 5-Fu treatment,the CCK8 assay showed that the cell viability and IC50 value of SW480-B7-H3 cells were higher than those of SW480-NC cells,and the cell viability and IC50 value of Caco-2-sh B7-H3 cells were lower than those of Caco-2-sh NC cells.Ed U assay and colony formation assay showed that the proliferation and colony formation ability of SW480-B7-H3 cells were higher than those of SW480-NC cells,while those of Caco-2-sh B7-H3 cells were lower than those of Caco-2-sh NC cells.The results of flow cytometry showed that the apoptosis rate of SW480-B7-H3 cells was lower than that of SW480-NC cells,and that of Caco-2-sh B7-H3 cells was higher than that of Caco-2-sh NC cells.In the animal experiment,the tumor volume and weight of SW480-B7-H3+5-Fu group were larger than those of SW480-NC group.In the second part,bioinformatics analysis was used to identify the possible downstream target of B7-H3,CYP1B1.Western blot analysis showed that B7-H3 was positively correlated with CYP1B1 protein expression in human CRC tissues.Western blot and showed that B7-H3 and CYP1B1 were higher in SW480-B7-H3 cells than in SW480-NC cells,and lower in Caco-2-sh B7-H3 cells than in Caco-2-sh NC cells.CO-IP experiment showed that there was a binding relationship between them.CCK8 assay showed that after CYP1B1 knockdown in SW480-B7-H3 cells,the cell viability was lower than that in SW480-B7-H3 cells after 5-Fu treatment.Cell fluorescence assay showed that B7-H3 co-localized with CYP1B1 in the cytoplasm.Animal experiments showed that the tumor weight and volume of SW480-B7-H3+si CYP1B1+5-Fu group were smaller than those of SW480-B7-H3+PBS,SW480-B7-H3+5-Fu,and SW480-B7-H3+si CYP1B1+PBS.In the third part,Western blot results showed that the expressions of B7-H3,CYP1B1,and p-AKT in SW480-B7-H3 cells were higher than those in SW480-NC cells,and there was no significant difference in AKT expression.The expressions of B7-H3,CYP1B1,and p-AKT in Caco-2-sh B7-H3 cells were lower than those in Caco-2-sh NC cells,and there was no significant difference in AKT expression.Western blot showed that the expression of CYP1B1 was decreased in SW480-B7-H3 cells and increased in Caco-2-sh B7-H3 cells after treatment with PI3K-AKT pathway inhibitor LY294002.Conclusions: In conclusion,B7-H3,an immune checkpoint protein,can affect the sensitivity of CRC to 5-Fu,and B7-H3 regulates CYP1B1 expression through the PI3K-AKT signaling pathway to increasing metabolic response to 5-Fu,thereby reducing chemosensitivity of CRC.This study provides new insights into the efficacy of 5-Fu in CRC and has important implications for the development of targeted agents to increase chemosensitivity in CRC. |
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