| Objective: Colon cancer is one of the most common malignancies in the digestive system.Chemokine receptors also play an important role in the development and metastasis of various tumors.CXCR7 is a 7-pass transmembrane G-protein coupled receptor superfamily with a typical structure of chemokine receptors and is expressed in many tumors.This study aimed to investigate the function of CXCR7 in colon cancer.Although there is evidence that CXCR7 promotes angiogenesis in colon cancer,the mechanisms involved in this process are unclear.This study was performed by in vivo and in vitro validation and overexpression or silencing of CXCR7 in mouse colon cancer xenografts using contrast-enhanced ultrasound imaging to investigate the correlation of CEUS perfusion parameters and microvessel density(MVD).Methods: This study focused on normal colon cells(HCoEpic)and colon cancer RKO,HCT116,SW480,and Caco-2 cell lines.First,the expression of CXCR7 in normal colon cells and colon cancer cells was detected by qRT-PCR and Western Blot.Secondly,the two cell lines with the highest expression and the lowest expression of CXCR7 were silenced or overexpressed,and their transfection efficiency was detected by qRT-PCR and Western Blot.Furthermore,the proliferation and migration ability of colon cancer cells after transfection were detected by CCK-8,EdU,and Transwell methods.In addition,the transfected cells were co-cultured with human umbilical vein endothelial cells(HUVEC),and the proliferation of HUVEC cells,effects of migration and angiogenesis.The transfected cells and their supernatants were collected to detect the expression of vascular endothelial growth factor(VEGF)in the cells and supernatants.At the same time,the inhibitors LY294002 and U0126 were added to the transfected cells to detect the AKT and ERK pathways.Finally,the stably transfected cells were inoculated subcutaneously in nude mice to form transplanted tumors and subjected to contrast-enhanced ultrasound detection.Western blot was performed for analysis of expression of various factors and their pathways.Results: 1.The expression of CXCR7 in normal colon cells and colon cancer cells was detected by qRT-PCR and Western Blot.The results showed that CXCR7 was highly expressed in all four colon cancer cells compared with normal colon cells,among which SW480 cells had the highest expression and Caco-2 cells had the lowest expression.2.Silencing SW480 cells and over-expressing Caco-2 cells.QRT-PCR and Western Blot results showed that the expression of SW480 cells that silenced CXCR7 was successfully reduced,and the expression of Caco-2 cells that over-expressed CXCR7 was successfully increased.The difference was statistically significant(P<0.05).3.The proliferation and migration ability of SW480 and Caco-2 cells were detected by CCK-8,EdU and Transwell experiments.The results showed that the proliferation and migration capacity of SW480 cells that silenced CXCR7 were reducedand Caco-2 cells overexpressing CXCR7 both increased proliferation and migration capacity compared with the negative control group.Secondly,the transfected cells were co-cultured with HUVEC cells,and the proliferation and migration capabilities of HUVEC were measured by CCK-8,EdU,and Transwell experiments.Compared with the negative control group,the proliferation of HUVEC co-cul tured with SW480 cells that silenced CXCR7 And the migration ability was significantly reduced;on the contrary,HUVEC co-cultured with Caco-2 cells overexpressing CXCR7 significantly increased the proliferation and migration ability,and the difference was statistically significant(P<0.05).Angiogenesis experiments showed that the number of vascular junctions of HUVEC co-cultured with SW480 cells that silenced CXCR7 was significantly reduced,and that of HUVEC co-cultured with Caco-2 cells that overexpressed CXCR7 compared with the negative control group.At the same time,further research found that the inhibitors LY294002 and U0126 can down-regulate cell proliferation,migration and lumen formation ability after overexpression.4.The VEGF concentration in the supernatant of the transfected cells was detected by ELISA.The results showed that the levels of VEGF in HUVEC co-cultured with SW480 cells that silenced CXCR7 were significantly reduced,and the levels of VEGF in HUVEC co-cultured with Caco-2 cells that overexpressed CXCR7 were significantly increased compared with the negative control group.At the same time,further research found that inhibitors can down-regulate VEGF concentrations in over-expressed cells.The difference was statistically significant(P<0.05).5.After transfection,the cells were tested for AKT and ERK pathways.Western Blot results showed that compared with the negative control group,SW480 cells that silenced CXCR7 reduced the phosphorylation of AKT and ERK and the expression of VEGF,and Caco that overexpressed CXCR7-2 cells increase AKT and ERK phosphorylation and VEGF expression,and AKT and ERK signaling can regulate each other in colon cancer cells.qRT-PCR results showed that compared with the negative control group,SW480 cells that silenced CXCR7 reduced VEGF expression,and Caco-2 cells that overexpressed CXCR7 increased VEGF expression.At the same time,further research found that inhibitors could down-regulate the VEGF expression in over-expressed cells.6.Four kinds of colon cancers,RKO,HCT116,SW480 and Caco-2,were inoculated subcutaneously in nude mice.The results showed that SW480 cells produced larger tumors and increased volume.Caco-2 cells produced smaller tumors and increased volume.slow.The comparison of contrast-enhanced parameters in colon cancer tumor tissues showed that the area under the curve(AUC)and peak intensity(PI)of SW480 and Caco-2 tumors were significantly different,and the PI(162.5±7.5)and AUC(1278.6±40.4)of SW480 tumors.The highest value,Caco-2 tumors have the lowest PI(5.5 ± 1.3)and AUC(96.2 ± 14.7)values.7.Inoculate SW480 cells that silence CXCR7 and Caco-2 cells that overexpress CXCR7 to inoculate subcutaneously in nude mice to form transplanted tumors.Detect differences in tumor growth after CXCR7 silencing or overexpression and parameters after contrast-enhanced ultrasound.Compared with the control group,the tumor tissue size and volume of SW480 cells silenced by CXCR7 were significantly reduced,and the contrast imaging parameters PI and AUC also significantly decreased,and the results were statistically significant.In contrast,the tumor tissue size and volume of Caco-2 cells overexpressing CXCR7 significantly increased,and the contrast imaging parameters PI and AUC also increased significantly.The results were statistically significant.8.The results of immunohistochemical staining showed that the tumor tissue staining of CXCR7,VEGF,Ki67,and p-ERK factors in tumor tissue of SW480 cells that silenced CXCR7 was significantly lower than that in the negative control group,and the staining score results were statistically significant.The tumor tissue staining of CXCR7,VEGF,Ki67 and p-ERK factors in the tumor tissues of Caco-2 cells overexpressing CXCR7 significantly increased compared with the negative control group,and the staining score results were statistically significant.9.Detection of tumor microvessel density(MVD)by CD34 immunohistochemical staining.The results showed that compared with the negative control group,the microvessels in tumor tissue of SW480 cells that silenced CXCR7 were significantly reduced,and that of Caco-2 cells that overexpressed CXCR7.Microvessels in tissues increased significantly.We compared the correlation between MVD and contrast-enhanced ultrasound parameters,and the results showed that peak intensity(PI)and area under the curve(AUC)were more correlated with MVD in colon cancer tissues that were overexpressed or silent,and other parameters were related to MVD values.There was no significant correlation between them.10.Finally,we tested the expression of CXCR7,VEGF,AKT,p-AKT,ERK,and p-ERK factors in tumor tissues by Western Blot.The expressions of p-ERK,p-ERK,CXCR7 and VEGF factors were increased compared with the negative control group.The expression of p-AKT,p-ERK,CXCR7 and VEGF factors in tumor tissues of SW480 cells that silenced CXCR7 were all lower than those of the negative control group and it was statistically significant.Conclusion: In summary,1.CXCR7 expression can promote colon cancer growth and angiogenesis by activating AKT and ERK pathways.CXCR7 provides a target for potential treatment of colon cancer.2.There are significant differences between ultrasound contrast parameters in colon cancer tumors that are silent or overexpressing CXCR7,so PI and AUC can be used as reliable indicators in CEUS to assess tumor perfusion.It may be useful to quantify tumor perfusion using CEUS for early detection of tumor angiogenesis. |
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