The Effect Of BRAF Gene Silencing On Angiogenesis Or Lymphangiogenesis Induced By CRC-derived Exosomes With BRAF^V600E Mutation

Part 1 Association between the mutation of BRAF^V600E and the density of micro vascular and lymphatic vessels in colorectal cancerObjective:To detect the relationship between the mutation of BRAF^V600Eand the density of micro vascular and lymphatic vessels respectively in CRC.Methods:1 The present study has collected the pathological data of 10 patients with BRAF^V600E-mutant and 20 patients with BRAF wild-type CRC,and investigated the relationship between the mutation of BRAF^V600E and clinicopathological characteristics in CRC.2 The present study has detected the density of micro vascular and lymphatic vessels in 30 patients in CRC by immunohistochemistry,and investigated the association between the mutation of BRAF^V600E and the density of micro vascular and lymphatic vessels in CRC.Results:1 The mutation of BRAF^V600E was associated with poor differentiation,and positively associated with the level of preoperative CA19-9,but not with gender,age,tumor location,mucinous histology,TNM stage,lymph node metastasis,and the levels of preoperative CEA and preoperative platelet count.2 In BRAF^V600E-mutant and BRAF wild-type CRC,the density of microvessels were?38.53±17.11?per high-power field and?27.54±9.54?per high-power field,respectively?P=0.03?;the density of lymphatic vessels were?9.67±5.63?per high-power field and?3.81±2.06?per high-power field,respectively?P<0.01?.Conclussion:1 The mutation of BRAF^V600E was associated with poor differentiation,and positively associated with the level of preoperative CA 19-9 in CRC.2 BRAF^V600E mutation was positively associated with density of micro vascular and lymphatic vessels in CRC.Part 2 The effect of BRAF gene silencing on angiogenesis or lymphangiogenesis induced by CRC-derived exosomes withObjective:To detect whether the silent of BRAF gene effects angiogenesis or lymphangiogenesis induced by CRC-derived exosomes with BRAF^V600Emutation.Methods:1 Exosomes derived by HT29(BRAF^V600E-mutant)or1627?BRAF-knockdown?cells were isolated by ultracentrifugation-based isolation techniques.The structure of exosomes were observed by transmission electron microscope.The expression of CD9 and CD63 which were specific markers of exosomes were detected by Western blot.2 Exosomes secreted from HT29 or 1627 cells were cultured with human umbilical vein endothelial cells?HUVECs?.The CCK-8 assay,wound healing assay and tube formation assay were used to detect the proliferation,migration and the ability of tube formation of HUVECs,respectively.3 Exosomes secreted from HT29 or 1627 cells were cultured with human lymphatic endothelial cells?HLECs?.The CCK-8 assay,wound healing assay and tube formation assay were used to detect the proliferation,migration and the ability of tube formation of HLECs,respectively.4 The expression of VEGF-A,b FGF,TGF-?1 and VEGF-C in exosomes secreted from HT29 and 1627 cells were detected by Western blot.5 Exosomes secreted from HT29 and 1627 cells were cultured with HUVECs for 24 h and with HLECs for 48 h.The expression of Claudin-5,Occludin or ZO-1 were detected by Western blot.Result:1 Exosomes were successfully isolated from HT29 and 1627 cells.Exosomes were approximately 30-150 nm in diameter with a lipid bilayer membrane structure.And exosomes from HT29 and 1627cells both expressed CD9 and CD63.2 The effect of BRAF gene silencing on angiogenesis induced by CRC-derived exosomes with BRAF^V600E mutation were detected.?1?Compared with HT29 group,the proliferation of HUVECs in 1627group was decreased?P<0.05?;?2?The percentage of wound healing area of HT29 and 1627 group were?82.863±3.095?%and?45.067±2.895?%?P<0.01?;?3?The number of tubes in HT29 group and 1627 group were?30.625±0.925?per low-power field and?12.750±1.887?per low-power field?P<0.01?.3 The effect of BRAF gene silencing on lymphangiogenesis induced by CRC-derived exosomes with BRAF^V600E mutation were detected.?1?Compared with HT29 group,the proliferation of HLECs in 1627group was decreased?P<0.05?;?2?The percentage of wound healing area of HT29 and 1627 group were?42.393±0.247?%and?23.327±1.434?%?P<0.01?;?3?The number of tubes in HT29 group and 1627 group were?26.750±2.016?per low-power field and?15.375±1.413?per low-power field?P<0.01?.4 The effect of BRAF gene silencing on the expression of VEGF-A,b FGF,TGF-?1 and VEGF-C in CRC-derived exosomes with BRAF^V600Emutation were detected.Compared with exosomes secreted form HT29 cells,the expression of VEGF-A,b FGF,TGF-?1 and VEGF-C in exosomes-derived from 1627cells were inhibited.5 The effect of BRAF gene silencing on the expression of ZO-1,Claudin-5 or Occludin of endothelial cells induced by CRC-derived exosomes with BRAF^V600E mutation were detected.?1?Compared with HT29 group,the expression of ZO-1 of HUVECs in 1627 group was increased at 24 h;?2?Compared with HT29 group,the expression of ZO-1,Claudin-5and Occludin of HLECs in 1627 group were increased at 48 h.Conclusion:1 Silencing BRAF gene inhibits angiogensis and lymphangiogenesis induced by CRC-derived exosomes with BRAF^V600Emutation.2 Silencing BRAF gene may pave a new path for developing clinical treatment methods for BRAF^V600E-mutant CRC.