| Background and ObjectiveColorectal cancer(CRC) is one of the most common gastrointestinal tumors with a high incidence and mortality. The recurrence and metastasis of CRC is the major factor leading to treatment failures while the traditional chemotherapy and radiotherapy have low efficacy and significant toxicity, thus falling far short of clinical demand. Though anti-angiogenesis has been widely applied to treat CRC in the clinic, the result was not satisfactory in prolonging the overall survival time. What’s more, it was often accompanied by such risks as increased stroke incidence and artery vascular events and some adverse reactions such as gastrointestinal hemorrhage, gastrointestinal perforation, delayed wound healing, etc. Thus, it would be of great significance to explore the new targets of anti-angiogenesis treatment so as to treat CRC in the clinic.Being an important component in the cell membrane, cholesterol plays an important role in the cells’ biological activities. It has been found in some researches that high cholesterol diet was a risk factor leading to CRC and CRC patients displayed hypocholesterolemia and high cholesterol level in the feces. Besides, there was an up-regulated expression of the key cholesterol metabolic enzyme-HMG-COA reductase in the CRC, which indicated that the CRC patients suffered from an disorder of cholesterol synthesis and absorption and that cholesterol metabolism played a key role in the development of CRC. And it has been reported with the discharge endothelial cell synthesis of cholesterol can affect angiogenesis, but the transport and metabolism of angiogenesis in the tumor cells themselves cholesterol yet reported in the literature.Cholesterol metabolism process is complicated which involving multiple metabolic enzymes and transporters, the specific effects of cholesterol metabolism on CRC is still unclear. Thus, we suspect that if cholesterol synthesis rate-limiting enzyme of other important related transporter proteins appears to change expression. 3β-hydroxysterol ?(24)-reductase(DHCR24, Seladin-1) catalysed the formation of the double bonds of sterol intermediates: C-24 and C-25. Besides, DHCR24 also participated in all the nineteen catalytic reactions involved in the catalytic synthesis of cholesterol from lanosterol, thus, it is a key enzyme at the last stage of cholesterol synthesis. At present, Niemann-pick type C1 like 1(NPC1L1) has been regarded as the key protein absorbed by cholesterol and plant sterols. 70% of the cholesterol in the diet is absorbed by NPC1L1 which is mainly expressed in the small intestine and liver. There were no published studies yet on transport protein-NPC1L1 and the key enzyme-DHCR24 expression in colorectal cancer and its effect on angiogenesis.Material and method1. To measure DHCR24 expression levels in CRC tissue specimens and explore the correlation between the values of DHCR24 expression levels and the clinical parameters.2. Establish HCT-116, SW480 cell DHCR24 deficient cell lines,and explore the impact of condition mediumon invasion and tube formation of endothelial cells.3. Methods such as Immunohistochemical, q RT-PCR,WB,etc. were employed to measure the expression level of NPC1L1 in the CRC tissues and in the cell lines,and explore the correlation between NPC1L1 expression level and vascular marker CD31.4. Establish HCT-116, SW480 cell NPC1L1 deficient cell lines,and explore the impact of condition mediumon proliferation, invasion, migration and tube formation of endothelial cells.5. A subcutaneously implanted tumor model in mice was established with CRC cell line HCT-116 cell line that contained interfered NPC1L1.) Immunohistochemical and Immunofluorescence method was employed to measure the expression level of CD31,NG2,Collagen IV and VEGFR2 in the subcutaneously implanted tumor of the mice.6. ELISA test was employed to compare CRC cells HCT-116 and SW480 and to explore the changes of various angiogenesis factors in the group where supernatant was cultured with a lower NPC1L1 content. WB technique was employed to compare CRC cells HCT-116 and SW480 and to explore the expression changes of such proteins as VEGFR2, p-VEGFR2, ERK, p-ERK, AKT and p-AKT in HUVEC cells which were processed by supernatant in the group with a lower NPC1L1 content. Observe the condition medium effect on endothelial cell tube formation and invasion supernatant after add in bevacizumab.Result1. Immunohistochemical staining showed that DHCR24 expression level was higher than that in the mucosa tissues adjacent to CRC. Statistical analysis showed that the up-regulation of DHCR24 in CRC tissues was related to patient’s clinical stage, pathologic differentiation degree, lymph node metastasis and survival prognosis. A higher DHCR24 expression indicated a worse prognosis and together with differentiation degree and clinical stage, DHCR24 could also be employed as a clinical prognostic index.2. There was a down-regulated expression of DHCR24 in two CRC cell lines: HCT-116 and SW480 which were transfected by lentivirus.The difference between effects of the invasion, and tube formation of endothelial cells in the group of HCT-116DHCR24 KD,SW480 DHCR24 KD and control group was not have statistically significant.3. The NPC1L1 expression level in the CRC tissues was lower than that in the mucosa tissues adjacent to CRC and a lower NPC1L1 expression level indicated a worse prognosis. The expression level of NPC1L1 and RNA in the normal intestinal epithelial cell line was higher than that in the CRC cell line.There was a negative correlation between the expression level of CD31 and that of NPC1L1 in the serial sections of the CRC paraffin-embedded tissues.4. There was a down-regulated expression of NPC1L1 in two CRC cell lines: HCT-116 and SW480 which were transfected by lentivirus. Compared with the control group, there were greater promoting effects in the proliferation, invasion, migration and tube formation of endothelial cells in the group where supernatant was cultured in the HCT-116NPC1L1 KD and SW480NPC1L1 KD cell lines with a lower NPC1L1 content.5. Immunohistochemical staining showed that compared with the control group, CD31 expression level was higher in the subcutaneously implanted tumor of the mice which was established by HCT-116NPC1L1 KD cell line with a lower NPC1L1 content.Both immunohistochemical staining and immunofluorescence staining showed that in the subcutaneously implanted tumor of the mice which was established by HCT-116NPC1L1 KD cell line with a lower NPC1L1 content, the expression level of CD31 and VEGFR2 was higher than that in the control group while the expression level of NG2 and Collagen IV was lower than that in the control group.6. ELISA test showed that in the group where supernatant was cultured in the HCT-116NPC1L1 KD and SW480 KD cell lines with a lower NPC1L1 content, the VEGF concentration was higher than that in the control group. WB test showed that in the group where supernatant was cultured in the HCT-116NPC1L1 KD and SW480 KD cell lines with a lower NPC1L1 content, the expression levels of such proteins as p-VEGFR2, p-ERK and p-AKT in HUVEC cells that were processed by supernatant were higher than those in the control group but there was no significant difference in the expression levels of VEGFR2, ERK and AKT between those two groups. Bevacizumab can rescue the greater promoting effects in the migration and tube formation of endothelial cells in the group where supernatant was cultured in the HCT-116NPC1L1 KD and SW480 KD cell lines with a lower NPC1L1 content.Conclusion1. DHCR24 expression level in the CRC tissues was significantly higher than that in the mucosa tissues adjacent to CRC. DHCR24 could also be employed as a clinical prognostic index. In vitro experiment confirmed that DHCR24 deficiency in CRC could not promote the migration and tube formation of human umbilical vein endothelial cell.2. The NPC1L1 expression level in CRC was lower than that in the mucosa tissues adjacent to CRC. The lower NPC1L1 expression level in the CRC tissues indicated a worse prognosis. There was negative correlation between the NPC1L1 expression level and microvessel density.. In vitro and vivo mouse experiment confirmed that NPC1L1 deficiency in CRC cell could promote tumor angiogenesis.3. NPC1L1 deficiency in CRC could promote angiogenesis by promoting VEGF-A secretion in the supernatant. Bevacizumab can rescue the effects of CRC angiogenesis with a lower NPC1L1 content. |
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