| Objective: The differential expression of THBS1 in lymph nodes of 52 patients with II-III colorectal cancer and its relationship with prognosis were studied by immunohistochemistry,and the regulatory effect of mir-19 a on THBS1 was preliminarily explored at the cellular level.In order to lay a theoretical foundation for the role of THBS1 in lymphatic metastasis of colorectal cancer.Methods: 1、From January 2015 to February 2017,paraffin specimens of lymph nodes from patients with colorectal cancer undergoing radical resection of colorectal cancer and pathological stage II or stage III were collected.the lymph nodes of all patients were stained with routine hematoxylin and eosin.Lymph node metastasis was observed under microscope.Among them,29 patients with stage II were included in the study,29 regional lymph node negative paraffin specimens were selected,and 23 patients with stage III were included in the study.there were 1 or 3 lymph node metastases in individual patients with stage III after operation.Therefore,33 positive paraffin specimens of all lymph nodes in 23 patients with III were selected.the average expression level of THBS1 in III patients with multiple lymph node metastases was analyzed.The paraffin sections of selected lymph nodes were examined by immunohistochemistry,and the expression of THBS1 in lymph nodes of patients with and without lymph node metastasis was detected by mean optical density analysis;2、The general clinical data of 52 patients with colorectal cancer were collected,and the relationship between the expression of THBS1 and the clinical features of colorectal cancer was analyzed by chi-square test,and the progression-free survival time(PFS)was used as the observation index.The survival curve of patients with colorectal cancer was drawn by Kaplan-Meier method,and the survival time of the two groups was compared by Log-rank test to study the relationship between the expression level of THBS1 and the prognosis of patients with colorectal cancer;3、Real-time fluorescence quantification was utilized to inspect the mi R-19 a expression in four different colorectal cancerous cell strains(which were LOVO,HT116,SW480 and HT29),and cell strains with highest and lowest mi R-19 a expression were selected;4、The two colorectal cancer cell lines selected from method 3 were performed mi R-19 a transfection silencing or overexpression,and then the expression of THBS1 was measured by Western blotting(Western Blot,WB);5、Two strains of colorectal cancer cells selected in method 3 and two strains of colorectal cancer cells transfected with silent and over-expression plasmids were cultured for cell scratch test,and the migration ability of the two cell lines before and after transfection was detected;6、Dual-luciferase experiment was adopted to verify the incorporation conditions between mi R-19 a and THBS1 3’UTR region.Results: 1、The results of immunohistochemistry showed that the relative expression levels of THBS1 in postoperative lymph nodes of colorectal cancer with and without lymph node metastasis were 0.261 ±0.053 and 0.451 ±0.014,respectively.The expression of THBS1 in patients with lymph node metastasis was significantly lower than that in patients without lymph node metastasis.The difference between the two was statistically significant(P < 0.05);2、The expression level of THBS1 in lymph nodes of patients with colorectal cancer was not correlated with age,sex,tumor location and pathological type(P > 0.05),but the expression level of THBS1 in lymph nodes of patients with colorectal cancer was related to lymph node metastasis(P < 0.05).PFS was used as the observation index.The survival curve was drawn by Kaplan-Meier method.the results showed that the survival time of patients with low expression of THBS1 was worse than that of patients with high expression of THBS1,and the difference was statistically significant by Log-rank test(P = 0.007);3、The expression levels of mi R-19 a in colorectal cancer cell lines LOVO,HT116,SW480 and HT29 were: 1.00±0.09,0.64±0.02,0.56±0.02,0.38±0.06,respectively.mi R-19 a was in colorectal cancer LOVO cell line.The highest expression level,HT29 mi R-19 a expression of the lowest;4、Before transfection of plasmid: the expression of THBS1 protein in HT29 cells was higher than that in LOVO cells;after transfection of plasmid: HT29 transfected the overexpression plasmid of mi R-19 a,the protein expression of THBS1 was significantly decreased;LOVO was transfected After mi R-19 a sh RNA,the protein expression of THBS1 was significantly increased;5 、 After transfection of mi R-19 a sh RNA,the migration ability of LOVO cells was significantly decreased;after HT29 cells were transfected with mi R-19 a overexpression plasmid,the migration ability was significantly increased;6、Compared with the transfected by THBS1 3’UTR WT,the luciferase activity of LOVO and HT29 was significantly improved when transfected by THBS1-3’UTR Mut.Conclusion: 1、The expression of THBS1 in metastatic lymph node tissues of colorectal cancer was significantly lower than that in metastatic negative lymph node tissues.The expression level of THBS1 may be related to the prognosis of patients with colorectal cancer.the lower the expression level of THBS1,the worse the prognosis of patients with colorectal cancer;2、Mi R-19 a in colorectal cancer cells may inhibit the expression of THBSl by directly combined with the 3’UTR region of THBS1,and further affect the occurrence and development of colorectal cancer. |
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