Function And Mechanism Research Of MiR-19a Downregulate THBS1 To Promote Metastasis In Colorectal Cancer

Objective:Like most solid tumors, the vascular metastasis and lymphatic metastasis of colorectal cancer are the main ways of metastasis. Vascular or lymphatic metastasis has occurred in about 50% of the surgical-treated patients, the majority of them relapsed soon, and their 5 years survival rate was only about 10%. Therefore, the metastasis situation has become one of the important factors in the prognosis of patients with colorectal cancer. This study was designed to reveal the rule and mechanism of metastasis of colon cancer cells by detecting the expression of miR-19a in colorectal cancer tissues and three colon cancer cell lines, verificating the function of miR-19a downregulate THBS1 on VEGF protein expression and abilities of proliferation and metastasis of colon cancer cells.Methods:This topic is divided into four parts. In the first part, the expression of miR-19a in colorectal cancer tissues and colon cancer cell lines was detected, including the following tests:The expression of miR-19a in colorectal cancer tissues. Colorectal cancer specimens were collected from the clinic, and were divided into two groups according to whether or not accompanied by metastasis. QRT-PCR method was applied to detect the expression of miR-19a in cancer tissues and colon cancer cell lines. Colon cancer cell lines (HT29, SW480 and LoVo) with different malignancy were selected to use in the study. In the second part, the expression of THBS1 and VEGF in colon cancer cell lines was detected, including the following experiments:QRT-PCR and Western-blot were used in detecting THBS1 gene and protein expression in colon cancer cell lines and Western-blot was used in VEGF expression. The binding reaction of THBS1 and VEGF protein in colon cancer cell lines was verified by coprecipitation method. The third part verifies the regulation of miR-19a on THBS1 expression in LoVo cell and its effects on the proliferation and migration of LoVo cells. SiRNA plasmid was constructed and transfected into LoVo cells, and the changes of the proliferation and metastasis of LoVo cells were verified before and after. And in the fourth part, reporter gene was used in verifiing the binding of miR-19a on THBS1 and it’s regulating mechanism. Through these experiments, the rule and mechanism of miR-19a promoting the metastasis of colorectal cancer cells through down-regulation of THBS1 is revealed.Results: ①MiR-19a was highly expressed in colorectal cancer tissues, and the expression of metastasis group was higher than that in non metastasis group. The expression level of miR-19a from high to low in colon cancer cell lines was LoVo, SW480 and HT29, the difference was significant. ②The results of QRT-PCR and Western-blot experiments for expression of THBS1 in colon cancer cell line showed that HT29 was the highest, SW480 was the second and LoVo was the lowest. The highest VEGF protein expression of colorectal cancer cell line is LoVo, the second was SW480 and the lowest was HT29. ③Immune coprecipitation of THBS1 and VEGF in LoVo, SW480 and HT29 showed all positive reaction. ④When the SiRNA plasmid of miR-19a was successfully constructed and transfected into LoVo cells, Wound healing assay and Transwell assay were all showed that proliferation and migration abilities decreased and THBS1 expression was significantly increased. ⑤Dual luciferase reporter assay and dual luciferase mutation assay showed that mir-19a can bind with 3’end of THBS1 and inhibited the expression of it, when mir-19a expression was inhibited, THBS1 expression increased.Conclusion:MiR-19a was highly expressed in colorectal cancer tissues and colorectal cancer cell lines, which was closely related to the occurrence and development of colorectal cancer. In colon cancer cell lines, the expression level of miR-19a was highest in LoVo, middle in SW480, and lowest in HT29, which was consistent with the malignant degree of these cell lines. This indicates that miR-19a expression is closely related to the malignant degree of colorectal cancer cells. In the colon cancer cell lines, the expression level of THBS1 was contrary to the malignant degree of them, like the expression of mir-19a. Immune coprecipitation reaction results of THBS1 and VEGF protein were all positive in the cell lines. These results shows that binding reaction occurs betreen THBS1 and VEGF protein in the colon cancer cell lines, and they are both closely related to the malignant degree of colorectal cancer. Luciferase experiments’results show that miR-19a plays an inhibitory effect by combing the 3’side of THBS1. In the colon cancer cells, mir-19a down-regulating the protein expression of THBS1 and reducing it’s combing reaction with VEGF, VEGF protein levels increased and it’s promoting function on formation of micro vascular and lymphatic tube is enhanced. This result provides convenient conditions for the colorectal cancer cells to transfer through the new micro vascular and lymphatic tube.