| Objective In this project,gastrointestinal tumor cell lines with high expression of LRP1 were screened out.We explored the effect on the growth of gastrointestinal tumor cells and metabolism-related proteins after knocking down LRP1.Through a series of experiments,we hope to establish a theoretical basis for clinical application and provide new solutions for the treatment of gastrointestinal tumors in the future.Methods 1.Screening out digestive tract tumor cell lines with high expression of LRP1 by Western blot and Q-PCR.2.Digestive tract tumor cell lines were transfected by LV-LRP1-RNAi lentivirus and negative control virus(NC),and establishing stable knocking down LRP1 cell lines.3.Detecting the impact of knocking down LRP1 and its associated signaling pathways by Western blot.4.CCK-8 assay was used to detect the growth of digestive tract tumor cells after LRP1 knockdown.EdU-594 Cell Proliferation Assay Kit was used to detect cell proliferation.Cell clone formation assay was used to detect population dependence and proliferation capacity of gastrointestinal tumor cells after LRP1 knockdown.5.Transwell assay and scratch test were used to detect invasion and metastasis of gastrointestinal tumor cells.Western blot analysis was used to detect the changes of invasion-related proteins6.Comparing the difference between human gastrointestinal tumor tissues and normal tissues by HE staining7.Immunohistochemistry was used to compare the expression of LRP1 in human tumor gastrointestinal tissues and normal tissues.8.Q-PCR was used to detect the changes of lipid metabolism-related gene expression after LRP1 knockdown.9.Oil red O staining was compared the lipid content of after LRP1 knockdown in pancreatic cancer BxPC-3 cells.Results 1.Western blot and Q-PCR results showed that the expression of LRP1 in gastric cancer SGC-7901 cells,hepatic carcinoma SMML-7721 cells and pancreatic cancer BxPC-3 cells increased significantly compared with human umbilical vein endothelial cells HUVEC cells.2.After transfection,Western blot results showed that the expression of LRP1 protein in gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells decreased significantly compared with shNC.3.After transfection,EdU-594 cell proliferation assay and CCK-8 assay results showed that the the proliferation of gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells decreased compared with shNC.Western blot results showed that the expression of p-AKT and EGFR proteins in gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells were down-regulated after LRP1 knockdown.4.The results of cell clone formation experiment showed that the colony forming ability of gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells decreased compared with shNC after LRP1 knockdown.5.The results of Transwell experiment and scratch test showed that the invasion and metastasis ability of gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells decreased compared with shNC after LRP1 knockdown.Western blot results showed that the expression of p-ERK and MMP-9 proteins in gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells decreased after LRP1 knockdown.6.The results of immunohistochemistry showed that the expression of LRP1 were significantly up-regulated in tumor tissues compared with normal gastrointestinal tissues.7.The results of Q-PCR showed that the expression of CD36 in liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells significantly decreased after knockdown of LRP1.8.The results of oil red O staining showed that lipid increased and lipid accumulated in pancreatic cancer BxPC-3 cells after LRP1 knockdown.Conclution 1.LRP1 was highly expressed in gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells,and pancreatic cancer BxPC-3 cells.2.LRP1 knockdown could inhibit tumor cell proliferation and inhibit related proteins expression in gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells,and pancreatic cancer BxPC-3 cells.3.LRP1 knockdown could inhibit tumor cell colony forming ability in gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells.4.LRP1 knockdown could inhibit tumor cell invasion and metastasis and inhibit invasion-related proteins expression in gastric cancer SGC-7901 cells,liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells.5.The expression of LRP1 increased in human digestive tract tumor tissues compared with normal tissues.6.LRP1 knockdown could inhibit CD36 expression,lipid uptake,and lead to lipid accumulation in liver cancer SMML-7721 cells and pancreatic cancer BxPC-3 cells. |
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