SREBP1 Mediates The Proliferation And Motility Of Human Esophageal Squamous Carcinoma Cells Via The Wnt/β-catenin Signaling Pathway

【Objective】To explore the mechanism of SREBP1 involved in the proliferation and metastasis of esophageal squamous cell carcinoma through Wnt/β-catenin signaling pathway.【Methods】(1)Based on two independent databases in Oncomine,the expression levels of SREBP1 in esophageal squamous cell carcinoma tissues and adjacent normal tissues were analyzed.H&E staining and immunohistochemical analysis were used to analyze the difference in the expression of SREBP1 between cancer tissues and normal tissues adjacent to the cancer in patients with esophageal squamous cell carcinoma.The expression of SREBP1 in cancer tissues and normal tissues adjacent to cancer was detected by PCR and Western blot.(2)CCK8 method was used to detect the effect of SREBP1 on the proliferation of esophageal squamous carcinoma cells.Western blot and immunofluorescence were used to detect the effect of SREBP1 expression on Ki-67 expression in esophageal squamous carcinoma cells.Western blot,immunofluorescence,and PCR were used to detect the effect of SREBP1 expression changes on EMT-related protein expression and EMT-related transcription factor formation in esophageal squamous cell carcinoma cells.(3)The effect of SREBP1 expression on the expression of VEGF-A,VEGF-C,MMP2,and MMP9 in esophageal squamous carcinoma cells was detected by PCR and ELISA;the effect of SREBP1 expression change on the migration and infiltration ability of esophageal cancer cell lines was detected by the scratch test and Transwell TM test;Western blot was used to detect the effect of SREBP1 expression on the activation of Wnt/β-catenin signaling pathway in esophageal squamous carcinoma cells;combined with Wnt/β-catenin signaling inhibitor ICG-001,Western blot was used to verify the change of SREBP1 expression in esophageal squamous carcinoma cells on Wnt /β-catenin signaling pathway regulation.(4)Western blot was used to detect the effect of SREBP1 expression on esophageal squamous cell carcinoma cells on SCD1 expression and SCD1 expression on Wnt/β-catenin signaling pathway activation.Combined with SREBP1 inhibitor Fatostatin(10 μM),Western blot was used to detect the nuclear translocation of SREBP1 and SCD1 and activation of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma cells.CCK8 and ELISA were used to detect the proliferation capacity of KYSE-150 cell line and the amount of VEGF-A and MMP9 in the supernatant after treatment with Fastostatin.(5)Immunohistochemistry was used to analyze the expression changes of SREBP1,p-GSK-3β,β-catenin,SCD1,Ki-67,MMP9 and VEGF-C in xenograft tumors between the sh NC and sh SREBP1 groups in nude mice.H&E staining analysis counted the number of lung metastases in the sh NC and sh SREBP1 groups in nude mouse metastasis models.【Results】(1)SREBP1 expression is increased in esophageal squamous cell carcinoma tissues and cell lines.Bioinformatic analysis and immunohistochemistry showed that SREBP1 expression in esophageal squamous cell carcinoma was higher than normal tissues adjacent to the cancer.The positive degree of SREBP1 in esophageal squamous cell carcinoma was positively correlated with tumor differentiation,lymph node metastasis and Ki67 expression level.Compared with normal tissues adjacent to cancer,the transcription and translation levels of SREBP1 in ESCC tissues increased significantly.(2)SREBP1 promotes the proliferation and EMT of esophageal squamous carcinoma cells.After inhibiting the expression of SREBP1,the proliferation of esophageal squamous cell carcinoma cells decreased,the expression of Ecadherin increased,the expression of N-cadherin and Vimentin decreased,and the expression levels of EMT-related transcription factors Slug and Snail1 decreased;while overexpression of SREBP1 was the opposite.(3)Down-regulation of SREBP1 inhibits cell proliferation and metastasis through Wnt/β-catenin signaling pathway.Knocking down SREBP1 can significantly reduce the expression of MMP9,VEGF-A and VEGF-C in ECA-109 cells,while up-regulating SREBP1 expression can be significantly enhanced.At the same time,whether up-regulating or down-regulating SREBP1 had no significant effect on the expression of MMP2 in esophageal squamous carcinoma cells.Inhibition of SREBP1 can enhance the invasion and metastasis ability of esophageal squamous cell carcinoma cells,but it is the opposite when SREBP1 is up-regulated.In esophageal squamous cell carcinoma,SREBP1 has a significant effect on Wnt/β-catenin pathway activation;the expression of SREBP1 in esophageal squamous cell carcinoma cells is not affected by ICG-001 treatment.(4)SREBP1 mainly activates the Wnt/β-catenin pathway through SCD1.SCD1 is significantly increased in esophageal squamous cell carcinoma cells that overexpress SREBP1,while interference with SREBP1 is the opposite.Regardless of whether SREBP1 is overexpressed,SCD1 knockdown inhibits β-catenin from entering the nucleus without affecting SREBP1 expression.Fatostatin(SREBP1 inhibitor)not only reduces the expression of SREBP1 and SCD1 in the nucleus,but also prevents the nuclear translocation of β-catenin in the cytoplasm.(5)In vivo experiments confirmed that SREBP1 is involved in ESCC proliferation and metastasis.Compared with the control group,the expression of SREBP1,β-catenin,MMP9,VEGF-C and Ki67 in transplanted tumors targeted to knock out SREBP1 was significantly inhibited,and the tumor volume of transplanted tumors was significantly smaller than that of the control group.Compared with the control group,the down-regulation of SREBP1 suppressed the number of metastases in the lung.【Conclusions】 SREBP1 is up-regulated in ESCC tumor tissues and cells.SREBP1 plays a key role in the proliferation,migration and invasion of ESCC cells,and causes the triggering of EMT and the up-regulation of VEGF and MMP.In addition,SREBP1 activates Wnt/β-catenin signaling pathway by up-regulating SCD1 to promote the proliferation,invasion and metastasis of esophageal squamous carcinoma cells.