Effects of S1P/S1PR1 Pathway Regulating LAMB3 Expression on Migration, Adhesion and Metastasis of Esophageal Squamous Cell Carcinoma Cells
MENG Chunmei1, REN Ling1, TANG Maolin1, XU Fengmin2, XIANG Tao3, LIU Lu4, LI Li5, HU Weimin1, 6*
This study aimed to investigate the effects of S1P (sphingosine 1-phosphate)/S1PR1 (sphingosine 1-phosphate receptor 1) pathway regulating the expression of LAMB3 (laminin subunit beta 3) on the proliferation, migration, adhesion and metastasis of ESCC (esophageal squamous cell carcinoma) cells. GEO database was used to analyze the difference of LAMB3 expression between ESCC tissues and normal tissues. Western blot was used to detect the expression of LAMB3 in human esophageal epithelial cell HEEC cells and human esophageal squamous cell carcinoma Eca109, TE-1 and KYSE-150 cells. TE-1 cells were treated with S1P, or knockdowned or overexpressed S1PR1 in Eca109 cells, the expression of LAMB3 was detected by Western blot. S1PR1-EGFP overexpression vector and LAMB3 siRNA (small interfering RNA) were co-transfected into Eca109 cells, and the migration ability was detected by scratch assay. LAMB3 siRNA or LAMB3 shRNA (short hairpin RNA) was used to down-regulate the expression of LAMB3 in ESCC cells, lentivirus with LAMB3 overexpression was used to upregulate the expression of LAMB3 in ESCC cells. The proliferation, migration and adhesion ability of ESCC cells were detected by CCK8 assay, scratch assay, and cell-matrix adhesion assay, respectively, and the expression of EMT (epithelial-mesenchymal transition) -related proteins and MMP9 (matrix metalloproteinase 9) were detected by Western blot. Eca109 cells with LAMB3 shRNA knockdown were injected into the tail vein of nude mice, and their metastatic ability and survival rate were evaluated. The results showed that the expression of LAMB3 gene and protein in ESCC cancer tissues and ESCC cells were higher than that in normal tissues and human esophageal epithelial cells. LAMB3 expression was increased in TE-1 cells treated with S1P. LAMB3 expression was downregulated in S1PR1 knockdown Eca109 cells, and conversely, overexpression of S1PR1 in Eca109 cells up-regulated LAMB3 expression, promoted cell migration, and knockdown of LAMB3 inhibited the effect. LAMB3 knockdown by siRNA or shRNA had no significant effect on the proliferation of Eca109 cells and TE-1 cells. However, the migration and adhesion of Eca109 cells and TE-1 cells were considerably reduced by LAMB3 knockdown. Overexpression of LAMB3 had no significant effect on the proliferation of Eca109 cells. However, the migration and adhesion of Eca109 cells were considerably enhanced by LAMB3 overexpression. EMT process was inhibited and MMP9 was significantly decreased in LAMB3 stable knockdown Eca109 cells, and the opposite effect were showed in LAMB3-overexpressing Eca109 cells. In the nude mice model of metastasis, there were higher survival rate and fewer metastases in LAMB3 knockdown group than that in the negative control group. In this study, it concludes that LAMB3 is up-regulated in ESCC cells and tissues, and S1P/S1PR1 pathway inducing the expression of LAMB3 promotes ESCC cells migration and adhesion in vitro and metastasis in vivo.
CN
EN