The Effect Of Resveratrol On The Invasion And Metastasis Of Mouse Melanoma Cell B16F10 Based On The S1P/S1PR1/STAT3 Signaling Pathway

Objectives:To explore the effects and mechanism of resveratrol on the invasion and metastasis of mouse melanoma cells B16F10 in tumor microenvironment.Methods: 1)After treating mouse melanoma cell B16F10 with different concentrations of resveratrol(25 ?M,50 ?M,100 ?M,200 ?M,400 ?M)for 24 hours,the CCK8 test was used to detect the inhibition of proliferation of mouse melanoma cell B16F10 by resveratrol;CCK8 test was used to detect the inhibition of resveratrol on the proliferation of mouse melanoma cells B16F10 under tumor microenvironment;the supernatant of mouse mononuclear macrophage Raw264.7 was collected as conditioned medium to culture B16F10 cells to simulate tumor microenvironment Healing test and Transwell invasion test were used to detect the migration and invasion ability of B16F10;Western blot was used to detect the expression of tumor-associated proteins E-cadherin and Vimentin and the expression of pathway protein p-STAT3 in B16F10 cells.2)B16F10 cells were transfected with S1PR1 plasmid and S1PR1 siRNA respectively,overexpressing and knocking down the expression of S1PR1 in cells;wound healing experiments and Transwell invasion experiments were used to detect the migration and invasion ability of B16F10;Western blot experiments were used to detect the pathway proteins S1PR1 and p in B16F10 cells.-STAT3 expression.3)Raw264.7 cells were transfected with viral vectors overexpressing S1PR1 protein,and the culture supernatant was collected to prepare conditioned medium.Conditionally cultivate B16F10 cells for 24 hours,collect the cells and inject B16F10 cells into the tail vein to establish a mouse lung metastasis model.From the first day of modeling,resveratrol 100 mg / kg was given by gavage twice a day for 21 days;after the last dose,the spinal dislocation was sacrificed,lung tissue was taken,lung metastases were counted,and pictures were taken.Western blot experiments were used to detect the expression of tumor-associated proteins E-cadherin,Vimentin,Twist and Snail in mouse lung tissue.Results : 1)The results of CCK8 show that resveratrol has an inhibitory effect on the proliferation of B16F10 cells and has a dose-dependent characteristic;at a concentration of less than 100 ?M,resveratrol does not significantly inhibit the proliferation of conditioned B16F10 cells.Three resveratrol concentrations of 25 ?M,50 ?M and 100 ?M were selected for the study.The wound healing experiment and Transwell invasion experiment showed that compared with the blank control group,the migration and invasion ability of B16F10 in the conditioned culture group was enhanced.After resveratrol treatment,the migration and invasion ability of the cells decreased.Western blot results showed that compared with the blank control group,the expression of E-cadherin decreased and the expression of Vimentin increased in the cells of the conditioned group,but after resveratrol treatment,the expression of E-cadherin increased and the expression of Vimentin decreased.2)The wound healing experiment and Transwell invasion experiment showed that the knockdown of S1PR1 reduced the migration and invasion ability of B16F10 cells.After resveratrol treatment,the migration of B16F10 cells further decreased;high expression of S1PR1 increased the migration and Invasive ability,and resveratrol inhibits this phenomenon.Western blot results showed that resveratrol inhibited the increase in the expression of p-STAT3 in the cells in the conditioned group;however,it was found in the mechanism study that the expression of p-STAT3 in the cells of the S1PR1 knockdown group decreased compared with the blank control group;The expression of p-STAT3 in the S1PR1 overexpression group increased,but after resveratrol treatment,the expression of p-STAT3 decreased;3)The mice were dissected and found that compared with the blank control,the lung metastasis nodules in the conditioned culture group increased,and after resveratrol treatment,the number of lung metastasis nodules decreased in the mice.Western blot results showed that compared with the blank control group,the expression of Vimentin,Twist and Snail in the lung tissue of the conditioned culture group increased,and the expression of E-cadherin decreased;after the resveratrol treatment,the expression of Vimentin,Twist and Snail Reduced,the expression of E-cadherin protein increased.Conclusion: Resveratrol inhibits invasion and metastasis of melanoma cell B16F10 in mice in vitro and in vivo and may function by S1P/S1PR1/STAT3 signaling.