LASS2 Regulates Invasion And Chemoresistance Via ERK/Drp1 To Modulate Mitochondrial Dynamics In Bladder Cancer Cells

Objectives:1.To discuss the effect of the overexpression and suppression expression of LASS2 on the invasion and chemoresistance in bladder cancer cells.2.To investigate the influence of mitochondrial dynamics/Drpl signal pathway to LASS2 expression on the invasion and chemoresistance of bladder cancer cells.3.To study the influence of LASS2 expression on mitochondrial membrane potential in bladder cancer cells.4.To investigate the role of LASS2 inhibiting Drpl signaling by regulating the ERK pathway.Methods:1.The expression levels of LASS2 protein in normal urethra epithelial cell line SV-HUC-1 and bladder cancer cell lines,such as BIU87,J82,5637 and T24 were detected by Western blot.LASS2 plasmids for overexpression and suppression expression were constructed,then expressed to the three bladder cells(BIU87,J82 and siRNA5637)by Lipo-3000.The expressions of LASS2 protein in the three cell lines were measured by Western blot.The changes of invasive ability for bladder cancer cells in overexpression and suppression expression were detected by Matrigel invasion assay.The cell proliferative ability for bladder cancer cells was detected by MTT assay.The viability and apoptosis of bladder cancer cell were tested by CCK-8 assay and Annexin V/PI staining.2.The proteins which can regulate mitochondrial dynamics directly,including p-Drpl,Drpl and Fisl,were detected by Western blot to verify the mechamism of LASS2 regulating mitochondrial function.The Mdivi-1,a specific blocker of the Drpl signaling pathway,was used in 5637 cells to clarify mitochondrial division/fusion balance and the role of Drpl signaling pathway in LASS2 regulating invasion and chemo-resistance of bladder cancer cells.The morphology of mitochondria was observed by laser scanning confocal microscope.The change of cell invasive ability for bladder cancer cells was investigated by Matrigel invasion assay.The apoptosis for bladder cancer cells was tested by Annexin V/PI staining.3.JC-1 staining was used by flow cytometry to investigate the role of LASS2 in controlling mitochondrial apoptosis by regulating mitochondrial membrane potential(??m).To investigate the role of LASS2 in mitochondrial homeostasis,mitochondrial morphology was observed by laser scanning confocal microscope.4.Then LASS2 experimental group,negative control group and PD98059 group were established.The expressions of p-ERK and ERK protein were detected by Western blot in LASS2 overexpression group and suppression expression group.PD98059,a MEK/ERK signaling pathway inhibitor,was applied to inhibit ERK phosphorylation.The expressions of p-ERK?p-Drpl protein were detected by Western blotting in order to observe the effect of Drpl signal on LASS2 expression by regulating ERK pathway.Results:1.The expression of LASS2 protein in 5637 cell showed relatively higher than that in normal urothelial cell line SV-HUC-1 and other bladder cancer cell lines,such as BIU87,J82,5637 and T24.The normal SV-HUC-1 showned more LASS2 expression level than that of the other three bladder cancer cell lines.The result by MTT assay showed that LASS2 overexpression significantly inhibited the proliferation of J82 cells.Invasion experiment results showed that LASS2 overexpression significantly reduced the number of invasion of J82 and BIU87 cells.In 5637 cells,LASS2 knockdown increased the number of invasion of bladder cancer cells.The results about chemotherapy resistance of LASS2 in bladder cancer cells showed that LASS2 overexpression reduced the cell vitality of J82 and BIU87 cells treated with doxorubicin for 24 and 48 hours.By comparision,the cell vitality was increased after LASS2 was knocked down in 5637 cells.The result from Annexin V/PI showed that the apoptosis of BIU87 and J82 cell lines was significantly increased after LASS2 overexpression and the 5637 cell apoptosis was remarkably lowered than control cells after LASS2 knockdown.The results showed that LASS2 can inhibit the invasion of bladder cancer cells and increase the sensitivity of bladder cancer cells to chemotherapeutic drugs.2.LASS2 overexpression in BIU87 and J82 cell down-regulated the expression of p-Drpl,Drpl and Fisl protein.LASS2 knockdown up-regulated the expression of p-Drp1,Drp1 and Fis1 protein.Confocal laser scanning microscopy revealed that knockdown of LASS2 in 5637 cells resulted in mitochondrial division.The cells were treated by Mdivi-1 induced mitochondrial fusion.Further validation found that Mdivi-1 reduced the number of invasive cells and up-regulated the percentage of apoptotic cells in the bladder cells.3.Our research showed that the percentage of red fluorescence decreased in LASS2 overexpressed J82 and BIU87 cells compared to the control group,which indicated decrease in mitochondrial membrane potential.However,the percentage of red fluorescence in LASS2 siRNA 5637 cells was up-regulated.These data indicated that LASS2 is a negative regulator of mitochondrial membrane potential.The results from laser scanning confocal microscope showed that mitochondria were more easily fused with elongated mitochondria in LASS2 overexpressed BIU87 and J82 cells,whereas mitochondrial division was showed in LASS2 siRNA 5637 cells.4.Our research found that LASS2 overexpression could inhibit ERK phosphorylation and LASS2 knockdown could up-regulate ERK phosphorylation.PD98059,a MEK/ERK inhibitor,down-regulated the amount of protein of p-Drp1 and p-ERK in LASS2 siRNA 5637 cells.Conclusions:1.LASS2 can not only inhibit the growth and invasion of bladder cancer cells,but also has a close relationship with the chemotherapy resistance of bladder cancer cells.LASS2 overexpression inhibited drug resistance of doxorubicin(DOX)in bladder cancer cells.2.LASS2 inhibits invasion and promotes apoptosis in bladder cancer cells by down-regulating mitochondrial/Drp1 pathway.3.LASS2 can control mitochondrial apoptosis by regulating mitochondrial membrane potential(??m),and LASS2 can induce mitochondrial fusion.The result confirms that LASS2 is involved in mitochondrial dynamic balance.4.The effect of LASS2 on bladder cancer cells may be implemented by inhibiting ERK/Drpl pathway.