The Effect Of BCL10 On The Biological Behavior Of Human Gastric Cancer Cells

Objective: to study the effects of BCL10 on cell proliferation,migration,invasion,apoptosis and cell cycle.The role of BCL10 in gastric cancer cells was preliminarily elucidated,providing theoretical experimental basis for the design of prevention and treatment strategies for gastric cancer.MethodsqRT-PCR and Western blot were used to detect the expression of BCL10 in normal human gastric epithelial cells(GES-1 cell line)and gastric cancer cells(SGC7901,BGC823 and MGC803 cell lines).After MGC803 and SGC7901 cells were transfected with lentivirus vector with BCL10 overexpression or with BCL10 knockdown by cell transfection technique respectively,MGC803 cell lines with stable BCL10 overexpression and SGC7901 cell line with stable BCL10 knockdown were constructed by purinomycin pressurized screening.Western blot was used to determine whether the transfection was successful.The biological characteristics of SGC7901 cell line with BCL10 knockdown and MGC803 cell line with BCL10 overexpression were detected by scratch assay,CCK-8 proliferation assay,Transwell migration and invasion assay,and flow cytometry.Results1.The expression of BCL10 in gastric epithelial cells and gastric cancer cells was detected by qRT-PCR and Western blot.qRT-PCR results showed that BCL10 was highly expressed in SGC7901 cells(P<0.05),while there was no significant difference in the expression of BCL10 in MGC803 and BGC823 cells,compared with GES-1 cell line.Western blot results indicated that BCL10 was more highly expressed in SGC7901 and BGC823 cells while.BCL10 was poorly expressed in MGC803 cells,respectively,compared with GES-1cell line(P<0.05).2.To construct MGC803 cell line with stable BCL10 overexpression.MGC803cells were transfected with HBLV-h-BCL10-3xflag-GFP-PURO,a lentiviral vector with BCL10 overexpression,and HBLV-GFP-PURO,an empty vector.After 24 hours,fluorescence microscopy illustrated that the number of fluorescent cells expressing GFP accounted for about 90% of the total number of cells.After 48 h,puromycin was added to screen out the total protein of cells after three generations for the detection of BCL10 expression.Western blot displayed that the expression level of BCL10 protein in MGC803 cells transfected with BCL10 overexpression vector increased significantly compared with that in the non-transfected group and the empty vector group(P<0.05),indicating that the MGC803 cell line withstable BCL10 overexpression was successfully constructed.3.Effects of overexpression of BCL10 on cell proliferation,migration,invasion,apoptosis and cell cycle in MGC803 cells.CCK-8 experiment revealed that OD value(450nm)of MGC803 cells with BCL10 overexpression at 24 h,48h and 72 h was higher than that of the non-transfected group and the empty vector group(P<0.05),suggesting that the proliferation capacity of cells was enhanced.The scratch test showed that the migration ability of MGC803 cells with BCL10 overexpression at 24 h and 48 h was stronger than that of cells in the non-transfected group and the empty vector group(P<0.05).Transwell migration and invasion experiments verified that the migration and invasion ability of MGC803 cells increased in the BCL10 overexpression group(P<0.05).The cell cycle detection results by flow cytometry displayed that the cell percentage in G0/G1 phase in BCL10 overexpression cell line was significantly lower while the cell percentage in S phase and G2/M phase was higher than that in the untransfected group and the empty vector group,which suggest that BCL10 overexpression could promote cell proliferation(P<0.05).The results of flow cytometry showed that overexpression of BCL10 inhibited apoptosis vs the untransfected group and the empty vector group(P<0.05).4.To construct SGC7901 cell line with stable BCL10 knockdown.SGC7901 cells were transfected with HBLV-h-BCL10shRNA1-GFP-PURO,HBLV-h-BCL10 shRNA2-GFP-PURO,HBLV-h-BCL10 shRNA3-GFP-PURO,which interfered with BCL10 and the control vector HBLV-h-GFP-PURO.Fluorescence microscopy suggested that the number of GFP fluorescent cells 24 h after transfected with BCL10 shRNAs accounted for about 90% of the total population of cells.After 48 h,puromycin was added to screen out the total protein of cells after three generations for the detection of BCL10 expression.Western blot verified that BCL10 protein in the BCL10 knockdown group was significantly decreased compared with that in the non-transfected group and the empty vector group(P<0.05),indicating that the stable BCL10 knockdown cell line was successfully constructed.SGC7901/HBLV-h-BCL10 shRNA3-GFP-PURO had the best interference effect,so the interference group was selected for subsequent experimental operation.5.Effects of BCL10 knockdown on cell proliferation,migration,invasion and apoptosis in SGC7901 cells.CCK-8 assay showed that the OD value(450nm)of BCL10 knockdown cells at 24 h,48h and 72 h was smaller than that of the non-transfected group and the empty vector group,suggesting that the proliferation capacity of cells was decreased(P<0.05).The scratch test revealed that the migration distance of BCL10 knockdown cells at 24 h,48h and 72 h was lower than that of cells in the non-transfected group andthe empty vector group(P<0.05).Transwell migration and invasion experiments showed that the migration and invasion ability of BCL10 knockdown cells was markedly reduced vs the non-transfected group and the empty vector group(P<0.05).Flow cytometry detection illustrated that the number of apoptosis increased after BCL10 knockdown(P<0.05).ConclusionOverexpression of BCL10 promoted proliferation,migration and invasion of gastric cancer cells and inhibited apoptosis,and down-regulation of BCL10 expression inhibited proliferation,migration and invasion of gastric cancer cells and promoted apoptosis.