| Objective:To observe the effect of altered expression of NDUFA4 on the growth of human colorectal cancer(CRC)cells;To explore the possible molecular mechanism of the effect of NDUFA4 on the growth of human CRC cells;To analyze the expression and its clinical significance of NDUFA4 in clinical CRC patients.Methods:1.Real time PCR and Western Blot were used to detect the expression of NDUFA4 in normal human colonic epithelial cells and colorectal cancer cell lines.Immunofluorescence were used to verify the expression of NDUFA4 in human colorectal cancer SW620 cells and SW480 cells.The study is divided into four groups,including p-NDUFA4 group,p-Cont group,sh-NDUFA4 group and sh-NC group.Previously constructed NDUFA4 overexpression plasmid and p-Cont control plasmid,sh-NDUFA4-plasmid and sh-NC plasmid,were transiently transfected into SW620 and SW480 cells in vitro using lipofectamine 3000,respectively.Real time PCR and Western Blot were used to detect the expression of NDUFA4 in cells.CCK-8 assay and clone formation assay were performed to analyze cell proliferation and colony formation ability.Flow cytometry was used to detect the cell cycle.Western Blot was used to detect the expression of AKT,ERK1/2,p-AKT,p-ERK1/2,p53,p-p53,c-Myc and the proliferation proteins Cyclin D1,Cyclin E1,CyclinA2,CDK4,CDK2,and p-Rb.Detection of Ki-67 expression,TUNEL in situ apoptosis,mitochondrial membrane potential change and mitochondrial ROS production by immunofluorescence,respectively.The colorimetric and chemiluminescence were used to detect the NAD/NADH ratio and ATP level changes.2.NDUFA4 overexpression plasmid and p-Cont control plasmid are transiently transfected into SW620 cells in vitro using lipofectamine 3000 nanoliposomes.Co-IP analysis and mass spectrometry were performed to analyze the interaction between the target protein and NDUFA4.The expression of LRPPRC was detected by Western Blot.Immunofluorescence staining was used to detect the colocalization of NDUFA4 and LRPPRC in human CRC cells.Real time PCR and Western Blot wereused to detect the expression of NDUFA4 in SW620 and SW480 cells.The proliferation and colony formation ability of SW620 and SW480 cells were detected by CCK-8 assay and clone formation assay.The chemiluminescence was used to detect the ATP level changes.Western Blot was used to detect the expression of AKT,ERK1/2,p-AKT,p-ERK1/2,p53,p-p53,c-Myc and the proliferation proteins Cyclin D1,CyclinA2,Cyclin E1,CDK4,and CDK2,as well as p-Rb,respectively.3.Immunohistochemical method was used to detect the expression of NDUFA4 in TMA cancer tissues and normal tissues of human colorectal cancer.According to the histochemical score of NDUFA4 expression,patients were divided into two groups: NDUFA4 high expression group and NDUFA4 low expression group.Chi-square test was used to analyze the correlation between the expression of non-invasive growth factor receptor 4 and its clinical significance.Kaplan-Meier method was used to analyze the overall survival rate of the two groups of patients.Immunohistochemistry was used to detect the expression of LRPPRC in human CRC TMA cancer and normal tissues.Pearson method was used to analyze of the clinical correlation between LRPPRC and NDUFA4.Results:1.Real time PCR and Western blot data showed that the level of NDUFA4 increased significantly in human colorectal cancer cell lines compared with FHC cells(P<0.05).Especially,among of these CRC cells,SW480 cells expressed highest level of NDUFA4.Immunofluorescence staining results further showed that compared with SW620 cells,SW480 cells expressed higher level of NDUFA4.Real time PCR and Western Blot showed that compared with the p-Cont group,the expression level of NDUFA4 in SW620 cells in p-NDUFA4 group increased significantly(P<0.05);Conversely,the expression level of NDUFA4 in SW480 cells in sh-NDUFA4 group was significantly down-regulated(P<0.05).CCK-8 and colony formation experiment data showed that compared with the p-Cont group,the proliferation and colony formation ability of SW620 cells in the p-NDUFA4 group was significantly accelerated(P<0.05);Conversely,the proliferation and clone formation ability of SW480 cells in sh-NDUFA4 group was decreased compared with that in sh-NC group(P<0.05).The results of cell cycle showed that the ratio of G0/G1 phase decreased(P<0.05),the proportion of S phase increased(P<0.05),and the proportion of G2/Mphase decreased(P<0.05)in SW620 cells of p-NDUFA4 group.But in SW480 cells,the ratio of G0/G1 phase in sh-NDUFA4 group increased significantly(P<0.05).The S phase ratio of cells decreased(P<0.05)and G2/M phase ratio decreased(P<0.05).Western Blot data showed that in SW620 cells,compared with p-Cont group,the expression levels of p-AKT,p-ERK1/2 and c-Myc in p-NDUFA4 group were significantly up-regulated(P<0.05),and the expressions of p53 and p-p53 were significantly down-regulated(P<0.05),In addition,the expression levels of growth and related proteins CDK4,CDK2,Cyclin D1,Cyclin E1,Cyclin A2 and p-Rb in the p-NDUFA4 group were higher than that in the p-Cont group(P<0.05).However,the expression levels of AKT and ERK1/2 had no significant difference(P>0.05).In SW480 cells,compared with sh-NC group,the expression levels of p53 and p-p53 in sh-NDUFA4 group were significantly up-regulated(P<0.05),and the expressions of p-AKT,p-ERK1/2 and c-Myc,as well as Cyclin D1,Cyclin E1,Cyclin A2,p-RB,CDK2 and CDK4 were significantly down-regulated(P<0.05).However,the expression levels of AKT and ERK1/2 had no significant difference(P>0.05).Immunofluorescence data showed that compared with p-Cont group,the expression of Ki-67 in SW620 cells of p-NDUFA4 group was significantly increased(P<0.05),while apoptosis was significantly reduced(P<0.05).The red fluorescence of mitochondrial membrane potential JC-1 increased significantly,and the production of mitochondrial reactive oxygen species decreased significantly.On the contrary,in SW480 cells,the expression of Ki-67 in sh-NDUFA4 group was significantly down-regulated,while the apoptosis was significantly increased.Mitochondrial membrane potential JC-1 enhances green fluorescence,and mitochondrial reactive oxygen species production is significantly up-regulated.The colorimetric method result showed that compared with the p-Cont group,the NAD / NADH ratio of SW620 cells in the p-NDUFA4 group reduced significantly(P<0.05),while the NAD/NADH ratio of SW480 cells in sh-NDUFA4 group increased obviously(P<0.05).The chemiluminescence method result showed that compared with the p-Cont group,the ATP expression of SW620 cells in the p-NDUFA4 group was increased(P<0.05),while the ATP expression of SW480 cells in sh-NDUFA4 group significantly decreased(P <0.05).2.CO-IP and mass spectrometry data showed that NDUFA4 could bind to LRPPRC.Western Blot showed that LRPPRC was expressed in SW620 cells.Immunofluorescence data showed that LRPPRC and NDUFA4 were co-localized inmitochondria of SW620 cells and SW480 cells.Reverse CO-IP data further showed that in SW620 cells,LRPPRC could bind to NDUFA4.Real time PCR and Western Blot data showed that compared with the p-NDUFA4 group,the expression level of LRPPRC was significantly down-regulated(P<0.05).The experimental data of CCK-8 and colony formation showed that compared with the p-NDUFA4 group,the proliferation and colony formation ability of SW620 cells and SW480 cells in the p-NDUFA4-si-LRPPRC group were significantly weakened(P<0.05).The chemiluminescence method data showed that compared with the p-NDUFA4 group,the ATP expression of SW620 cells and SW480 cells in the p-NDUFA4-si-LRPPRC group decreased significantly(P<0.05).Western Blot data showed that in SW620 cells and SW480 cells,Compared with the control group,the expression level of p-AKT was significantly decreased,p-ERK1/2 and c-Myc in p-NDUFA4 group were significantly downregrulated(P<0.05)and the expression levels of p53 and p-p53 was obviously upregulated(P<0.05).In addition,the expression level of growth related proteins including CDK4,CDK2,CyclinD1,CyclinE1,CyclinA2,and p-Rb in p-NDUFA4-si-LRPPRC group were obviously lower than those in p-NDUFA4 group,respectively(P<0.05).However,there was no significant difference in the expression levels of AKT and ERK1/2(P>0.05).3.The TMA data of human colorectal cancer showed that NDUFA4 was expressed in 93 cancer tissues and 87 normal tissues.The expression level of NDUFA4 was significant higher in cancer tissues than in adjacent normal tissues(P<0.05).Based on the H-Score of NDUFA4,patients were divided into NDUFA4 high expression group and NDUFA4 low expression group.Further analysis shows that there is no significant difference between the two groups regarding Age,sex,histological differentiation,pathological type,tumor size,TNM stage and clinical stage respectively(P>0.05).Cox regression analysis showed that histological grade and Clinical staging is an independent risk factor affecting patients' survival(P<0.05).Kaplan-Meier analysis showed that there was no significant difference in the overall survival rate between the two groups(P>0.05).TMA data of human colorectal cancer showed that the expression level of LRPPRC was significant higher in cancer tissues than in adjacent normal tissues(P<0.01).Pearson analysis showed that NDUFA4 was positively correlated with LRPPRC expression.Conclusions:1.Up-regulation of NDUFA4 expression promotes cell proliferation and mitochondrial energy metabolism of human colorectal cancer SW620 cells in vitro,While,Down-regulation NDUFA4 expression inhibits the growth and mitochondrial energy metabolism of human colorectal cancer SW480 cells growth in vitro.2.LRPPRC is a target molecule that interacts with NDUFA4.Down-regulating LRPPRC reverses the effect of NDUFA4 on the growth of human colorectal cancer cells.3.NDUFA4 is highly expressed in human colorectal cancer specimens,and its high expression has no significant correlation with clinical index and overall survival time,but its high expression is positively correlated with LRPPRC expression. |
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