The Role Of KLF13/ACOT7 Signaling Pathway In The Prolifetation And Migration Of Hepatoma Cells

Objective:Hepatocellular carcinoma has abnormal lipid metabolism.Acyl CoA thioesterase 7(ACOT7)plays an important role in fatty acid metabolism.This study will clarify the effects of ACOT7 on the proliferation,migration and fatty acid production of hepatoma cells and the mechanism of Krüppel like factor 13(KLF13)regulating AC OT7.Methods:The expression levels of ACOT7 and KLF13 mRNA were obtained from the Cancer Genome Atlas(TCGA)database.The mRNA expression was detected by real-time quantitative PCR,and the protein expression was detected by immunohistochemistry and Western blotting.CCK-8 and EdU were used to evaluate the proliferative capacity of HCC cells.The migration ability of HCC cells was detected by wound-healing assay and Transwell assay.Flow cell cycle was used to detect the effect of ACOT7 on cell cycle.The subcutaneous tumorigenesis experiment in nude mice was used to evaluate the effect of ACOT7 on the proliferation of hepatoma cells in vivo.The effect of ACOT7 on the content of free fatty acids in hepatoma cells was analyzed by gas chromatography-mass spectrometry.JASPAR database was used to analyze the potential binding sequence of KLF13 and ACOT7.The transcriptional regulation of ACOT7 by KLF13 was verified by luciferase reporter experiment and Chip-qPCR.Results:The expression of ACOT7 was up-regulated in HCC,and ACOT7 was a risk factor for the survival of HCC patients.Overexpression of ACOT7 in HCC cells enhanced cell proliferation,migration,invasion and G1/S phase transition,while knockdown of ACOT7 inhibited cell proliferation,migration,invasion and G1/S phase transition.Meanwhile,overexpression of ACOT7 enhanced the expression of EMT related proteins in hepatoma cells.Moreover,overexpression of ACOT7 enhanced the proliferation of hepatoma cells in nude mice.In addition,ACOT7 overexpression mainly increased the production of monounsaturated fatty acid oleic acid(C18:1),and oleic acid also enhanced the proliferation and migration of hepatoma cells.In mechanism,ACOT7 was transcriptionally activated by KLF13.Conclusion:ACOT7 is transcriptionally activated by KLF13 to promote the progression of liver cancer.Part I:the expression and clinical significance of ACOT7 in hepatocellular carcinomaObjective:The aim is to investigate the expression and clinical significance of acyl CoA thioesterase 7(ACOT7)in hepatocellular carcinoma.Methods:The ACOT7 mRNA differential expression was analyzed between normal and primary HCC tissues in UALCAN website(http://ualcan.path.uab.edu/)based on The Cancer Genome Atlas(TCGA)database,and the expression of ACOT7 for HCC survival was analyzed.The expression of ACOT7 in liver cancer tissues was detected by quantitative Real-time PCR(qRT-PCR),western blotting and immunohistochemistry.Results:The expression level of ACOT7 mRNA was collected in TCGA database.It was found that the expression of ACOT7 mRNA in liver cancer was significantly higher than that in normal tissues,and the high expression of ACOT7 mRNA was a risk factor for poor survival of liver cancer patients.The expression of ACOT7 mRNA in 26 liver cancer patients was detected by qRT-PCR.It was found that the expression of ACOT7 mRNA in 18 patients’ cancer tissues was higher than that in adjacent tissues.Western blotting also confirmed that the expression of ACOT7 in cancer tissues was higher than that in adjacent tissues in 7 of the 8 liver cancer patients.The expression of ACOT7 in cancer tissues and adjacent noncancerous tissues of 20 liver cancer patients was detected by immunohistochemistry,and the cancer tissues of 13 patients showed high expression.Conclusions:ACOT7 is highly expressed in hepatocellular carcinoma.The high expression of ACOT7 is a risk factor for the survival of hepatocellular carcinoma patients and ACOT7 can be used as an important molecular marker to predict the survival for hepatocellular carcinoma.Part Ⅱ:ACOT7 promotes the proliferation,migration and invasion of liver cancer cells and the production of monounsaturated fatty acidObjective:The aim is to investigate the effect of ACOT7 on proliferation,migration and invasion for hepatoma cells and the role of ACOT7 in fatty acid metabolism in hepatoma cells.Methods:We constructed overexpressing ACOT7 lentivirus and ACOT7 knockdown plasmids sh#1 and sh#2.Two hepatoma cell lines with overexpression of ACOT7 and low expression of ACOT7 were screened by qRT-PCR and Western blotting.Overexpressing ACOT7 lentivirus infected low expressing ACOT7 cell line,and ACOT7 knockdown plasmid was transfected into high expressing ACOT7 cell line.In vitro,experiments such as CCK-8,EdU,wound-healing assay,Transwell and flow cell cycle were used to verify the effect of ACOT7 on the proliferation,migration,invasion and cycle of hepatoma cells.Western blotting was used to detect the effect of ACOT7 on the expression of CyclinDl,CDK2 and CDK4 in hepatocellular carcinoma cells.The expression of N-cadherin、E-cadherin、vimentin were detected by Western blotting.The effect of ACOT7 on the proliferation of hepatoma cells in vivo was verified by subcutaneous tumor formation model in nude mice,and the expression of cell proliferation indexes Ki-67 and PCNA was detected in subcutaneous tumor of nude mice.The effect of ACOT7 on fatty acid production of liver cancer cells was detected by gas chromatography combined with mass spectrometry.Results:The protein expression of ACOT7 in hepatoma cell lines infected with ACOT7 overexpression lentivirus was significantly higher than that in the control group.The protein expression of ACOT7 in hepatoma cell lines transfected with ACOT7 knockdown plasmid sh#1 and sh#2 was significantly lower than that in the control group.Compared with the control group,overexpression of ACOT7 significantly enhanced the proliferation,migration and invasion of hepatoma cells.Compared with the control group,knockdown of ACOT7 significantly inhibited the proliferation,migration and invasion of hepatoma cells.Western blotting showed that overexpression of ACOT7 significantly increased the expression of N-cadherin and vimentin and decreased the expression of E-cadherin.On the other hand,knockdown of ACOT7 significantly decreased the expression of N-cadherin and vimentin and increased the expression of E-cadherin.Overexpression of ACOT7 significantly increased the expression of CyclinDl and CDK2,while knockdown of ACOT7 significantly inhibited the expression of CyclinDl and CDK2.Flow cytometry showed that overexpression of ACOT7 promoted G1/S transition and knockdown of ACOT7 inhibited G1/S transition.For overexpression and knockdown of ACOT7 in hepatoma cell lines,it was found that ACOT7 mainly increased the content of C18:1(oleic acid)by gas chromatography combined with mass spectrometry.C18:1 enhanced the proliferation and migration of hepatoma cells.Conclusions:1.ACOT7 can promote the progression of liver cancer and increase the content of monounsaturated fatty acid C18:1 in liver cancer cells.2.Monounsaturated fatty acid C18:1 enhances the malignant ability of liver cancer cells.Part Ⅲ:ACOT7 is transcriptionally activated by KLF13Objective:The aim is to explore the regulatory mechanism of KLF13 for ACOT7.Methods:The expression level of KLF13 in hepatocellular carcinoma was analyzed through TCGA database,and meanwhile,the expression of KLF13 in hepatocellular carcinoma was detected by Western blotting.The expression of KLF13 mRNA in hepatocellular carcinoma was detected by qRT-PCR,and the correlation between KLF13 mRNA and ACOT7 mRNA was analyzed.Overexpression KLF13 lentivirus was constructed to infect hepatoma cells,and the effect of KLF13 on the expression of ACOT7 mRNA and protein was detected.The sequence sites of KLF13 binding to ACOT7 promoter were predicted by bioinformatics website JASPAR database.Chip-qPCR was used to verify the combination between KLF13 and ACOT7.KLF13 control plasmid,KLF13 overexpression plasmid,wild-type ACOT7 promoter firefly luciferase plasmid,mutant ACOT7 promoter firefly luciferase plasmid and renilla luciferase plasmid were constructed and co-transfected into 293T cells:(1)wild-type ACOT7 promoter firefly luciferase plasmid+KLF13 control plasmid+renilla luciferase plasmid.(2)Wild type ACOT7 promoter firefly luciferase plasmid+KLF13 overexpression plasmid+renilla luciferase plasmid.(3)Mutant ACOT7 promoter firefly luciferase plasmid+KLF13 control plasmid+renilla luciferase plasmid;(4)Mutant ACOT7 promoter firefly luciferase plasmid+KLF13 overexpression plasmid+renilla luciferase plasmid.The luciferase activities of firefly and renilla were detected by multifunctional enzyme labeling instrument,and the ratio of firefly/renilla signal in each hole was calculated to determine the regulation of KLF13 on ACOT7 promoter.ACOT7 interference shRNA was transfected into KLF13 overexpressed hepatoma cell line.The recovery experiment was carried out by CCK-8,wound-healing assay and Transwell assay to further verify that KLF13 regulates ACOT7.Results:TCGA database showed that the expression of KLF13 mRNA in liver cancer tissues was significantly higher than that in normal tissues.Western blotting also showed that the expression of KLF13 in 6 of 8 cancer tissues was higher than that in adjacent tissues.KLF13 mRNA was positively correlated with ACOT7 mRNA expression in 26 patients.Overexpression of KLF13 in hepatoma cells increased the expression of ACOT7 mRNA and protein,and knockdown of KLF13 also decreased the expression of ACOT7 protein.The results of luciferase reporter experiment showed that compared with the control group,the luciferase activity of wild-type ACOT7 promoter co-transfected with KLF13 overexpression plasmid was increased,while the luciferase activity of mutant ACOT7 promoter group did not change significantly.Chip-qPCR showed that overexpression of KLF13 enhanced the enrichment of ACOT7 compared with the control.ACOT7 interference RNA can reverse the effect that KLF13 promoted the proliferation and migration of hepatoma cells.Conclusions:ACOT7 is transcriptionally activated by KLF13.