Investigation Into The Molecular Mechanism Of FABP4 In Colorectal Cancer

Part One FABP4 Expression in Colorectal Cancer Tissues and CellsObjective:The aim of this study was to detect the expression of FABP4 in colorectal cancer(CRC)tissues and cells,as well as the relationship with clinicopathological features and to explore potential effect of FABP4 on the occurance and progression of CRC.Methods:1.After being approved by the Ethics Committee of The First Hospital of Hebei Medical University,66 matched pairs of human CRC tissues and adjacent normal tissues(over 5cm from the edge of tumor tissues)were collected from July 2018 to May 2019 with permission of the Ethics Committee of the First Hospital of Hebei Medical University.All patients were free of any therapeutic intervention and signed an informed consent form before the procedure,and all cancer tissues were verified to be either colorectal adenocarcinoma or mucinous adenocarcinoma histologically.2.HT29,SW480 and NCM460 cell lines were provided by the central laboratory of the First Hospital of Hebei Medical University.3.Total RNA was extracted from tissue samples and CRC cells by Trizol reagent,and reverse transcriptional synthesis was carried out to obtain c DNA.Expression of FABP4 in colorectal cancer tissues and adjacent normal tissues were tested by RT-q PCR.Results:1.FABP4 expression of CRC tissues was significantly higher than that in paired adjacent normal tissues in our cohort [0.0019(0.0005,0.0068)vs.0.0006(0.0001,0.0020),P<0.001].2.The m RNA expression of FABP4 was higher in clinical stage Ⅲ-Ⅳcancer tissues compared to stage Ⅰ-Ⅱ cancer tissues in CRC patients(P<0.001).The m RNA expression of FABP4 was higher in T3+T4 stage cancer tissues compared to T1+T2 cancer tissues in CRC patients(P<0.05).The m RNA expression of FABP4 was higher in cancer tissues with lymph node metastasis compared to those without(P<0.05).The m RNA expression of FABP4 was higher in cancer tissues with distant metastasis compared to those without(P<0.05).3.FABP4 m RNA expression of CRC cells(HT29 and SW480)was higher than that in normal colon epithelial cell lines(NCM460)(P<0.001).Conclusions:FABP4 was overexpressed in colorectal cancer and is associated with the depth of invasion,lymph node metastasis,distant metastasis,and clinical TNM staging of colorectal cancer.Part Two FABP4 Functions in Human Colorectal Cancer cellsObjective:We aim to investigate the potential biological effects of FABP4 by knockdown FABP4 in CRC cells in vitro.Methods:1.To deplete FABP4 expression,si RNA specifically targeting FABP4(si-FABP4)sequence was transfected into HT29 and SW480 cells,with si-NC as a negative control.Lipofectamine 3000 Reagent was applied for transient transfection in cells.The transfection efficiency was verified by RT-q PCR.2.The cell proliferation ability of each group was detected by CCK-8 kit and Ed U kit.3.Sphere formation assay was used to compare the effect of FABP4 knockdown on the stemness of CRC cells.4.The flow cytometry was used to compare the effect of FABP4 knockdown on the apoptosis of CRC cells.5.The expression of proliferation-associated protein(PCNA),stemness markers(Sox2,Oct4,and ALDHA1),and apoptosis-correlated proteins(Bax and Bcl-2)was assessed via performing western blot.Results:1.Compared with the negative control group,depletion of FABP4 restrained cell proliferation of SW480 and HT29 cells by CCK-8 kit and Ed U experiments.2.The number of spheres was memorably lessened by knocking down FABP4 in HT29 and SW480 cells.3.Apoptosis of HT29 and SW480 cells was dramatically boosted after FABP4 depletion by flow cytometry.4.FABP4 depletion remarkably decrease PCNA and Bcl-2 expression,but increase Bax expression.The level of stemness markers,including Sox2,Oct4 or ALDHA1,was all reduced by FABP4 knockdown in SW480 and HT29 cells.Conclusions:FABP4 played a pro-carcinogenic role in colorectal cancer,which included promotion of cell proliferation,migration and stemness,as well as suppression of apoptosis.Part Three Molecular Mechanism of FABP4 in Human Colorectal Cancer cellsObjective:The purpose of this part of the study was to explore the potential molecular mechanism of FABP4 in CRC cells.Methods:1.The influence of glycolysis after knocking down FABP4 by ECAR,lactate production,glucose uptake,and ATP/ADP ration in colorectal cancer cells.And glycolysis-related proteins(LDHA and Glut1)were evaluated via performing western blot.2.The flow cytometry was used to compare the effect of FABP4 knockdown on ROS level in CRC cells,and ROS level after used NAC,a ROS scavenger.3.The effects of FABP4 knockdown on the proliferation,stemness,cell apoptosis and glycolysis by NAC treatment in SW480 and HT29 cells was analyzed.4.The expression of the ERK/mTOR signaling pathway-associated protein(p-ERK,ERK,p-mTOR and mTOR)was assessed via performing western blot.5.The nude mice were subcutaneously inoculated with HT29 cells stably transfected with sh-FABP4 or sh-NC,tumor size of the murine xenograft model was measured.Results:1.FABP4 downregulation could markedly repress ECAR in HT29 and SW480 cells.Furthermore,FABP4 downregulation obviously reduced lactate production,glucose uptake,and ATP/ADP ration in colorectal cancer cells.Knockdown of FABP4 dramatically lowered the protein level of Glut1 and LDHA in colorectal cancer cells.2.ROS level was obviously boosted by downregulating FABP4 in both HT29 and SW480 cells,which was restored by NAC,a ROS scavenger.NAC could reverse the effect of FABP4 depletion on ROS level in colorectal cancer cells.3.The repressive impacts of FABP4 knockdown on cell proliferation,stemness,and the promotion impact on cell apoptosis were reversed by NAC treatment in HT29 and SW480 cells,which were further confirmed through western blot,as evidenced by enhanced PCNA and Bcl-2 expression,weakened Bax expression,and upregulated Sox2,Oct4,and ALDHA1 expression in SW480 and HT29 cells.4.FABP4 knockdown-caused inhibition effect on glycolysis was retarded by NAC treatment,as proved by increasing ECAR,acceleration of lactate production,glucose uptake and ATP/ADP ration,as well as upregulated expression of Glut1 and LDHA in HT29 and SW480 cells.5.FABP4 depletion markedly increase p-ERK expression,but decrease p-mTOR expression,suggesting FABP4 depletion could activate the ERK/mTOR signaling pathway.The ERK/mTOR pathway activation induced by FABP4 knockdown was inhibited upon treatment with NAC.6.In the murine xenograft model,the volume of tumors was obviously suppressed by FABP4 depletion.Moreover,the protein expression of FABP4 was markedly downregulated by knocking down FABP4 in the nude mice.Conclusions:FABP4 could function as a pro-carcinogen via the ROS/ERK/mTOR pathway,which included the promotion of cell proliferation,stemness and glycolysis,as well as inhibition of apoptosis in CRC cells.