| Background:Colorectal cancer(CRC)is one of the most common human digestive system malignancies.In recent years,the incidence and mortality rate of CRC have showed a clear upward trend.Tumor cells need to regulate their own energy metabolism to meet their need for rapid growth,invasion and metastasis,that is,metabolic re-programming,in which "aerobic glycolysis" is the main way of tumor glucose metabolism.Inhibition of glycolysis can significantly inhibit tumor proliferation,invasion and metastasis.6-phosphofructokinase-2/fructose-2,6-bisphosphatase(PFKFB)catalyzes synthesis reaction Fru-6-P + ATP ? Fru-2,6-BP + ADP and hydrolysis reaction Fru-2,6-BP + H2O?Fru-6-P + P.2,6-bisphosphate fructose is an allosteric activator of 6-phosphate fructokinase-1(PFK-1)and the most potent stimulant for glycolysis.PFKFB family are widely expressed in a variety of biological cells,and they regulate the level of intracellular glycolysis by regulating the expression of 2,6-bisphosphate fructose(F-2,6-BP).PFKFB family are currently known to have 4 kinds of isoenzyme,which are PFKFB1,PFKFB2,PFKFB3 and PFKFB4.These isozymes are encoded by four different genes PFKFB1-4,which are located on different chromosomes.The expression level and activity of these enzymes are regulated by hormones and metabolites.All isozymes contain highly conserved core structures of kinase and phosphatase activity.Previous studies have shown that hypoxia-inducible factor-1?(HIF-1?)can induce the expression of PFKFB in tumor,and PFKFB can play a role in carcinogenic factors,but its expression in colorectal cancer and its relationship with the prognosis of patients is not yet clear.Therefore,our study will examine the expression of PFKFB in colorectal cancer and adjacent tissues,evaluate the correlation between PFKFB and HIF-1,and explore its relationship with clinicopathological features and prognosis.The most important prognostic gene will be chosen to be our target gene,and we will observe the changes in glucose metabolism,proliferation and invasion and metastasis of colorectal cancer cells in vitro after knockdown the target gene.These may provide new strategies and ideas on prognost ic indicators and glucose metabolism targeted therapy for colorectal cancer..Objective:To investigate the expression level of 6-phosphate fructose-2-kinase(PFKFB)in CRC and adjacent tissues,and to explore its clinical significance and its effect on the glucose metabolism,proliferation,invasion and metastasis of CRC cells in vitro.Methods: 90 cases of CRC tissues and corresponding 90 cases of adjacent tissues which abtained from the Southwest Hospital of the Third Military Medical University,and normal colonic epithelial cell lines(FHC,CCD841)and CRC cell lines(Lovo,HT-29,HCT-116,RKO,SW620,SW480)were used as research objects.This research was divided into three parts:The first part:To investigate the relationship between the expression level of PFKFB1,PFKFB2,PFKFB3,PFKFB4 and HIF-1? in CRC and the relationship between the expression of PFKFB and HIF-1? in CRC tissues:(1)Using tissue microarray combined with immunohistochemistry to investigate the expression level of PFKFB1,PFKFB2,PFKFB3,PFKFB4 and HIF1-? in 90 cases of colorectal cancer tissues and corresponding 90 cases of adjacent tissues from the Southwest Hospital of the Third Military Medical University.(2)Evaluate the relationship between the expression of PFKFB3 protein and the clinical prognosis of CRC.(3)Detect the correlation between the expression of PFKFB protein and the expression of HIF-1? protein.All statistical analysis using SPSS 19.0 statistical software,if P <0.05,the results were considered that they have statistically significant differences.The second part:Establish a stable cell line with low expression of PFKFB3,and detect the effect of knockdown PFKFB3 on cellular glucose metabolism.(1)Detect the expression of PFKFB3 in normal colonic epithelial cell line s(FHC,CCD841)and CRC cell lines(Lovo,HT-29,HCT-116,RKO,SW620,SW480):PFKFB3 mRNA expression level in the above cell lines were detected by Real-time quantitative PCR,and Western Blot was used to detect PFKFB3 protein expression.(2)Transfect the CRC lines with lentivirus to knockdown the expression level of PFKFB3.Cells which transfected with no lentivirus were used as wild type group,with blank control lentivirus were control group,and with knowdown PFKFB3 gene lentivirus were PFKFB3 low expression group.And the PFKFB3 low expression stable CRC cell lines were identified by Western blot and Real-time quantitative PCR.(3)To explore the effect of PFKFB3 on glucose metabolism in CRC cells,the amount of glucose and lactic acid in the supernatant of CRC cells were detected in the control group and PFKFB3 low expression group.The third part: Detect the impact of PFKFB3 on proliferation,invasion and metastasis in colorectal cancer cells in vitro:(1)The CCK8 kit test and cell clone formation test were us ed to detect the proliferation activity of CRC cells in control group and PFKFB3 low expression group.(2)Transwell assay and invasion experiment were used to detect the ability of invasion and metastasis of colorectal cancer cells in control group and PFKFB3 low expression group.Results:The first part:(1)The number of high expression cases of PFKFB1,PFKFB2,PFKFB3,PFKFB4 and HIF1-? in 90 cases of CRC were 57,59,59,51 and 60 respectively and the expression level was obviously higher than their adjacent tissue(P<0.05).(2)The high expression of PFKFB3 in CRC was closely related to tumor size,clinical stage,lymph node metastasis and prognosis(P<0.05).The prognosis of high expression group was worse than that of low expression group(P = 0.008).The high expression of PFKFB3 showed poor clinical outcome in CRC patients with high clinical stage(P= 0.009),but not in patients with low clinical stage(P= 0.490).(3)The expression of PFKFB3 was correlated with the expression of HIF-1?(r = 0.627,P <0.001).(4)The expression of PFKFB1,PFKFB2 and PFKFB4 had no significant correlation with tumor size,clinical stage,lymph node metastasis and prognosi s of CRC(P>0.05).According to the results,PFKFB3 was selected as the target gene for the following experiment.The second part:(1)The expression level of PFKFB3 protein in different colon cell lines was different.The PCR results showed that compared with normal colonic epithelial cell line(FHC,CCD841),the expression of PFKFB3 m RNA was low in low invasive CRC cell lines RKO and Lovo,but the expression was high in high invasive CRC cell lines HT-29,HCT-116,SW620 and SW480.The Western Blot results showed that the expression level of PFKFB3 protein was low in CRC cell lines RKO and Lovo compared with normal colonic epithelial cell lines(FHC,CCD841),but was high in HT-29,HCT-116,SW620 and SW480,which were consistent with the PCR results.(2)SW480 and SW620 CRC cell lines were chosen to be transfected with lentivirus which can know down the expression of PFKFB3,and the PFKFB3 low expression stable cell lines were identified by drug screening,Real-time quantitative PCR and Western blot.(3)SW480 and SW620 cells had a change in the ability of cell glycolysis after knocking down PFKFB3.Compared with the control group,the amount of glucose and lactic acid in the supernatant of PFKFB3 low expression group significantly decreased,and the difference was statistically significant(P<0.05).The third part:(1)By CCK8 kit test and cell clone formation test,the results prove that after knocking down PFKFB3,the cell proliferation of SW480 and SW620 cells lines changed:the cell proliferation state of SW480 and SW620 cells was significantly lower than that of the control group(P<0.05).(2)By transwell test and cell invasion experiment,the results prove that after knocking down PFKFB3,the invasion and metastasis ability of SW480 and SW620 cell lines changed:compared with the control group,the ability of invasion and metastasis of PFKFB3 cells significantly decreased(P<0.05). |
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