| Background and purposeMetastasis is the main cause of death in clinical colorectal cancer patients,it already has 20%?40%of cases occur distant metastasis when diagnosed as colorectal cancer,and the recurrence of liver metastatic rate is about 50%after the radical cure;hepatic metastasis rate in colorectal cancer death patients is as high as 45%?71%[1].Therefore,strengthening basic research on colorectal cancer development mechanism,clarifying the molecular mechanism of colorectal cancer metastasis,colorectal cancer metastasis forecast,diagnosis and prognosis judgement is the frontier subject of colorectal cancer research,it has important scientific significance such as to increase colorectal cancer cure rate and reduce mortality.This earlier experiments found that miR-223 promote colorectal cancer metastasis through its downstream target genes FBX08,FBXO8's expression decreases in colorectal cancer tissues,but the invasion and metastasis mechanism of FBX08 gene is unclear.FBX08 is a new member of the F-box protein family belongs to the ubiquitin-proteasome system,the system can mediate in most eukaryotic cells regulating the degradation of proteins,and plays its important role in maintaining the intracellular calcium homeostasis,the process of regulation of cell proliferation,differentiation,cycle and apoptosis[2].Ubiquitin for protein post-translational modification mediated by three enzyme:E1 activated enzyme,E2 crosslinked enzyme and E3 ligase.F-box protein family is characterized with about 40 amino acids F-box sequence,F-box family play its role as subunits in the ubiquitin E3 ligase(Skpl/Cullin/F-box)SCF protein complexes,F-box protein determines the substrate specificity of ubiquitin mainly through combined with substrate directly[2].There are only three articles which reported FBX08's role in tumor.FBX08 inhibits invasion ability mediated by ARF6 in breast cancer cells[3]FBX08 were found to be a new c-Myc binding protein,c-Myc improves the ability of cancer cells invasion by inhibiting FBX08 related functions,[4].Our research has found that FBX08 inhibits the ability of invasion and metastasis of liver cancer cells and associated with the low survival rate of liver cancer patients[5].All above suggest FBX08 is closely·related to the evolution of the tumor.Our topic selected FBX08 as targets to further explore FBX08's function and related mechanism of invasion and metastasis in colorectal cancer,the main contents are as follows:(1)FBX08's role in invasion and metastasis of colorectal cancer;(2)Reveal the new mechanism FBX08 involved in invasion and metastasis of colorectal cancer by Ubiquitin degradating mTOR.This topic aims to expound the molecular mechanism of colorectal cancer metastasis and invasion,to provide a new target gene for clinical resistance to colorectal cancer's invasion and metastasis.Methods1.FBX08's effect on colorectal cancer cell biology behavior(1)Designed and builded FBX08 virus overexpressional vector,packaged the virus supernatant conducted by company to infect colorectal cancer cell lines HT29 and SW480,builded stable overexpression cell lines after the flow separation,used fluorescence quantitative PCR and Western blot to test FBX08 protein expressional change.(2)Used the method of cationic liposomes to transiently transfect colorectal cancer cell line SW620 with three commercial FBX08 SiRNAs fragment,used fluorescence quantitative PCR and Western blot to test FBXO8 expression change and got Sil and Si3 efficient interference fragment.Designed and builded the Si3 FBX08 slow virus interference carrier,virus supernatant packaged by company,infected colorectal cancer cell line SW620,builded stable expression cell lines after flow separation technology sorting and used fluorescence quantitative PCR and Western blot to test FBX08 protein expression change.(3)Used MTT colorimetric method,plate clone formation experiment,cell cycle,the invasion experiment in vitro,tested the effect after overexpression and inhibition of FBX08 on proliferation and invasive ability in vitro of colorectal cancer cell lines.(4)Used of the overall visual animal models to observe the transfer ability by nude mice subcutaneously into tumor and the tail intravenous injection after FBX08 overexpression and interference.2.The discussion about invasion and metastasis mechanism of FBX08 in colorectal cancer(1)Used Western blot experiment to observe the ubiquitin degradation for FBX08 targeted mTOR.(2)Used confocal and CO-IP to detect FBX08 interaction with mTOR and builded truncated gene to find the mutual binding site.(3)Used MTT colorimetric method and the invasion experiment in vitro to test FBX08 regulation of invasion and metastasis through ubiquitin degradation mTOR in colorectal cancer.Results1.FBX08's effect on cell biology behavior of colorectal cancer cell1.1 Established FBX08 stable overexpression and interference cell lines(1)After stable overexpression of FBX08,used the fluorescent quantitative PCR to detect FBX08 expression quantity change in colorectal cancer cell lines HT29 and SW480,Mock group for reference(mean ± SD:1 ±0.000),the independent sample T test analysis showed that FBX08 has significant differences between Mock and overexpression groups in the two cell lines.Pairwise comparison showed FBXO8 expression in SW480 and HT29 cell is higher than the other group,and each group is significantly higher than the Mock set expression(P<0.001,P<0.001).(2)After FBXO8 instantaneous interference,used the fluorescent quantitative PCR to detect FBXO8 expression quantity change in SW620 cell lines,used NC group as control(mean ± SD:1 ± 0.000),one-way analysis of variance result showed that FBXO8 expression has significant difference in group 3 and group 1 compared with NC group(mean = 0.141,mean = 0.141)and has no significant difference between group 2 and NC group(mean = 0.73),and the NC group is significantly higher than interference group 1 and group 3(F = 10323.340,P<0.001).Elected the interference fragment 3,after stable FBBO8 interference,used fluorescent quantitative PCR to detect FBXO8 expression in SW620 cell lines,NC group as control(mean ±SD:1 ±0.000),the independent sample T test analysis showed that FBXO8 has a significant difference between the interference group 3 and NC group,and NC group expression is significantly higher than interference group 3(T = 238.247,P<0.001).(3)After overexpression of FBXO8,used Western blot to detect FBXO8 protein expression in two kinds of colorectal cancer cell lines HT29 and SW480,the results showed that compared with the mock group,FBXO8 expression in HT29 and SW480 cells was higher.(4)After FBXO8 transient interference,used Western blot to detec FBXO8 protein expression in SW620,the results showed FBXO8 expression in NC group was highest,the interference group 1 and group 3 expression was low,it had no significant difference between group 2 and NC group.Choosed the interference section 3 to stable interfered,results showed that compared with NC group,FBXO8 expression was lower in interference group 3.1.2 FBXO8 overexpression's effect on biological behavior in vitro of colorectal cancer cell(1)Used MTT colorimetric method to detect the change of proliferation ability in vitro in HT29/MOCK,HT29/FBXO8,SW480/MOCK,SW480/FBXO8 tumor cells,and drew the growth curve.Factorial analysis results showed that there was no significant difference on the level of growth time in HT29 cell lines(F = 2490.517,P<0.001),the ability of cell proliferation comparence had significantly differences(F=4459.680,P<0.001);Time and group interactional effect had no significant difference(F = 525.859,P<0.001);There was no significant difference on the level of growth time in SW480 cell lines(F = 624.042,P<0.001),the ability of cell proliferation comparence had significantly differences(F = 1173.557,P<0.001),and time and group interactional effect had no significant difference(F = 131.971,P<0.001).The result shows that compared with the Mock group,FBX08 overexpression group proliferation speed slowed significantly.(2)Used plate clone forming experiment to observe change of cell proliferation in vitro in HT29/MOCK,HT29/FBX08,SW480/MOCK,SW480/FBX08 cells.Independent samples t-test statistics showed cell clone formation ability existed significant differences between the two groups in HT29/MOCK,HT29/FBX08 and SW480/MOCK,SW480/FBX08 cell lines(T=7.379,P=0.002;T=30.765,P<0.001).The results suggest that compared with the Mock group,FBX08 proliferation ability of overexpression group was obviously lower(P=0.002;P<0.001).(3)Used-Boyden chamber to observe the change of invasion ability in vitro in HT29 and SW480 cell line after FBX08 overexpression.Independent samples t-test showed that the cell invasion ability of HT29/FBX08 and SW480/FBX08 group had a significant difference compared with HT29/MOCK,SW480/MOCK groups(T = 11.515,P<0.001,T = 0.001,P<0.001).Compared with the Mock group,HT29/FBXO8 and SW480/FBXO8 cell number passed through the membrane decreased significantly(P<0.001;P<0.001),the results showed that FBX08 inhibited tumor cell invasion ability in vitro.1.3 The effect on biological behaviour in vitro of colorectal cancer cells after FBX08 interference(1)MTT colorimetric method wass used to detect proliferation change of SW620NC,SW620Sil,SW620Si3 cells in vitro,and drew the growth curve.Factorial analysis results showed that there was no significant difference of SW620 cell lines on growth time levels(F=1517.886362321,P<0.001),it had significant difference on the cell proliferation ability among groups(F=268.6163860441,P<0.001);Time and interactional effect was no significant difference between groups(F=1517.886362321,P<0.001);The results suggested that compared with NC group,FBX08 interference group significantly increased cell proliferation rate.(2)Used plate clone formation experiment to observate the change of cell proliferation of SW620NC,SW620Si1,SW620Si3 in vitro,according to the results of one-way analysis of variance statistics,the cell clone formation ability had significant differences between SW620NC and SW620Si or SW620Si3(F = 0.19,P = 0.011;F ?0.24,P = 0.009).Results showed that compared with NC group,the proliferation ability of FBX08 interference group was obviously higher.(3)The use of the cell cycle experiment to observe the change of cell cycle in SW620NC,SW620Si3 cells.Two independent samples T test showed it had significant differences in G1 phase between SW620NC and SW620Si3 group(T ?9.624,P = 9.624),S and G2phase also existed significant difference between two group(T=14.970,P<0.001;T=8.060,P=0.001).Results showed that compared with SW620NC,SW620 Si3 group's cell proliferation number increased significantly.(4)Used Boyden chamber to observe the change of invasion ability in vitro of SW620 cell lines after FBX08 interference.According to one-way analysis of variance,it had significant difference in cell invasion ability between SW620NC and SW620Sil,SW620Si3(F = 199.20,P<0.001;F = 305.6,P<0.001).Compared with NC group,cells number of two interference group passed the membrane increased significantly(P<0.001;P<0.001),the results showed that after FBX08 overexpression,invasion ability in vitro of colon cancer cells enhanced.1.4 The effect on biological behaviour of colorectal cancer cells in vivo after FBX08 overexpression and inhibitionUsed SW480/MOCK,SW480/FBXO8 SW620NC,SW620Si3 4 different cell lines to conduct subcutaneous tumor and tail intravenous injection transfer experiment in vivo,nude mice subcutaneously into tumor individual repeated measurement factor variance analysis results showed that:Two kinds of cells had obvious difference within the group of subcutaneous tumor ability(F=9.552,P=0.01487710397974;F=28.525,P<0.001).After comparison,we found that subcutaneous tumor growth speed of MOCK group was obviously faster than FBX08 overexpression group,subcutaneous tumor growth speed of NC group was obviously slower than FBX08 interference group.Nude mouse tail intravenous injection transfer experiment in vivo results showed that two kinds of cell lines appeared apparent metastases in the lung surface,In SW480/MOCK group nude mous's lung metastasis rate was 100%(6/6);In SW480/FBXO8 group nude mouse's lung metastasis rate was 66.7%(4/6);In SW620NC group nude mouse's lungs in lung metastasis rate was 66.7%(4/6);In SW620Si3 group nude mouse's lung metastasis rate was 100%(6/6).The above results showed that FBX08 inhibited the ability of the body into a tumor and metastasis in colorectal cancer cell.2.The discussion on invasion and metastasis mechanisms of FBX08 in colorectal cancer2.1 FBX08 targets mTOR for ubiquitin degradation.(1)Used Western blot to detect the expression change of target genes and Akt/mTOR signaling pathways protein in SW480/MOCK,SW480/FBXO8 cells,the results showed that compared with SW480/MOCK group,Akt,p-Akt protein expression in SW480/FBXO8 group had no obvious change,but the mTOR,p-mTOR,P70S6K,p-P70S6K protein expression level decreased obviously.(2)Used MG-132 treat colorectal cancer cell lines SW620 and breast cancer cell line MCF-7,Western blot results showed:after MG 132 processed,mTOR,the P-mTOR protein expression level of two kinds of cell lines increased obviously.(3)Utilized MG-132 treat colorectal cancer cell lines SW480/FBXO8,Western blot results showed:after MG132 processed,the protein expression level of mTOR,P-mTOR in the cell lines increased obviously.(4)The test about FBX08 targeting mTOR for ubiquitin degradation is still in progress.2.2 The detection on FBX08 interaction with mTOR(1)Using laser confocal microscope to observe the co-localization condition of FBX08 and mTOR in SW480/MOCK and SW480/FBXO8 cells,the result showed that FBX08 and mTOR co-located in the cytoplasm of cancer cells,FBX08 and mTOR co-localization fluorescence in SW480/FBXO8 cells was much higher than the SW480/MOCK group.(2)Used the CO-IP experiment to detect the situation FBX08 combined with mTOR in SW480/MOCK and SW480/FBXO8 cells,the results showed:FBX08 could directly combined with mTOR.(3)Builded FBX08 two truncated gene:FBX-Sec7(1132-1700a)and FBX-1(851-1247a),used CO-IP method to detect their molecular combination with mTOR,results showed FBX-Sec7 truncated gene could combined with mTOR.2.3 FBX08 regulated proliferation and invasion ability through targeting mTOR for ubiquitin degradation in control colorectal cancer.Used rapamycin to treat colorectal cancer cell lines SW620Si3,MTT colorimetric method was used to test the proliferation ability change of SW620Si3cells in vitro,Boyden invasion chamber to observe the invasion ability change of SW620Si3 cells in vitro.The results showed that after rapamycin processed,the proliferation speed of SW620Si3 cells decreased significantly;The invasion ability of SW620Si3 cells in vitro weakened significantly.The above results suggested that FBX08's inhibition function on invasion and metastasis in colorectal cancer was by ubiquitin degradatiing mTOR.Conclusion1.FBXO8 can inhibit proliferation,invasion and metastasis ability in colorectal cancer cells.2.Put forward a new regulatory mechanism that FBXO8 inhibits colorectal cancer invasion and metastasis ability by ubiquitin degradation mTOR. |
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