The Molecular Mechanism Of FMNL2 Regulating Colorectal Cancer Invasion And Metastasis By Targeting COMMD10

Background and ObjectiveColorectal cancer is one of the most common diseases in the modern time, about 1.2 million people are diagnosed with colorectal cancer per year, of which around 600 thousand of them were died. The Grade is the most important prognosis factor for colorectal cancer, for instance the 5 year survive rate for Grade I patients is up to 90%, while which is only about 10% for Grade IV. The tumor metastasis caused the main reason for high morbidity of colorectal cancer patients, to better understand the mechanism of colorectal cancer development helps reducing the morbidity of clinical patients.FMNL2 was previously screened as one of the proteins expressed higher in colorectal cancer tissues when compared to the normal mucosa, which is also positively correlates with the lymphatic metastasis and promotes the activity, invasion as well as migration of colorectal cancer cells. Moreover, we previously preliminary analyze the mechanism FMNL2 regulates the colorectal cancer migration and invasion which showed that FMNL2 induced the colorectal cancer cell migration through positively regulates the EMT(Epithelial Mesenchyme Transformation) process Meanwhile, colorectal cancer metastasis could also be regulated by HMGA1/miR-137/FMNL2 signaling pathway.Yeast two hybrid test was previously used to select the direct interaction partner proteins of FMNL2 and it showed that COMMD10 interacts specially and directly with FMNL2. COMMD is a newly discovered protein family which is characterized by the possess of a specific COMMD domain. COMMD has been reported to be in the regulation of numerous biology processes like the transportation of copper and sodium, the transcription activity of NF-κB and cell proliferation also adaption to hypoxia and so on.Only 6 articles about the relationship between COMMD and tumor development have been reported world widely so far COMMD1 was reported to be down regulated in numerous human tumors which positively regulates the tumor invasion and metastasis. Moreover COMMD1 inhibits the transcription activity of NF-κB, which signaling pathway was participate in a lot biology processes like the tumor cell proliferation, the formation of neovascularization, and tumor cell invasion and migration. COMMD5 expressed less in glioblastoma, nephroma and hepatocellular carcinoma tissues when compared with the normal tissues indicating that it might regulates the tumor cell cycle, the proliferation ability of HEK293 cells reduced after COMMD5 was transferred into, the cell G2/M cycle was blocked meanwhile P21ciP1/WAF1 p27KIP1 were down regulated. Gkiafi. ect screened out 7 proteins who directly interact with COMMD, of which BL41 is targeted by EB virus in Burkitt’s lymphoma. Agglutinin promotes the degradation of COMMD1 and IκB protease through interacting with SCF-βTrCPE3. None report on the relationship between COMMD10 and tumor development has been done so far.In this research, we identified COMMD10 as a FMNL2 interaction partner protein and analyzed the function COMMD10 has on FMNL2 regulated colorectal cancer invasion and metastasis.Moreover, the potential mechanism was further analyzed to reveal a new way FMNL2 regulates colorectal cancer metastasis which might provide a potential clinical molecular directed target to coloreatal cancer.Methods1、Identification of COMMD10 as a FMNL2 interaction partner(1) Using CO-IP (Co-immunoprecipation) to detect the FMNL2 physical interaction with COMMD10 and IF (immunofluorescence) to testify the co-location of them inside colorectal cancer cell lines SW480 and HCT116.(2) GST-COMMD10 protokaryon truncates were constructed and GST-pull down assay and CO-IP were further used to detect the interaction region between the genes (FLAG-FMNL2 eukaryon truncates were constructed previously by DOC. Qiao Yudan).(3) To extract the RNA and protein of stable colorectal cancer cell lines SW480/ FMNL2 and SW620/SiFMNL2, followed by using QPCR and Western blotting to detect the expression changes in these cells. Transiently transferring the COMMD10 over-expression vector and COMMD10 siRNA fragments into the colorectal cancer SW480 and HCT116 cell lines, extracting the RNA and protein of these four groups of cells and using QPCR and Western blotting to detect the FMNL2 expression changes in them.2、COMMD10 regulates colorectal cancer invasion and metastasis through NF-κB signaling pathway.(1) Using QPCR and western blotting to detect the COMMD10 expression in 6 colorectal cancer cell lines and choose out two of them with the highest, lowest as well as one with middle expression of COMMD10 respectively.(2) The COMMD10 forced over-expression vector was constructed with pEGFP-C1 as a negative control vector followed by being transiently transferred into colorectal cancer cell lines SW480 and SW620. Using QPCR and Western blotting to detect the COMMD10 expression changes between recombinant cell lines and negative control ones.(3) Transiently transfer 3 COMMD10 siRNA fragments which were purchased as commercial goods into colorectal cancer SW480 and HT29 cell lines, QPCR and western blotting were then carried out to detect the COMMD10 expression changes among groups which turned out that COMMD10 was knocked down most efficiently by SI-1. SI-1 was hence choose to be packaged into virus supernatant and used to infect colorectal cancer cell line SW480, FlowCytometer was used to select the ones that were successfully infected and expressing GFP. Using QPCR and Western blotting to detect the expression changes between grouped cell lines.(4)Using CCK8(cell counting kit 8), Boyden cell invasion detection chambers, cell colonies formation assays, cell cycle detection assays and cell apoptosis detection assays to detect the influence over-expression and knock down of COMMD10 have on colorectal cancer cells biology behaviors.(5)Observing the influence of COMMD10 knocked down has on subcutaneous tumor sizes and in vain tumor migration ability in SPF class BALB/c nude mice models.(6) 31 pairs of flesh colorectal cancer tissues and matched normal mucosa were collected to detect the COMMD10 mRNA expression level by QPCR, and 10 of them which were randomly picked to detect the COMMD10 protein expression level by western blotting.(7)Immunohistochemistry was carried out in 120 clinical colorectal cancer parafin sections to detect the COMMD10 expression level and analyze its clinical relevance with tumor pathology.(8)Using luciferase assay to detect the COMMD10 influence on NF-κB transcription activity.(9)Using western blotting to detect the COMMD10 influence on the expression level of NF-κB signaling pathway proteins.3、The mechanism analysis of FMNL2 regulation on colorectal cancer invasion and metastasis through COMMD10(1) Knock down COMMD10 in FMNL2 stably knocked down colorectal cell lines or over-express COMMD10 in FMNL2 stably forced over-expressed cell lines and using CCK8, Boyden cell migration ability detection chambers, subcutaneous tumor size and in vain tumor migration detection assays to observe the influence COMMD10 has on FMNL2 regulated colorectal cancer biology behaviors.(2)Using western blotting to detect the COMMD10 influence on FMNL2 regulated NF-κB signaling pathway protein expression levels.Results1、Identification of COMMD10 as a FMNL2 interaction partner(1) CO-IP result showed that FMNL2 interacts directly with COMMD10 in SW480 and HCT116 cell lines.(2) Con-focal result showed that FMNL2 co-localize with COMMD10 in the cytoplasm of colorectal cancer SW480 and HCT116 cell lines.(3) COMMD10 truncates pGEX-6p-1-COMMD10 (1-132a) and pGEX-6p-1-COMMD10 (133-202a) were successfully constructed in which fused GSP protein GST-COMMD10 (1-132a) and GST-COMMD10 (133-202a) were expressed respectively. GST-pull down result showed that FLAG was detected in SW480/FMNL2 (525-616a) but not in SW480/FMNL2 (1-524a)、 SW480/FMNL2(617-1092a) or SW480 cell lines indicating that COMMD10(1-132a) binds with FMNL2(525-616a).(4) Extract the RNA and protein of FMNL2 and COMMD10 stably forced over-expressed or knocked down cell lines, and the QPCR and western blotting result showed that COMMD10 expressed less in SW480/FMNL2cell lines when compared with negative control groups and the expression difference was statistically significant (p<0.01), COMMD10 expressed much higher in SW480/SiFMNL2 and SW620/SiFMNL2 cell lines when compared with negative control groups (p<0.05, p<0.01) indicating that FMNL2 regulates COMMD10 expression. There was not obvious difference in the expression of FMN12 in SW480/COMMD10 SW620/COMMD10 cell lines when compared with negative control ones (p>0.05,p >0.05), meanwhile there was either not obvious difference in the FMNL2 expression in SW480/SiCOMMD10 and HT29/SiCOMMD10 cell lines when compared with negative ones (p>0.05,p>0.05) indicating that COMMD10 failed to regulates FMNL2 expression.2、COMMD 10 regulates colorectal cancer invasion and metastasis through NF-κB signaling pathway.2.1 The influence of COMMD 10 has in colorectal cancer biology behaviors2.1.1 Detection of COMMD10 expression level in colorectal cancer cells(1) Western blot and QPCR results showed that there was statistically significant differences in the expression of COMMD 10 among the 6 colorectal cancer cell lines (F=131.316,p<0.001); COMMD10 expressed the most in HT29 cell line and it was statistically higher than that in LS174T, SW480, HCT116, SW620 and LoVo cell lines (p<0.001, p<0.001, p<0.001, p<0.001); COMMD 10 expressed the lowest in SW620 cell line, and it was statistically lower than that in HT29、 LS174T、SW480、HCT116 cell lines (p<0.001, p<0.001, p<0.001,p<0.001). (2) Transiently transfer the COMMD 10 vectors into colorectal cancer cell lines SW480 and SW620, QPCR and Western blotting carried out in which showed that there was statistically significance in COMMD 10 expression between grouped cell line (t=-4.756, p=0.017; t=-466.661, p=0.158) and it expressed much lower in MOCK groups than the other groups detected by Independent-Sample T tests (p <0.05, p<0.001).(3) Using QPCR and western blot to detect the COMMD10 expression changes in HT29 and SW480 cell lines after COMMD10 was transiently knocked down, One Way-ANOVA result showed that there was statistically significant differences in COMMD10 expression among NC, SI-1 and SI-2 groups (p<0.001, p<0.001, p<0.001, p<0.001,p<0.001, p<0.001), but there was not obvious difference in SI-3 group, and COMMD 10 expressed much higher in NC group than that in SI-1 and SI-3 groups; Using QPCR and western blot to detect COMMD 10 expression level after SW480SI1 stable cell line was constructed. Independent-Sample T Test result showed that COMMD10 expressed much less in COMMD10 knocked down cell lines than that in NC cell groups (p<0.001, p<0.001).2.1.2 COMMD 10 influence on colorectal cancer cells in vitro biology behaviors(1) Used CCK8(cell counting kit 8) method to detect the change of proliferation ability in vitro in SW620/MOCK and SW620/COMMD1, SW480/MOCK and SW480/COMMD10, SW480/NC and SW480/SiCOMMD10, HT29/NC and HT29/SiCOMMD10 tumor cells respectively, and drew the growth curve. Factorial analysis results showed that there was significant difference on the level of growth time between COMMD10 forced over expressed and negative control cell lines (F=304.467, p<0.001; F= 1874.713, p<0.001), however the ability of cell proliferation had significantly differences between cell groups (F=133.700, p 0.001; F= 753.713, p<0.001); Time and group interaction effect had significant difference (F= 24.504, p<0.001; F= 102.071, p<0.001); There was significant difference on the level of growth time in COMMD10 knock ed down cell lines (F=612.674, p<0.001; F= 505.402, p<0.001), the ability of cell proliferation had significantly differences between cell groups (F=110.541, p<0.001; F= 966.668, p<0.001), and time and group interaction effect had no significant difference (F= 15.385, p<0.001; F= 71.803, p<0.001). The result shows that compared with the Mock group, COMMD10 over-expression group proliferation speed slowed significantly, and in consist, the proliferation ability in COMMDIO knocked down cell groups were promoted significantly when compared to the NC control group.(3)Using cell clone formation experiment to observe changes of colorectal cancer cell proliferation in vitro after COMMD10 was forced over-expressed or knocked down. Independent-Sample T test statistics showed that cell clone formation ability existed significant differences between the two groups in SW620/COMMD10 and SW480/COMMD10 cell lines as well as SW480/SiCOMMD10 and HT29/ SiCOMMD10 cell lines (F= 8.291, p>0.05; F=0.3435,p>0.05).There were much less cells transported out of the chamber membrane in SW620/COMMD10 and SW480/COMMD10 cell groups when compared to that in MOCK cell lines.And in consist, there were much more cells transported out of the chamber membrane in SW480/SiCOMMD10 and HT29/SiCOMMD10 cell groups when compared to that in NC cell lines (p<0.001; p<0.001). The results suggest COMMD10 inhibits the invasion ability of colorectal cancer cells.(4) The cell cycle and apoptosis results showed that COMMD10 failed to regulate the colorectal cancer cell cycle or apoptosis.(p>0.05).2.1.3 The influence COMMD10 has on colorectal cancer in vivo biology behaviorsUsing SW480/NC and SW480/SiCOMMD10 2 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 no obvious difference within the group of subcutaneous tumor ability (t=-7.166,_p<0.001). After comparison,we found that subcutaneous tumor growth speed of NC group was obviously slower than COMMD10 knocked down group. Nude mice tail intravenous injection transfer experiment in vivo results showed that there were vision available transferred colorectal cancer tumor spots in mice lungs, and in SW480/NC group nude mice lung metastasis rate was 80%(4/5);In SW480/SiCOMMD10 group nude mice’s lung metastasis rate was 100%(5/5) indicating that COMMD10 inhibits the subcutaneous tumor genesis and tumor metastasis in colorectal cancer cells.2.1.4 The expression of COMMD10 in colorectal cancer tissues and its relevance with clinical pathology duvelopment.(1) Using QPCR to detect the COMMD10 mRNA expression in 31 pairs of flesh colorectal cancer tissues and matched normal mucosa, which turned out that COMMD10 was down regulated in the cancer tissues when compared to that in the normal mucosa (t=5.226, p<0.001).(2) Immunohistochemistry was carried out in 120 cases of colorectal cancer paraffin sections to detect the COMMD10 expression and analyze its relevance with clinical pathology, which showed that there was no obvious relativity between COMMD10 expression and age, differentiation, invasion depth, tumor size or tumor Dukes stage (p>0.05, p>0.05, p>0.05, p>0.05, p>0.05), however it did showed that COMMD10 expression statically relevant with the lymphatic as well as distant metastasis (p=0.013, p=0.014)..3、The mechanism analysis of FMNL2 regulation on colorectal cancer invasion and metastasis through COMMD103.1 COMMD10 inhibits the activation of NF-κB signaling pathway(1) Luciferase detection assay result showed that the luciferase activity was significantly promoted after being exited by TNF-α in SW480 cells when compared with the negative control group (p=0.005; p=0.028). The luciferase activity was down regulated after both COMMD10 vector and TNF-α were transiently transferred into SW480 cells when compared to the cell group that was only transferred with TNF-α.The luciferase activity was promoted after COMMDIO was knocked down when compared with the NC cell group which however would then be reversed (p< 0.001; p< 0.001) by the adding of IκBα negative mutates indicating that COMMD10 inhibits the transcription activity of NF-κB signaling pathway.(2) Western blot was used to detect the expression changes of NF-κB signaling pathway proteins which showed that p-IKKα/β, p-IκBα and nucleus located P65were up regulated after the stimulation of TNF-α in SW480 cell lines when compared to the negative control group and the IKKα/β, IκBα and p65 located in cytoplasm were down regulated. However the p-IKKα/β, p-IκBα and nucleus located P65were down regulated after both the COMMD10 and TNF-α were over-expressed and p-IKKα/β, p-IκBα and nucleus located P65were up regulated. And in consist, p-IKKα/β, p-IκBα and nucleus located P65were up regulated after COMMDIO was knocked down in SW480 cell lines when compared to the negative control group and the IKKα/β, IκBα and p65 located in cytoplasm were down regulated. And the p-IKKα/β, p-IκBα and nucleus located P65were down regulated after the COMMD10 was knocked down and IκBα negative mutates were added into the cells meanwhile p-IKKα/β, p-IκBα and nucleus located P65were up regulated. The above results indicated that COMMD10 inhibits the activation of NF-κB signaling pathway.3.2 FMNL2 regulates the activation of NF-κB signaling pathway through interacting with COMMD10Western blot was used to detect the expression changes of NF-κB signaling pathway proteins in SW480/MOCK, SW480/FMNL2, SW480/FMNL2/COMMD10 and SW620/NC, SW620/siFMNL2, SW620siFMNL2/siCOMMD10 cell lines which turned out that p-IKKα/β and p-IκBα were up regulated in SW480/FMNL2 cell lines when compared with the MOCK cell group, meanwhile IKKα/β and IκBα were down regulated. And p-IKKα/β and p-IκBα were down regulated in SW480/FMNL2/COMMD10 cell lines wen compared with SW480/FMNL2 cell groups, meanwhile IKKα/β and IκBα were up regulated. When compared to SW620/NC cells, p-IKKα/β and P-IκBα were down regulated and IKKα/β、IκBα were up regulated in SW620/siFMNL2 celllines. And when compared to SW620/siFMNL2 cell lines, p-IKKα/β and p-IκBα were up regulated and IKKα/β and IκBα were down regulated in SW620/siFMNL2/siCOMMD10 cells indicating that FMNL2 activates the NF-κB signaling pathway by interacting with COMMD10.3.3 FMNL2 regulates colorectal cancer cells invasion and migration(1)CCK8(cell counting kit8) results showed that when compared with SW480/MOCK group, the in vitro proliferation speed of SW480/FMNL2 was increased (p<0.001), and when compared with SW480/FMNL2 group, the in vitro proliferation speed of SW480/FMNL2/COMMD10 cells was statistically decreased (p<0.001). When compared with SW480/NC and SW620/NC groups, the in vitro cell proliferation speed of SW480/SiFMNL2 and SW620SiFMNL2 cells were decreased (p<0.001), and when compared with SW480/SiFMNL2、 SW620SiFMNL2 cell groups, the in vitro cell proliferation speed of SW480/SiFMNL2/SiCOMMD10 and SW620SiFMNL2/SiCOMMD10 were significantly increased (p<0.001)(2) The Boyden cell invasion detection chamber results showed that when compared with SW480/MOCK group, the in vitro migration ability of SW480/FMNL2 was increased (p<0.001), and when compared with SW480/FMNL2 group, the in vitro migration ability of SW480/FMNL2/COMMD10 cells was statistically decreased (p <0.001). When compared with SW620/NC groups, the in vitro migration ability of and SW620SiFMNL2 cells were decreased (p<0.001), and when compared with SW620SiFMNL2 cell groups, the in vitro migration ability of SW620SiFMNL2/SiCOMMD10 were significantly increased (p<0.05).(3)The nude mice subcutaneous tumor genesis results showed that when compared with SW620/NC group, the weight of subcutaneous tumors of SW620SiFMNL2 group were significantly lighter (p< 0.05), and the weight was recovered in SW620SiFMNL2/SiCOMMD10 cell group when compared with SW620SiFMNL2 cell groups (p<0.05).(4) The nude mice tail intravenous injection transfer experiments results showed that the transferred colorectal cancer spots were vision available in all three groups of mice lungs, when compared with SW620/NC group, there were less transformation spots in the lungs of SW620SiFMNL2 group and when compared with SW620SiFMNL2 group, the less transformation spots in the lungs of SW620SiFMNL2/SiCOMMD10 mice were increased indicating that the migration ability was recovered.Conclusion1.FMNL2-(525-616a) domain directly combines to COMMD-(1-132a) domain.2. COMMD10 was down regulated in colorectal cancer tissues and it inhibits the proliferation, invasion and migration ability of colorectal cancer cells.3.COMMD10 participates in the FMNL2 regulated NF-κB signaling pathway and effect on the colorectal cancer invasion and metastasis.