| BackgroundColorectal cancer (CRC) is one of the most common malignant tumors in our country, the incidence rate and mortality rate of CRC in China is increasing fast during the past decades. Metastasis is one of the basic charaxteristic of malignant tumors and is crucial for the death of CRC patients. Elucidating molecular mechanism of metastasis and represings its mastastasis in colorectal cacer, is the main research orientation of the researchers.In previous research, three CRC cell lines which have different metastatic potentials to analyse gene-expression profiles. By comparing gene-expression profiles of SW620(high metastatic.derived form the lymph node metastases of SW480)> SW480/M5(a hepatic metastatic subline of low metastatic SW480) between SW480(low metastatic), we selected FMNL2,one of the members of Formins which are the key regulators for cell migration and cell invasion,as a new target gene to investigate CRC invasion and matastasis, because FMNL2expression increased in high metastatic potency cell lines.The present study results indicated FMNL2is closely related with tumorigenesis and metastasis of colorectal cancer,and the possiblely promote cancer invaded and metastasis by regulated cell movement. PhD Zhu xiling first investigated the relationship between FMNL2and colorectal cancer metastasis, and its molecular basis. Found that FMNL2overexpression in metastatic cell lines and tissues of colorectal carcinoma is associated with more aggressive tumour behaviour. FMNL2RNAi significantly reduced the ability of proliferation, movement, invasion and in vivo hepatic metastasis of CRC cell lines.by using bioinformatics, Katoh etc. found FMNL2mRNA was expressed in highly malignant tumors such as diffuse-type gastric cancer, breast cancer, chondrosarcoma, melanoma, glioblastoma., and might be implicated in polarity control, invasion, migration, or metastasis through regulation of the Rho-related signaling pathway. Kitzing etc. identified formin-like2(FMNL2) as a specific RhoC effector, showing selective interaction of FMNL2with active RhoC, and could regulat cell motility by mediated ameboidmovementamebism. Li Y etc. researches identify a novel EMT and tumor promoting function for FMNL2, which is involved in TGF-beta-induced EMT and colorectal carcinoma cell invasion via Smad3effectors, or in collaboration with MAPK/MEK pathway.FMNL2is a positive regulator of cell motility, invasion, and metastasis of colorectal carcinoma. But how does(do) the protein(s) regulate FMNL2specifically? Which position is FMNL2in the cell movement signal pathway? Which target protein(s) does(do) FMNL2interact with? The mentioned above is.still unresolved. We aim to explore the regulation of FMNL2in the cell motility signal pathway, and be helpful to reveal the molecular basis of FMNL2regulating CRC metastasis.ObjectiveAs a key regulator of the member of Formins for migration and metastasis of cancer,FMNL2is closely related to metastasis of colorectal cacer, It could change the assembling actin cytoskeleton to affect Cell polarization, cell adhesion and movement. In fact, it is unrealized that the mechanism of metastasis and the transition of focal adhesions, in our study, we overexpressed FMNL2lentivirus was infected into SW480and HT29cell lines, RNAi plasmid that can express siRNA targeting FMNL2was designed, to study the role of FMNL2in regulating the migration of colorectal cancer sells,and to evaluate the correlation between the expression of FMN12gene and that of Src and Talin.Methods 1Overexpressed and silencing FMNL2in CRC(1) Two short hairpin RNAs were designed and recombinant plasmid vectors to silence FMNL2experssion(provided by PhD Zhu xiling.)(2) Cell culture and transfectionSW620was provided by Pathology Laboratory of Southerm Medical University. The cells were cultured at37℃,5%CO2in DMEM medium containing5%FBS, penicillin and streptomycin. All of the constructed plasmids were transfected into SW620cells by Lipofectamine2000(Invitrogen, California, USA). The oligofectamine reagent was composed of0.5μg/ul plasmids2ul, Lipofectamine20002μl and DMEM96ul. Stable transfects were selected for two weeks under G418。(3) Western blot analysisExpression of SW620protein was detected by western blot assay. Cells were harvested and lysed. Protein concentration of the supernatant was determined by the BCA protein assay procedure. Protein was boiled for5min, electrophoresed on an8%polyacrylamide gel, and then transferred to a polyvinylidene difluoride membrane using semi-dry transfer apparatus. After being blocked with3%BSA+7%fat-free dry milk, the membrane was treated for2h at room temperature with primary antibodies(1:500-diluted antibody of FMNL2), followed by incubation with horseradish peroxidase-labeled secondary antibody (Beijing Biosynthesis Biotechnology Co. Ltd) for1h at room temperature, and then stained with reagent.(4) Overexpressed FMNL2-CT recombination lentivirus from genechem company in shanghai, named pGC-FU-EGFP-3Flag-FMNL2-CT, was infected into SW480and HT29cell lines, which lowexpressed FMNL2, as well as control lentivirus pGC-FU-EGFP-3Flag infection. The independent colonies of FMNL2-CT overexpressed stable cell lines were harvested with screening method of finite dilution in2-3weeks. The FMNL2-CT expression in the stable cell lines was determined by real-time PCR and western blot.(provided by PhD Zeng yuanfeng)2In vitro invasive assay were performed to investigate the influence of FMNL2expression on colorectal cell invasive power。Using24-well transwell units with8μm pore size polycarbonate inserts, ECM1(70μl)。Cells that were suspended in DMEM uncontaining FBS were added to each upper compartment of the transwell units, DMEM containing20%FBS were added to each lower compartment of the transwell units. After being cultured for48h, cells migrating through the matrigel-coated polycarbonate membrane were fixed by paraformaldehyde, stained with crystal violet and counted in five different fields, selected randomly.3Western blot was used to detect the expressions of FMNL2、Src、Talin.we assessed the effects of PP1on the Talin, Src, FMNL2by Western blot.(1) Cell cultureSW620,SW620/FMNL21;SW480, SW480/FMNL2T;HT29, HT29/FMNL2T cells were cultured at37℃,5%CO2in DMEM medium containing5%FBS,PP1(10μmol/L)for24h.(2) Western blotSW620, SW620/FMNL2↓,SW480, SW480/FMNL2↑,HT29, HT29/FMNL2T protein was detected by western blot assay. Cells were harvested and lysed. Protein concentration of the supernatant was determined by the BCA protein assay procedure. Protein) was boiled for5min, electrophoresed on an8%polyacrylamide gel, and then transferred to a polyvinylidene difluoride membrane using semi-dry transfer apparatus. After being blocked with3%BSA+7%fat-free dry milk, the membrane was treated for12h at4℃with primary antibodies(1:500-diluted antibody of FMNL2,1:1000-diluted antibody of Src,1:5000-diluted antibody of Talin), followed by incubation with horseradish peroxidase-labeled secondary antibody (Beijing Biosynthesis Biotechnology Co. Ltd) for1h at room temperature, and then stained with reagent. Protein bands were also quantified by Quantity One.4Co-immunoprecipitate assay and Immuno-colocalization assay were used to analysis the interactive relationship between FMNL2and Src,Talin,F-actinSW620,SW480and HT29cells were cultured on coverlips and fixed with4%paraformaldehyde for lo min,after15min in1%PBS The cells were permeabilized in0.1%Triton-100for8min. A fter15min in1%PBS.the cells were incubated for12h at4℃with primary antibodies(1:100-diluted antibody of FMNL2,1:100-diluted antibody of Src,1:100-diluted antibody of Talin), followed by a secondary antibody DyLight488Conjugated Goat anti-Mouse IgG(H+L),and DyLight549Conjugated Goat anti-Rabbit IgG(H+L).F-actin was visualized with Rhodamine-phalloidin.The mounting media contained DAPI for staining nuclei. The cells were imaged with FSV-100confocal microscope.5Statistical analysisData were analyzed using SPSS13.0software. Results are presented using means±SD. Comparison of means between two samples was performed using Student’s t test. Statistical comparisons of more than two groups were performed using one-way analysis of variance (ANOVA), and then multiple comparisons were performed using least-significant difference (LSD). In all cases, P<0.05was considered statistically significant.Results1Down-regulation of FMNL2expression by RNAiSW620cells were infected with pGenesil-FMNL21shRNA and pGenesil-FMNL22shRNA, and incubated with G418to select cinresistant single clone.2Western blot examined FMNL2protein expressionWe examined FMNL2protein expression in clones and found clone2silented a dramatic knock down of FMNL2protein expression.(Designated SW620/FMNL2↓)3. Down-regulation of FMNL2inhibits cell invasionMore cells penetrated the matrigel-coated membrane in SW620, HT29/FMNL2↑, SW480/FMNL2↑than in SW620/FMNL2↓, HT29, SW480, which provided evidence that down-regulation of FMNL2may reduce cell invasion ability.4Effec of FMNL2silencing or overexpressing on expression of Src and TalinFMNL2expression was positively correlated with Src expression (F=73.56, P<0.01, SW480F=147.567, SW480P<0.01; HT29F=147.567, HT29P<0.01)), but negatively correlated with Talin expression (F=7.772, P<0.05, SW480F=1631.542, SW480P<0.01; HT29F=150.603,HT29P<0.01)5Western blot examined FMNL2、Src and Talin protein expression in CRC treated with PP1We found that FMN12expression was no change after treating with PP1in all cells, Src expression showed no changes after treating with PP1in HT29/FMNL2T/SW480/FMNL2↑,SW480, HT29.,but a little down in SW620and SW620/FMNL2↓. Treatment with PP1prominently decreased Talin expression (SW480F=1631.542, SW480P<0.01; HT29F=150.603, HT29P<0.01)6Co-immunoprecipitate assay and Immuno-colocalization assay analysis the interactive relationship between FMNL2and Src,Talin,F-actinBy the immuno-colocalization assay we found that FMNL2and Talin or Src were colocalized in cytoplasm and plasma membrane. In bipolar cell SW620, FMNL2and Talin or Src were prominent colocalized in both ends of cells.Conclusion1FMNL2gene significantly promotes invasion CRC cells.2FMNL2may act upstream of Src and Talin to regulatiaon expression of Talin and src,and though Src signal pathway affect expression of Talin to Control the transition of focal adhesions indirectly. |
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