Effect Of Non-Smad Pathway In Mouse Colorectal Cancer Liver Metastasis

Objective: To establish CT26-Smad4KD cell line, and determine it. Methods: We used thelentivirus system to establish the stably knockdown Smad4in wild type CT26cell line.Article-validated shRNA sequence was used as interference sequence, while common negative sequence was used as negative control to construct the lentiviral vectors.293T cellwas transfected to harvest lentiviral particles. After selected by puromycin, positive cellclones were detected by western blot and co-immunoprecipitation to determine theexpression of Smad4protein and the level of Smad2/3/4complex, and the MAPK pathwayof the non-Smad pathway. Results: We got the best infect efficiency on the condition of theMOI was50and the concentration of polybrene was5μ g/ml. In the result of western blotassay, Smad4#1, Smad4#8and Smad4#18cell clone expressed very low level of Smad4protein, while wild type CT26cell and the negative control cell expressed sufficient Smad4protein. In the co-immunoprecipitation assay, neither given exogenous TGF-β or not,CT26-Smad4KD cell couldn’t product sufficient Smad2/3/4complex. Conclusion:CT26-Smad4KD cell line and its negative control were successly established. Objective: To evaluate the role of Smad4in the colorectal cancer cell CT26proliferation,apoptosis and tumorigenicity. Methods: The ability of proliferation of Wild type CT26cell,Negative control (NC), and Smad4KD cell was evaluated by cell counting kit-8(CCK-8)assay. Then, we use soft agarose assay to detect the ability of contact independent colonyformation of these three types cell. Annexin V-FITC/PI assay was be used to determine theapoptosis in parent CT26cell and its negative control and Smad4KD cell. At last, we usedtumorigenicity assay to evaluate the capability of tumor growth in vivo. Results: Withoutexogenesis TGF-β, the CCK-8assay showed that those cell thich were knocked downSmad4protein behave a disadvantage on cell proliferation compared to wild type CT26celland the negative control cell, while there is no significant difference between these threetypes cell when given exogenesis TGF-β. Also we got the same pattern in the soft agaroseassay, that is, Smad4KD cell showed a decline of colony formation both in size and numberin soft agarose. The result of Annexin V-FITC/PI assay showed that the apoptosis rate ofSmad4KD cell was increased, compared with the wild type CT26and negative control. Atlast, the effect of interference of Smad4in vivo was examined by tumorigenicity assaywhich showed that the loss of Smad4protein reduced tumor growth in Balb/c-nu mice,compared with the wild type CT26cell. Conclusion: Loss of Smad4inhibits mouse colorectal cancer cell CT26proliferation and colony formation, and promotes apoptosis. Objective: To investigate the role of Smad4in the colorectal cancer cell CT26migration, and the relationship between the Smad-dependent pathway and the Smad-independentpathway. Methods: We evaluated the role of TGF-β/Smad signaling on cell mobility,migration, invasion in Smad4KD colorectal cancer cell CT26-Smad4KD by wound-healingassay, transwell cell migration assay and transwell cell invasion assay, respectively. Then,we combined TGF-β and JNK and ERK pathway inhibitors, detected the migrativephenotype when the JNK and ERK pathway were blocked. Results: The wound-healingassay showed that the mobility of wild type CT26cell can be promoted by exogenesis, andthe Smad4KD cell has a increased mobility than its parent. In transwell cell migration assay,loss of Smad4enhanced the migration of CT26cells. Used the inhibitors against theMAPK/JNK or MAPK/MEK pathway, the enhanced migration can be decreased.Conclusion: TGF-β/Smad signaling suppresses migration of CRC cell CT26. Loss ofSmad4promote CT26Cell mobility and metastasis. The enhanced metastasis phenotype byshe loss of Smad4could be attenuated by the inhibitors against JNK and ERK pathway.Inhibitor of JNK and ERK signaling might be a potential therapeutic approach for the CRCpatients.