Nuclear Localization Of Costimulatory Molecule 4-1BBL In Colorectal Cancer Cells And Its Biological Effects

The costimulatory molecule 4-1BBL belongs to the TNF family.The interaction of 4-1BBL with its receptor 4-1BB mediates bi-directional signaling and plays an important role in the initiation and regulation of immune responses.Previous studies have shown that 4-1BBL expressed on the membrane of activated T cells,B cells,dendritic cells,monocyte macrophages,natural killer cells and a variety of tumor cells.The cell membrane 4-1BBL molecule can be cleaved off by metalloproteinase to form the soluble 4-1BBL molecules.Previous studies of our research group suggest that 4-1 BBL molecules may be localized in the nucleus of colorectal cancer cells.Therefore,this study confirmed the expression and cell subfraction distribution of 4-1 BBL in colorectal cancer cells by RT-PCR,gene sequencing,laser confocal microscopy and western blot assay.The roles of nuclear localization of 4-1 BBL on the biological behavior of colorectal cancer cells were explored in vitro and in vivo with the 4-1 BBL gene knockout colorectal cancer cell lines.Part Ⅰ Distribution of 4-1 BBL in colorectal cancer cellsObjective:To confirm the expression of 4-1 BBL molecule in colorectal cancer cells,and to investigate the distribution of 4-1 BBL in subcomponents of colorectal cancer cells.Methods:Human colorectal cancer cells DLD1,HCT 116,RKO,SW480,HT29 were selected as cellular models,and the 4-1BBL mRNA expression of colorectal cancer cells was detected by RT-PCR.The RT-PCR products were sequenced and compared with wild type 4-1 BBL gene.The distribution of 4-1 BBL molecules in the subcomponents of colorectal cancer cells was detected by western blot assay and immunofluorescence staining combined with laser confocal microscopy.Results:① RT-PCR results showed that 4-1 BBL was expressed in colorectal cancer cells at the mRNA level.The RT-PCR product sequencing showed that the mutation of 4-1 BBL gene from colorectal cancer cells was not detected.② Western blotting and immunofluorescence staining combined with laser confocal microscopy showed that 4-1BBL of colorectal cancer cells were expressed in the nucleus,with little or no expression in the cell membrane and cytoplasm.Conclusion:The 4-1BBL molecules were expressed in colorectal cancer cells and were predominantly distributed in the nucleus of colorectal cancer cells.The 4-1BBL gene did not mutate in colorectal cancer cells.Part Ⅱ.Effect of 4-1BBL gene knockout on biological behavior of colorectal cancer cellsObjective:To investigate the effect of nuclear localization of 4-1BBL on the biological behavior of colorectal cancer cells.Methods:The 4-1BBL knockout human colorectal cancer cell line DLD1/4L KO and the control cell line DLD1/Cas9 only,which were established in our research group by using CRISPR/Cas9 technology,were selected as cell models to analyze the biological function of nuclear 4-1BBL in colorectal cancer cells.The cell counting experiment,scratch healing test and transwell assay were applied to detect the ability of cell proliferation and migration of colorectal cancer cells.Realtime PCR was used to detect the expression of the Wnt pathway related genes in cancer cells.Tumor bearing nude mice was used to observe the tumor formation rate,tumor growth curve,tumor size and weight of colorectal cancer cells.Results:① The growth curve assay showed that the proliferation rate of DLD1/4LKO cell was significantly slower than that of the DLD1/Cas9 only cells,P=0.0003.② The scratch healing experiments showed that the unhealing percentage of DLD1/4LKO group was higher than that of the DLD1/Cas9 only group.Transwell assay detected that the migrated cells of DLD1/4LKO group were significantly less than that of the DLD1/Cas9 only group,P=0.0002.③ Compared with DLD1/Cas9 only group,the slug,c-myc,CD44,cyclinD1,mmp10 and cox2 genes were downregulated in DLD1/4LKO cells.④ The tumor formation rate in nude mice of DLD1/Cas9 only group was 100%,and the tumor formation rate of DLD1/4LKO was 85.7%.The tumor growth rate of DLD1/4LKO group was significantly slower than DLD1/Cas9 only group,P<0.01.The tumor weight of DLD1/4LKO group was 21.71±7.393 mg,and the tumor weight of DLD1/Cas9 only group was 162.3±33.16 mg.The weight of DLD1/4L KO group was significantly lighter than that of the DLD1/Cas9 only group,P=0.0014.Conclusion:Deletion nuclear 4-1BBL could impair the proliferation and migration ability of colorectal cancer cells,and slow down their growth in vivo.The nuclear 4-1BBL molecule may participate in the malignant biological behavior of colorectal cancer by regulating the Wnt pathway.