| Costimulatory molecule 4-1BBL plays an important role in the process of immune response,and participates in immune homeostasis.Previous studies showed that 4-1BBL molecule was transmembrane molecule that expressed on the surface of activated macrophages,B cells,monocytes,DCs,activated T cells and a certain of tumor cells.The soluble 4-1BBL was released from activated immune cells and tumor cells was evidenced as well.The preliminary study found that the nuclear distribution of 4-1BBL molecule in the colorectal cancer cells,hence this study discussed the clinical significance of 4-1BBL localized in the nuclei of colorectal cancer cells.And then,on the basis of constructing the 4-1BBL gene knockout cell line,this study discussed the biological effects of the nuclear 4-1BBL on the growth and migration of colorectal cancer cells.Part ? The localization and its clinical significance of the costimulatory molecule 4-1BBL in colorectal cancerObjective: to explore the distribution of 4-1BBL in colorectal cancer cells,and to explore its clinical significance.Methods: tissue microarray with tumor tissues and tumor adjacent tissues from 90 colorectal cancer patients was selected.Immunohistochemistry staining and tissue microarray assay was performed to analyze the distribution form of 4-1BBL in colorectal carcinoma and evaluate its clinical significance.The distribution of 4-1BBL was analyzed with multiple colon cancer cell lines to further confirm its nuclear localization.Result:1)The expression of 4-1BBL was significantly higher than that of adjacent tissue in clinical specimens of colorectal carcinoma(colon cancer cell nucleus 4-1BBL staining score was 6.517 ± 0.1910,significantly higher than the comprehensive score of expression of 4-1BBL in nucleus carcinoma 3.584 ± 0.153,P < 0.0001.2)4-1BBL molecules predominantly distributed in the nucleus of colorectal cancer cells(the comprehensive score of 4-1BBL expression in colorectal carcinoma is 6.517 ± 0.1910,which is significantly higher than that of the adjacent cancer tissue 4-1BBL expression 3.528 ± 0.124,P < 0.0001.3)There is no significantly difference between 4-1BBL nuclear localization and cytoplasmic distribution in different age groups and different gender groups of colorectal cancer patients.There no significant difference between 4-1BBL nuclear and cytoplasmic expression in different tumor size and pathological grading groups of colorectal carcinoma patients as well.4)Nuclear distribution of 4-1BBL of lymph node metastasis group was significantly higher than that of group without lymph node metastasis.Nuclear 4-1BBL staining score of tumor tissue in lymph node metastasis group was 6.804 ± 0.242,significantly higher than that in the group without lymph node metastasis(6.000 ± 0.32),P < 0.05.5)The 4-1BBL expression of colorectal cancer with clinical stage III-IV was significantly higher than that of group with clinical stage I-II.Tumor tissue 4-1BBL nuclear staining score was 6.837 ± 0.230 in clinical stage III-IV,significantly higher than that in tumor tissues of patients with stage I-II,its nuclear 4-1BBL staining score 5.900 ± 0.341,P < 0.05.6)The survival time of 4-1BBL high nuclear distribution group was 38.53 ± 3.16 months,survival time of 4-1BBL low group was 48.22 ± 4.51 months.The survival curve of two groups were compared by the method of Kaplan-Meier,the survival time of 4-1BBL high nuclear distribution group was significantly lower than that of 4-1BBL low expression group,P <0.05.7)By immunofluorescence staining and flow cytometry analysis,it is showed that 4-1BBL molecule was not expressed on the membrane of colon cancer cell lines.Interestingly,4-1BBL was intracellular located.By immunofluorescence staining and laser confocal microscopy observation,the nuclear expression of 4-1BBL was further confirmed.Conclusion:(1)The form of nuclear distribution of costimulatory molecule 4-1BBL in colorectal cancer cells was found.(2)The nuclear expression of 4-1BBL might be a new prognostic index.(3)The nuclear 4-1BBL expression may be involved in malignant biological behavior of cancer cell,such as the metastasis and the invasion.(4)In addition,this study further confirmed the nuclear expression of 4-1BBL in colorectal cancer.Part ? 4-1BBL gene knockout and the effect on migration ability of colon cancer cell lineObjective: To investigate the effect of nuclear 4-1BBL on the malignant biological behavior of colorectal cancer cells by knocking out 4-1BBL gene.Methods: By the method of bioinformatics,gene sequence encoding human 4-1BBL exon first(exon1)was identified.gRNA sequence to exon1 was selected according to the design principle of Crisper/Cas9.The primers targeting human 4-1BBL gene were inserted into Crisper/Cas9 gene knockout vector PGK1.2,and the recombinant vector named PGK1.2/H4LK-7.After being transfected with PGK1.2/H4LK-7,the GFP positive HCT116 cells were sorted and subcloned for further cultivate.The monoclonal cells were picked up for intracellular staining and flow cytometry analysis to screen 4-1BBL negative cell lines.Result:1)The gene knockout vector PGK1.2/H4LK-7 that targeting human 4-1BBL was successful constructed.2)PGK1.2/H4LK-7 was successfully introduced into the colon cancer cell line HCT116,and GFP protein was successfully expressed.HCT116 cells were transfected with PGK1.2 vector to establish the parallel control cell line.3)The colon cancer cell line HCT116 was successfully knocked out 4-1BBL,named HCT116/4LKO.And the parallel control cell line named HCT116/cas9 only.4)Knock out nuclear 4-1BBL could affect the migration ability of human colorectal cancer cell lines.HCT116/cas9 only cells,serum starved for 12 h,migrated to serum containing medium was 22.60 ± 3.14 / high magnification,significantly higher than that of HCT116/4LKO cells that was 10.60 ± 0.51 / high magnification after 24 h cultivated,P < 0.01.5)The growth of colorectal cancer cells were inhibited by 4-1BBL knockout.In vitro data showed that doubling time of HCT116/cas9 only cell was 18.1 h,and that of HCT116/4LKO cells was 16.2 h;Wound healing experiment results show that 4-1BBL gene knockout can significantly reduce the wound healing time of colorectal cancer cells;The tumor bearing nude mice model further confirmed that 4-1BBL gene knockout can reduce the colorectal cancer cell growth in vitro.Conclusion: The results preliminarily verified that the nuclear 4-1BBL in colorectal carcinoma cells was related to the growth and migration of colorectal cancer cells,which was consistent with the results from the clinical study in part I and provided clues for the mechanism exploration.How the nuclear 4-1BBL to influence the invasion and growth and other biological behavior on colorectal cancer cell? Which is the signal transduction mechanism related with the nuclear 4-1BBLin colon cancer cells? Does the 4-1BBL nuclear localization is universal in other types of tumor cells? These scientific problems need to be further explored. |
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