| There are many genes involved into the development process of malignant tumors. In this process ,the depletion and deactivation of anti-oncogene act as an important role. WWOX is one of many anti-oncogenes . People have gradually realized its inhibition on the generation of malignant tumors. Study on the suppression mechanism of WWOX on malignant tumors will be very significant to illuminate the pathogenesy of malignant tumors and cure malignant tumors.WWOX gene maps to the common fragile site FRA16D region in chromosome 16q23.3-24.1. The full-length WWOX (46KD) possesses two N-terminal Wwdomains (containing conserved tryptophan residues) and a C-ternimal short-chain SDR domain. The WW domain is rarely deleted and its amino acid sequence is highly conserved among human , mouse and rat. WW domains are protein-protein interaction modules that bind to short proline-rich motif in proteins that operate in a variety of signaling pathway, and regulate expression of many genes.We do not comprehend the function of WWOX in normal cells. Someone has already found that WWOX could enhance TNF cytotoxicity in L929 fibroblasts via its WW and SDR domain as determined using stable cell transfectants. Blocking of WWOX expression or loss-of-function by antisense mRNA or by a dominant negative WWOX abolished p53 apoptosis, indicating that WWOX is an essential partner of p53 in apoptosis in NIH3T3 cells. These findings implied WWOX is related whith apoptosis. WWOX expression was found to be reduced and deletion in several cancer types, including breast, prostate, esophageal, lung pancreatic, et al. But in normal tissuses WWOX expression is normal. It also sugessted WWOX has relationship with the differentiation and maturation of cell. Studing the mechanism of WWOX suppressing tumor and recovering normal WWOX expression are very important to suppress malignant tumors.In order to explore the mechanism of WWOX suppressing tumor and regulating immunosuppression, we obtained the full-length encoding sequence of WWOX and constructed its eukaryotic expression vector by the molecular-biologic method. Then we detected the mRNA and protein expression of WWOX by RT-PCR and Western blot. We detected the effect of WWOX gene-transfection on the generation, apoptosis and cell cycle of Lewis cells through MTT method and FACS. Investigated the effect of supernatant of WWOX gene-transfected cells on the proliferation of mouse lymphocytes by MTT method. Analyze the expression change of genes related to apoptosis, cell cycle and immunosuppressors by semi-RT-PCR. Detected the location and expression change of c-jun protein in cell by immunohistochemistry staining after WWOX was transfected. Constructed Leiws lung cancer model in C57 mouses and observed the action of WWOX gene suppressing tumors and the effect of regulating immunosuppression. The results is followed:Section 1: Construct of eukaryotic expression vector of WWOX and its expression in Lewis lung cancer cells We obtained the encoding sequence of WWOX from HEK293 cells by RT-PCR, and constructed eukaryotic expression vector of WWOX, pcDNA4.0/Myc-His-WWOX. We confirmed the sequence obtained is right by restriction enzyme and DNA sequencing. Also the sequence has the whole open reading frame and can form a fusion protein with the following tag protein . Transfected pcDNA4.0/Myc-His-WWOX into Lewis lung cancer cells, and detected the expression of WWOX in cells by RT-PCR and Western blot.Section 2: Biological effect of WWOX gene transfecting into Lewis lung cancer cells1.The effect of WWOX gene transfection on generation of Lewis lung cancer cellsTransfected the recombinated plasmid pcDNA4.0/Myc-His-WWOX and empty vector pcDNA4.0/Myc-His into Lewis cells, compared with entransfected cells. In the following 5 days, we observed the generation of three groups .The result was untransfected cells and empty vector-transfected cells grew fast and the curve of cell growth was ascendant. But the recombinated plasmid- transfected cells appeared to grow slowly from the third day after transfected and the curve of cell growth was descended. This suggested WWOX gene could inhibit the generation of Lewis lung cancer cells.2.The effect of WWOX gene transfection on apoptosis and cell cycle of Lewis lung cancer cellsTransfected the recombinated plasmid pcDNA4.0/Myc-His-WWOX and empty vector pcDNA4.0/Myc-His into Lewis cells, compared with entransfected cells, Detected apoptosis and the change of cell cycle of Lewis cells after 72 h. The results displayed the apoptosis percence of the recombinated plasmid-transfected cells(18.21%) was higher than empty vector-transfected cells(3.76%) and untransfected cells(2.85%). The cell cycle also changed after WWOX gene was transfected and the cell cycle stopped at G1 step, the percence of cells which was at G0-G1step was 63.82% , which is higher than empty vector-transfected cells(53.61%) and untransfected cells(45.37%). This demonstrated WWOX gene could induce Lewis lung cancer cell apoptosis and make the cell cycle stopped at G1 step.3.The effect of WWOX gene transfection on gene expression related with apoptosis and cell cycleThe results of semi-RT-PCR displayed, the mRNA expression of cyclinD1 was descendent, otherwise the mRNA expression of p21 was adscendent after WWOX gene was transfected . The mRNA expression of bax was adscendent, otherwise the mRNA expression of bcl-2 was descendent after WWOX gene was transfected. It is significant compared with empty vector-transfected group and untransfected group. The expression of Fas had no change among three groups. This suggested WWOX gene could regulate the mRNA expression of genes realted with apoptosis and cell cycle.4.The effect of tumor cell supernatant after WWOX gene transfection on proliferation of mouse lymphocytesThe supernatant selected from transfected cells was cultured with mouse lymphoctes for 48h and detected proliferation of lymphocytes by MTT method. The OD value of different groups was following: the recombinated plasmid-transfected group was 0.278±0.01, the empty vector-transfected group was 211±0.05, untransfected group was 0.191±0.07, the lympocyte group was 0.313±0.02. The result displayed the supernanant from the empty vector-transfected group and untransfected group could inhibit the proliferation of lymphocytes, it was significant compared with lymphocytes group, P<0.05. When the supernanant was attenuated, the inhibition disappeared. The supernanant from the recombinated plasmid-transfected group had no inhibition on generation of lymphocytes. This demonstrated WWOX gene could rivalried the immunosuppression of tumor cells .5 . The effect of WWOX gene transfection on mRNA expression of immunosuppressorsDetected the mRNA expression of four immunosuppressors by semi-RT-PCR. The result displayed the mRNA expression of TGF-β,VEGF,FasL reduced after the recombinated plasmid pcDNA4.0/Myc-His-WWOX was transfected into Lewis lung cancer cells, compared with control group , p<0.05. The mRNA expression of IL-10 had no change. This suggested WWOX gene could reduce the mRNA expression of immunosuppressors.6.The effect of WWOX gene transfection on protein expression of c-jun in Lewis lung cancer cellsTransfected the recombinated plasmid pcDNA4.0/Myc-His-WWOX and empty vector pcDNA4.0/Myc-His into Lewis cells, compared with untransfected cells. After the recombinated plasmid was transfected, the location of c-jun in Lewis cells changed: the expression in cytoplasm was much more than in cell nucleus, it was significant compared with lymphocytes group, p<0.05. It suggested WWOX could inhibit the transcriptive activity of c-jun entering into cell nucleus.Section 3: The effect of WWOX gene transfection on immunological function of bearing cancer-mouse 1.The effect of inhibiting tumor mediated by WWOX in vivoInjected 0.2ml tumor cell suspension at the left oxterof every mouse and then divided 25 into 5 groups. It is following:⑴PBS control group;⑵empty vector group (pcDNA4.0/Myc-His);⑶cyclophosphamide group (CYP);⑷recombinated plasmid group (pcDNA4.0/Myc-His-WWOX);⑸association group (pcDNA4.0/Myc-His-WWOX + CYP). We could touch the tumor lump after injected tumor cell suspension about 6-8 days, the diameter was about 0.5cm. Injected respectively PBS, pcDNA4.0/Myc-His, CYP, pcDNA4.0/Myc-His-WWOX and pcDNA4.0/Myc-His-WWOX + CYP. Injected once in every two days and continued 6 times. After the last injection, we killed mouse and obtained spleen and tumor lump , measured the volumn of tumor lump and accumulated the inhibition ratio.The result displayed: the volumn of tumor lumps in the groups which recived therapy was smaller than groups which didn,t recived therapy, there was significant difference amang groups, p<0.05. Inhibition ratio of different groups was following: association group was 71.1%; recombinated plasmid group was 50.5%; cyclophosphamide group was 51.3%, which was all higher than PBS control group and empty vector group. This illustrated injecting WWOX gene into tumor lumps could inhibit tumor growth and raise Inhibition ratio.2.The effect of WWOX on the proliferation activity in vivoCompared the proliferation activity of 5 groups, the result displayed: the proliferation activity of cyclophosphamide group(1.158±0.10) was weaker than PBS group(1.410±0.18), p<0.05. The proliferation activity of the recombinated plasmid group (1.712±0.11) was stronger than PBS group(1.410±0.18), p<0.05. There were no difference between the association group and PBS group. This suggested WWOX could strengthen the proliferation activity of mouse lymphocytes in vivo.3.The effect of WWOX on the killing activity of NK in vivoCompared the killing activity of 5 groups, the result displayed: when the rate of effector cell and target cell was 20:1 and 50:1, the killing percence of cyclophosphamide group (20.0±0.24, 20.0±0.34) was lower than PBS group(25.8±0.20, 42.3±0.27), p<0.05; the killing percence of recombinated plasmid group (30.9±0.31,47.5±0.35) was higher than PBS group, p<0.05. When the rate of effector cell and target cell was 20:1 , the killing percence of association group (41.6±0.16) was higher than PBS group, p<0.01. This suggested WWOX could strengthern the killing activity of NK in vivo.4.The effect of WWOX on the killing activity of CTL in vivoCompared the killing activity of 5 groups, the result displayed : when the rate of effector cell and target cell was 20:1 and 50:1, the killing percence of cyclophosphamide group(14.1±0.31, 15.0±0.38) was lower than PBS group(17.8±0.08, 24.6±0.16), p<0.05; the killing percence of recombinated plasmid group (25.0±0.19, 38.4±0.25) was higher than PBS group, p<0.05. the killing percence of recombinated plasmid group (35.0±0.26, 35.1±0.29) was higher than PBS group, p<0.01. This suggested WWOX could strengthern the killing activity of CTL in vivo.All together, the results of our subject indicated that WWOX revealed the ability of inducing inhibiting tumor growth and regulating immunosuppression through stopping the process of cell cycle, inducing tumor cell apoptosis, regulating the expression of immunosuppressors. In this process of regulating growth, WWOX act as an transcription factor, WWOX regulated not only transcription of some genes directly, such as bcl-2 and bax, but also indirectly the expression of the other genes by interfered in the transcription of c-jun, including cyclinD1, p21, VEGF, TGF-β, FasL.In our study , we realized the successful expression of WWOX gene in Lewis lung cancer cells for the first time, and we also found WWOX had immunoregulation activity for the first time. These outcomes riched our comprehension about biological activity of WWOX. Also provided some experimental proof and theory supportion for studing mechanism of inhibiting tumor of WWOX and biological therapy targetting at WWOX gene. |
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