5’-Aza-CdR Induced Inhibition Of CHFR Expression And Its Inhibitory Effect On Methylation Status In Raji Cells In Vivo And On The Growth Of Xenografted In Nude Mice

Objective To determine the inhibitory effect of 5’-Aza-Cd R on the growth of Burkitt Lymphoma’s xenografts in nude mice, to observe its effect on C HFR gene expression and methylation in the xenografts, and to explore the possible mechanisms.Methods ①Raji cell suspension of the live cell concentration about 3×107/200 ul were inoculated subcutaneously in the right armpit of 18 male nude mice about 4-6 week old,which to establish xenograft models of Burkitt Lymphoma. When the tumor’s shorter diameter was more than 0.40 cm, the modeling was successful. ②After successful modeling, 18 nude mice were randomly divided into experimental group and control group. 5’-Aza-Cd R was in injected intraperitoneally in experimental group, 1ug/g every two days and with equal normal saline in control group in the same way. The tumor volumes were measured every 3 days until 24 days. According to the tumor volume size drew tumor growth curve. All the mice were killed after 24 days observation end, stripping out the xenografts, which were compared tumor volume and weight. ③DNA was extracted from tumor tissue and applied the methyl of specific polymerase chain reaction method(MSP) for the detection of the methylation status of CHFR gene in the two groups of transplanted tumor tissue. ④RNA was extracted from tumor tissue and applied the real- time quantitative PCR for the detection of the expression of CHFR m RNA in the two groups of transplanted tumor tissue.Results ① When the Raji cells were inoculated subcutaneously for 1 weeks, the naked eye could observe the size of a small grain of the nodules, two weeks later the tumor’s shorter diameter was more than 0.42 cm. At this time, the tumor volume of transplantation tumor in experimental group was(44.11±4.01) mm3 and control group was(47.11±2.71) mm3, no difference between the two groups of volume(p>0.05). ②The 7th day after the treatment, we observed that tumor volume of the experimental group was smaller than the control group, and we also from tumor growth curve observed the difference of tumor volume between the two groups increased gradually and the tumor growth was slow in the expre imental group. At the end of the 24 days observation, the tumor volume of transplantation tumor in the experimental group was(290.11±23.86) mm3 and control group was(674.56±25.25) mm3. The tumor volume in the experimental group was significantly smaller than that in the control group, and the difference was significant(p<0.01). ③The tumor weight of transplantation tumor in the experimental group was(762.00±87.87) mg and control group was(1058.00±104.62) mg. The tumor weight in the experimental group was significantly decreased compared with the control group, and the difference was significant(p<0.01), and the inhibitory rate of tumor was 27.98%. ④In the control group only methylatio n stripe of CHFR gene promoter were amplified by PCR. Methylation stripe in experimental group compared with control group obviously dim, and had the non methylation stripe. ⑤The expression of CHFR m RNA in the experimental group was 3.29 times higher than the control group. Compared with the control group(1.04 ±0.30), the expression level of CHFR m RN A in the experimental group(3.69±1.20) was significantly increased, and the difference was significant(t=3.94,P=0.001).Conclusion 5’-Aza-Cd R can inhibit in vivo growth of Burkitt Lymphoma’s xenografts in nude mice, and induces the cancer cells to re-express C HFR in nude mice. The mechanism may be that 5’-Aza-Cd R makes the methylated CHFR demethylate and increase the expression of CHFR m RN A by reactivation of CHFR, to play its function of cancer suppressor gene which inhibits cell proliferation. The experiment may provide a theoretical and experimental basis for the targeted therapy of the demethylation gene treatment of malignant lymphoma.