| Objective:We design and Synthesis of PU22 fragments. we investigated that PU22 influence on the expression of VEGF, cell proliferation, cell apoptosis and angiogenesis case, in vitro.Methods:(1)We are designed and synthesized oligonucleotide fragments PU22 by bioinformatics, which analysis the VEGF gene promoter region. MUTPU22 sequence formed by disrupting PU22 sequence as a control sequence. They contrast with the gene database in order to ensure that specific.(2)After the oligonucleotide PU22 bring FITC fluorescence labeling join HCT116 cells and SW480 cells, we analysis of colon cancer cell uptake of oligonucleotides PU22 case.(3)The total RNA extracted colon cancer HCT116 and SW480 cells which were treated with different concentrations PU22 or MUTPU22. By Real-time PCR technology we detect m RNA expression of VEGF gene in order to identify the optimal concentration.(4)Follow-up of the experiment were divided into three groups: PU22 treated experimental group, MUTPU22 treated control group and untreated control group.(5)The total protein of colon cancer cells was extracted. Detect VEGF gene protein which by Western blotting technique.(6)Analysis of apoptosis in colon cells using Annexin V-FITC method by flow cytometry. Detect cases of colon cancer cell proliferation by MTT assay.(7)Research PU22 impact on angiogenesis by human umbilical vein endothelial cells(HUVEC) tube formation in vitro.Results:(1)By bioinformatics database successfully screened and design out PU22 acting sequences, which possible role of the promoter region of VEGF and MUTPU22 control sequences. They contrast with the gene database in order to ensure that specific.(2)We have detected that PU22 successfully uptake into cancer cells, and the uptake rate greater than 90%, by fluorescent staining.(3)q RT-PCR results showed that human colon cancer cell line HCT116 and SW480 which effect with different concentrations PU22 vegf gene RNA expression levels are reduced. Among them, the most obvious effect concentration 8μl / L, the optimum time is48 hours, SW480 cells better than the effect of HCT116 cells. the m RNA expression level was 0.027 ± 0.002. RNA expression levels of VEGF genes in cells by MUTPU22 is no significant change, no statistically significant difference(P>0.05).The results show PU22 can inhibit the expression of VEGF gene of m RNA.(4)The results detected by Western blotting show that HCT116 cells treated 48 h by different conditions, the gray value of treatment group(0.074±0.007) was significantly less than the normal group(0.737±0.034) and control group(0.513±0.013), and there is significant difference(P <0.05).And after processing 72 h, gray value of treated group(0.252±0.094) is slightly less than the normal group(0.466±0.063) and control group(0.396 ± 0.119),and there is significant difference(P <0.05).SW480 cells treated 48 h by different conditions, the gray value of treatment group(0.02±0.001) was significantly lower than the normal group(0.572±0.127) and control group(0.488±0.034), and there is significant difference(P <0.05).And after processing 72 h, gray value of treated group(0.113±0.009) was significantly less than the normal group(0.635±0.061) and control group(0.481±0.007),and there is significant difference(P <0.05).The results showed that protein expression levels of VEGF genes in human colon cancer cell line HCT116 and SW480 in different conditions have decreased.Illustrate the design of exogenous Gquadruplex PU22 can inhibit the expression of VEGF gene protein.(5)Detect cell apoptosis after treatment 48 h under different conditions by Annexin VFITC/PI assay.Treatment group of HCT116 cell apoptosis rate(42.46%±2.03%) is higher than the control group(28.78%±1.87%),and there is significant difference(P<0.05).Treatment group of SW480 cell apoptosis rate(69.14%±1.77%) is higher than the control group(32.95%±2.18%),and there is significant difference(P <0.05). The results showed that PU22 promote apoptosis of SW480 and HCT116 cells.(6)MTT results showed that, 72 h after, colon cancer cells proliferation of the experimental group is significantly lower than that value and control groups.At this time,OD450 of SW480 in the experimental group, the control group and the blank group relative are 0.247±0.067, 0.408±0.034, 0.423±0.115; OD450 of HCT116 in the experimental group, the control group and the blank group relative are 0.402±0.193,0.896±0.074,0.921±0.127. Description PU22 can inhibit HCT116 and SW480 colon cancer cells proliferations.(7)By HUVEC tubule formation assay the results show that, after PU22 role in colon cancer cells,HCT116 cells in the experimental group, the control group and the blank group of the number of blood vessels formed relative are 11.5±1.81, 24.5±0.97, 26±2.62;SW480 cells in the experimental group, the control group and the blank group of the number of blood vessels formed relative are 8.27±2.04, 21.3±1.66, 21.5±0.91. Among them, the angiogenesis rate of the experimental group was significantly lower than the control group and the control group, and the lumen is incomplete. The show that PU22 can inhibit angiogenesis in vitro.Conclusion:After the successful uptake of PU22 oligonucleotide sequences, colon cancer cells HCT116 and SW480 can inhibit the expression of vegf genes, induce apoptosis, inhibit proliferation. Their cell secretion inhibit HUVEC of angiogenesis in vitro. Description oligonucleotide sequences PU22 could kill colon cancer cells, inhibit angiogenesis. The mechanism may be related to G-quadruplex which formed in the promoter region of the gene. This study provides new ideas and methods for the treatment of colon cancer. |
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