Study On Effects Of Cyclooxygenase-2 RNAi-mediated Gene Silencing On HT-29 Cells Of Human Colon Carcinoma

The First Section: Study on designing and synthesis of COX-2 siRNA and optimizing transfection conditionsObjective To design and chemically synthesize siRNA targeted on human COX-2 and optimize transfection conditions. Methods To follow the principle of selecting RNA interference target sequence, to utilize the web resource,design four potential small interference RNA (siRNA) of COX-2,and then were annealed. The FAM- negative-siRNA was transfected to HT-29 cells by lipofectamineTM2000. To optimize transfecting conditions, the fluorescence microscope was used for deciding the efficiency of transfection and the activity of cells were measured by MTT after transfecting. Results The four siRNA targeted on COX-2 were successfully constructed. The best transfection conditions was the combination with 1.5ul lipofectamineTM2000 and 40pmol siRNA in the 24 wells board. Conclusions The four siRNA targeted on COX-2 were successfully designed and constructed. The study on effects of COX-2 siRNA on HT-29 cells will be performed in the most optimal transfection conditions.The Second Section: Study on effects of COX-2 siRNA-mediated gene silencing on the expression of COX-2 and proliferation of human colon cancer cellsObjective To investigate the effect of synthesized COX-2 specific siRNA on the cell proliferation and apoptosis of COX-2 overexpressing human colon cancer cells. Methods Human colon cancer cells of the line HT-29 were cultured and transfected with the most optimal COX-2 siRNA by screening from four siRNA. RT-PCR and Western blot were used to detect the expression of COX-2 mRNA and protein. The method of MTT was used to examine the proliferation of the cells. The apoptosis of the cells was detected by Hoechest staining. Results The expression of COX-2 mRNA and protein in the COX-2 siRNA group decreased remarkably(P<0.05),especially 72 hours after transfecting. The proliferation of HT-29 cells transfected with COX-2 siRNA did not changed significantly 24 hours after transfecting, however, decreased 48 hours after and especially 72 hours and 1 week after(P<0.05), Meanwhile, the apoptosis of the HT-29 cells transfected with COX-2 siRNA was significantly higher than that of the cells transfected with the negative COX-2 siRNA and that of the untransfected cells. Conclusions COX-2 is closely related to the proliferation and apoptosis of tumor cells, transfection of the specific siRNA targeting on COX-2 helps inhibiting the expression of COX-2, thus inhibiting the growth and enhancing the apoptosis of the tumor cells.The Third Section: Study on construction and identification of the eukaryotic expression plasmid for cyclooxygenase-2 specific shRNA and optimizing transfection conditionsObjective To clone the recombinant eukaryotic expression plasmid of specific small interfering RNA (siRNA)against COX-2 gene and optimize transfection conditions. Methods The COX-2 siRNA template DNA sequence for short hairpin RNA (shRNA)was designed and synthesized. The annealed siRNA template was inserted into pGPH1-GFP-Neo plasmid. The recombinant plasmid (pshCOX-2) was transformed into DH5αstrain and identified by restrictive enzyme digestion and sequence analysis. The effect of the recombinant plasmid on the COX-2 expression of human colon cancer HT-29 cells was detected by RT-PCR and Western blot after optimizing transfection conditions. Results It was confirmed by restrictive enzyme digestion and sequence analysis that the recombinant plasmid was cloned and the aim sequence was obtained. The COX-2 expression of HT-29 cells was inhibited at mRNA and protein levels 72 hours after transfected with the recombinant plasmid. The difference is significant between the group transfected with the recombinant plasmid and the control groups (F=349.58,P=0.0001), but the difference between the control groups is not significant(P>0.05). Conclusion COX-2 shRNA expression plasmid pshCOX-2 successfully constructed can lastingly inhibit the expression of COX-2 gene in human colon cancer HT-29 cells. It may be a tool to study the relations between COX-2 and colon carcinoma in vitro and in vivo.The Fourth Section: Study on effects of gene silencing of COX-2 on the biological activity of HT-29 cellsObjective To observe the effect on proliferation of HT-29 cells with short hairpin RNA targeted on COX-2 gene in vitro, and to explore the potential mechanisms. Methods After HT-29 cells were transient transfected with pshCOX-2 plasmid, growth curves were detected by MTT methods, and the methods of RIA and ELISA were respectively used to estimate the content of PGE2 and VEGF in supernatant. Cell clones which stably expressed pshCOX-2 were obtained in the medium which contained G418 after 3-4 weeks. The time of healing the wound was utilized to observe the effect on cell migration, invasion and locomotion of HT-29-pshCOX-2 cells. The numbers of clone formation were used to observe the ability of proliferation of HT-29-pshCOX-2 cells.The expressions of COX-2 and EGFR of HT-29- pshCOX-2 cells was judged by immunohistochemistry and the expression of VEGF, MMP-2 and EGFR mRNA was detected by RT-PCR. Results The growth of HT-29 cells was inhibited at 72h after transient transfected with pshCOX-2 and the content of PGE2 and VEGF in supernatant decreased. The expression of COX-2 of HT-29 cells which stably expressed pshCOX-2 down-regulated. Time when cells healed the wound was (7.67±1.53) d, which is longer than control groups (F=9.50 , P=0.0138). The number of clone formation was 257.67±39.53, which is few than control groups(F=37.07,P=0.0004)The expression of COX-2 and EGFR of HT-29-pshCOX-2 decreased and VEGF, MMP-2 and EGFR mRNA down-regulated significantly. Conclusions Inhibiting COX-2 expression can decrease the proliferation and invasion in HT-29 cells, downregulate the expression of VEGF,MMP-2 and EGFR mRNA and decrease the synthesis of PGE2; It may potentially inhibit angiopoiesis of tumor, which is foundation to the next in vivo experiment.The Fifth Section: Study on blocking COX-2 protein expression to promote HT-29 apoptosisObjective To observe the effect on chemotherapy- sensibility and apoptosis of HT-29 cells with short hair-pin RNA targeted on COX-2 gene in vitro, and to explore the potential mechanisms. Methods After HT-29 cells were transient transfected with pshCOX-2 plasmid, 5-Fu was sequently added or singly used, growth curves were detected by MTT methods, apoptosis rate were analyzed by flow cytometry. Morphocytology of apoptotic HT-29-pshCOX-2 cells were observed by TUNEL methods, and the expression of Bcl-2/Bax and Fas/FasL of these cells was measured by RT-PCR and Western blot. Results The growth of HT-29 cells was inhibited at 72h after transient transfected with pshCOX-2 in vitro, and it is significant compared with control groups(P>0.05). The synergistic inhibition can be found in the HT-29-pshCOX-2 cells 7d after 5-Fu used(F=231.94,P=0.0001). FCM show the apoptosis ratio is (7.07%) in pshCOX-2 group, it is statistically significance between pshCOX-2 group and control groups(0.3 % and 0.27%)(F=24.71,P=0.0013), but lower than combination group(13.57%). The results of TUNEL were the same as the FCM. The results of RT-PCR and Western blot showed that the expression of Bax and Fas up-regulated, Bcl-2 down-regulated and FasL had no changes in pshCOX-2 group. Conclusions: The stably expression of COX-2shRNA in HT-29 cells can inhibit the proliferation of HT-29 cells and promote the apoptosis of HT-29 cells. Down-regulating the expression of COX-2 by pshCOX can increase the chemotherapy- sensibility of 5-Fu.The Sixth Section: The therapeutic effect of COX-2 shRNA on colon carcinoma HT-29 cells in vivoObjective To investigate the effect of COXsiRNA gene therepy in vivo, and to explore the potential mechanisms. Methods We carried out vivo study with BALB/C/nu/nu nude mice. 25 nude mice were randomly divided into five groups, and 5×106 cells per mouse was subcutaneously injected on the right HS abdomen of mice(A)control group: 5 mice injected with HT-29 cells;(B)negative control group: 5 mice injected with HT-29pshNC cells;(C) pshCOX-2 group: 5 mice injected with HT-29pshCOX-2cells;(D)5-Fu group:5 mice injected in the abdomen with 5-Fu 50ug/kg/d×5d;(E)5-Fu+HT-29pshCOX-2 group(combination group):C+D. All mice were sacrificed 42 days later and the changes of weight of all nude mice were observed. The volume and weight of tumor were measured and the rates of tumor inhibition were evaluated. The growth curves and stage of latency of tumor were observed, and morphocytology of apoptotic cells were detected by TUNEL and TEM. The expression of COX-2, EGFR and CD34 were stained by immunohistochemistry. The microvessel density (MVD) of tumor tissue was evaluated. Results Stage of latency lengthen and growth of tumor in group C was inhibited. The volume and weight of tumors were significantly different between group C and control groups(P<0.05). The effect of inhibition of tumor in group C was weaker than combination group (P<0.05).The apoptosis in situ of tumors in group C increased, it is statistically significance compared with the control groups (P<0.05). The expression of COX-2 and EGFR decreased in group C,and the MVD of group C were scarce and punctiform. But in control group, MVD are intensive and annuliform, it is statistically significance between group C and control groups (P<0.05). Under TEM,more apoptosic cells in groupC can be found. Conclusions: By inhibiting the expression of COX-2 of mice, the growth of tumor can be inhibited; more apoptosic cells can be found in tumor, Inhibiting COX-2 expression can decrease vascularization of tumor and improve the sensibility of chemotherapy. The therapeutic effect of COXsiRNA in vivo suggested that COX-2 gene be a good target for siRNA gene therapy of colon carcinoma. We obtain a useful theoretical basis and measure for gene therapy of colon carcinoma.