Effect Of Dcr3 Antisense RNA On Liver Cancer And Observation Of Synergies With Cisplatin

PART Ⅰ CONSTRUCTION AND EXPRESSION OF THE ANTISENSE RNA OF DCR3Objective: DcR3(decoy receptor 3,DcR3) was a member belonging to the super family of tumor necrosis factor receptors,could help tumor cell surving and escaping from immunologic survival,immunologic killing through competitively combinding with LIGHT ,FasL so as to inhibite cell apoptosis induced by both LIGHT and FasL. We took use of antisense RNA technology to observe whether apoptosis of hepotoma cell group SMMC-7721 would increase after combindination of DcR3 gene fragment and antisense DcR3 gene fragment? Which was more efficient in inducing apoptosis comparing with tranditional chemical therapy medicine—cisplatin? Whether they had coordinational function when they were unitedly used? First,in part I we constructed antisense RNA of DcR3, transfected it into hepatoma cellular,then compared their protein expression in different groups. Methods: Firstly, total RNA was abstracted from fresh hepatoma tissue,RT-PCR was processed under constriction of DcR3 gene primer and DNA fragment of DcR3 were obtained,secondly the gene fragment were procedured electrophoresis and gene sequence analysis to demonstrate that it just was DcR3, after that DcR3 fragment was excised by both BamH Ⅰ and Xhol Ⅰ to make nicked end of DcR3 naked;While vecctor PVAX Ⅰ (-) was excised at the same time; PVAX I -antisense-DcR3 was obtained after the two (DcR3,PVAX Ⅰ )with nicked end were ligated in antisense direction through T4 DNA ligase , then PVAX Ⅰ -as-DcR3 was transfected into hepatoma cellular group SMMC-7721. Western Blot was used to measure differences of protein expression among 3 groups while β -actin being constrast group:group SMMC-7721,group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 transfected PVAX Ⅰ (-). Result: It was demonstrated that there was an anticipated electrophoresis belt of 620bp in electrophoresis of DcR3 fragment,while the fragment was just DcR3 fragment through gene sequencing; the recombinant vector, PVAX Ⅰ -as-DcR3,was successfully constructed and demonstrated by obtaining 2 anticipated electrophoresis belt—DcR3 (620bp) PVAX Ⅰ (3000bp) after PVAX Ⅰ -as-DcR3 was excised by both BamH Ⅰ XhoL Ⅰ and electrophoresed;Western Blotwas showed color depth of group SMMC-7721 and group SMMC-7721 transfected PVAX Ⅰ (-)was deeper comparing with group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,it revealed that protein expression was relatively decreased for translation of DcR3 mRNA being inhibited while that of the other two groups were not infiuenced,furtherly it showed that antisense RNA of DcR3 could obviously decrease the expressionof DcR3 protein. Conclusion: DcR3 gene fragment could be accurately amplificated under constriction of DcR3 primer ; The complexed vector, PVAX Ⅰ -as-DcR3,could be obtained through ligation of DcR3 fragment and vector PVAX Ⅰ (-) in antisense direction after the both were digested by BamH Ⅰ XhoL Ⅰ ; Western Blot was shown that PVAX Ⅰ -as-DcR3 could make DcR3 protein expression of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3 obviously decreased.PART Ⅱ EFFECT AND COMPARISON OF INDUCING APOPTOSISBY ANTISENSE DCR3 AND CISPLATINObjective: Apoptosis was a kind of biological regulating mechanism and a kind of automatic suicide processed by gene procedure,some chemical therapy medicine ,for example cisplatin,could kill tumor cell through inducing their apoptosis.So MTT,Tunel and FCM were used to measure whether antisense DcR3 could increase inducing apoptosis of hepatoma cellular SMMC-7721 through complemental conjugation with DcR3 mRNA in nucleus? Which was more effective in inducing apoptosis comparing with cisplatin? Whether there was coordinative function when the both were united and used? Methods:On the basis of experiment Part Ⅰ ,first liposome was used to swallow and transfect PVAX I -as-DcR3 and PVAX Ⅰ (-) into hepatoma cellular SMMC-7721 respectively. MTT was used to measure OD(optical density),tumor inhibiting rate and statistical analysis was being done among the different groups: group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 effected by cisplatin(4mg/ml),group SMMC-7721 transfected and effected by both of PVAX Ⅰ -as-DcR3 and cisplatin(2mg/ml),some contrast groups;TuneI was used to observe picture of hepatoma cellular apoptosis in different groups.Apoptosis Index(AI) was calculated and Antomatic Picture Analysis System was used to analyse and compare Picture Unit(PU) of different groups;FCM was used to accuratively calculate apoptosis rate,observe whether cell cycle of apoptosis cell was blocked at G0G1 phase? compare apoptosis peak in different groups ;Finally statistical analysis was taken to compare whether there were statistical differences in different groups. Results:MTT shouwed that the apoptosis of group SMMC-7721 effected by cisplatin and group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin were more than that of the other two groups(p<0.05) during the first 24h ,apoptosis of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3 ,group SMMC-7721 effected by cisplatin and group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin were more than that of the constrast group respectively during the second 24h(p<0.05),while there was no statistical difference of apoptosis among that of the 3 groups(p>0.05).There was no any difference in situation between that of the second and third24h.During the forth 24h,there was no any statistical difference of apoptosis among the whole groups;Tunel showed that Apoptosis Index(AI)of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 effected cisplatin,group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin was much higher than that of the constrast group(p<0.05)while that of group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin was highest among those of the whole groups and there was statistical difference of apoptosis index in this group comparing with those of the other 3 groups(p<0.05);FCM showed that cell cycle of the group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 efected by cisplatin ,group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin was all blocked at G0G1 phase.Apoptosis peaks appeared in different stage among the 3 groups and apoptosis rate of the 3 groups was statistical different comparing with that of the constrast group respectively(p<0.05) while there was no statistical difference of apoptosis rate among the 3 groups(p>0.05). Conclutions:MTT,Tunel,FCM were taken used to demonstrate that antisense DcR3 could obviously inhibite the expression of DcR3 gene and increase apoptosis of procedured hepatoma cellular; DcR3 acted as an important role during the apoptosis of hepatoma cell.Decrease of its protein expression and lost of its function could induce increase of apoptosis in hepatoma cell for scarcity of DcR3 protection; There was a kind of coordinating effect when antisense DcR3 and cisplatin were united and used.intensity of chemical therapy medicine could be decreased while function inducing apoptosis of hepatoma cell was not influenced,so that tolerance to chemical therapy medicine could be increased.PARTⅢ STUDY OF ANTISENSE DCR3 IN TUMOR TRANSPLATED SUBCUTANEOUSLY IN NUDE MICEObjective: Those were detected : invasiveness and chemotaxis motility assay of hepatoma cell in group SMMC-7721 transfected PVAX Ⅰ -as-DcR3 ,group SMMC-7721 effected by cisplatin,group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin ,and some influenced factors were compared for transplanted tumor subcutaneously injected by different treated hepatoma cell—SMMC-7721 in nude mice. Methods: On the basis of experiment Part Ⅰ and Ⅱ ,artificial basement membrane and Transwell Chamber were taken use to simulate basement membrane in vivo and being measured quatitively invasiveness and chemotaxis mobility assay of hepatoma cell SMMC-7721 treated by PVAX Ⅰ -as-DcR3,cisplatin and PVAX Ⅰ -as-DcR3+cisplatin while time of tumor forming ,tumor weight,tumor volume were calculated in different nude mice groups subcutaneously injected hepatoma cell SMMC-7721 treated by PVAX Ⅰ-as-DcR3,cisplatin(4mg/ml), PVAX Ⅰ -as-DcR3+cisplatin(2mg/ml) respectively. Results: Invasiveness statistically decreased in group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 effected by cisplatin,group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin comparing with contrast group(p<0.05) while that of group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin decreased mostly and was statistically different comparing with those of the other groups (p<0.05);The situation in chemotaxis motility assay was the same as ininvasiveness; There was no statistical difference for time of tumor forming of nude mice between group SMMC-7721 effected by cisplatin(4mg/ml) and group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin(2mg/ml) but with statistical longer time than that of group SMMC-7721 transfected PVAX I -as-DcR3 and contrast group(p<0.05).Nude mice injected with hepatoma cell SMMC-7721 treated by PVAX I -as-DcR3+cisplatin(2mg/ml) had lightest tumor weight and there was no statistical difference from that of group SMMC-7721 effected by cisplatin(p>0.05),but statistically different from that of the both---group SMMC-7721transfected PVAX Ⅰ -as-DcR3 and contrast group while there was no statistical difference between the two groups(p>0.05);Still,nude mice injected with hepatoma cell SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin had the smallest tumor volume,there was no statistical difference from that of group SMMC-7721 effected by cisplatin (p>0.05),while contrast group nude mice had the biggest tumor volume and there was no statistical difference from that of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,but tumor volume of the two groups were statistically different from that of the other two groups(p<0.05). Conclusions: The effect of antisense DcR3 was not very satisfied in vivo experiment and was not different from that in vitro experiment,maybe because antisense DcR3 was degraded more in vivo or had relatively lower transfecting efficiency; Enviroment and factors in vivo were much complicated than that in vitro,it couldn't be concluded that antisense DcR3 was not fit to clinical need,but a very hopeful candidate treatment when antisense DcR3 were decorated to escape from being degraded or were improved transfecting efficiency; The union of antisense DcR3 and cisplatin showed more effective function in inhibiting hepatoma cell while elevating tolerance to chemical therapy and decreasing intensity and toxicity of chemical therapy medicine.