An Experimental Study On Biological Effect On Human Choriocarcinoma Cell Line JAR/MTX Induced By Antisense Oligonucleotide Targeting Survivin

The choriocarcinoma with special histologic source and biologic behavior is one of highly malignant trophoblastic tumors. It is the one of the malignant tumors which can be cured by antineoplastic drug. But multidrug resistance and recur is the main cause of the died patients of trophoblastic tumor. The technology of antisense oligonucleotide (ASODN) is a hope of curing tumors. Based on the principle of base complementation, the synthetic antisense oligonucleotide can combine with the specific sequence on the purposal gene or mRNA to block the information from gene to protein. It can block or inhibit replication, transcription or translation of the gene. Survivin,a novel inhibitor of apoptosis protein,deserves attention as a selective target for cancer therapy because it lacks expression in diffefentiated adult tissues but is expressed in a variety of human tumors.The study designed an antisense oligonucleotide specifically targeting nucleotides 232-251 of survivin mRNA.By several methods, the biologic functions of the antisense oligonucleotides to human choriocarcinoma cell line JAR/MTX were observed.Objective: This study was designed to explore the effects of liposome-survivin antisense oligonucleotide on the growth and apoptosis, the apoptotic mechanism of inducing JAR/MTX and drug-resistant mechanism of JAR/MTX.Methods: culture cells in vitro and divide them into 4 groups:①control group:culture fluid(same volume without FBS and antibiotics);②Lip group:liposome+culture fluid;③SODN group:SODN+liposome+culture fluid;④ASODN group:ASODN+liposome+culture fluid。Survivin-ASODN were transfected into JAR/MTX cells mediated by lipofectamine 2000.The inhibiting rate of JAR/MTX cells was assessed by MTT assay. The concentration whose inhibiting rate was almost 50% was used in the following experiment. After JAR/MTX cells were disposed for 24h,the change of cytomorphology was observed by HE stain. The protein expression of survivin was determined by SABC immunocytochemistry method. The mRNA expression of survivin was determined by RT-PCR.The apoptotic rate was estimated by flow cytomerty Annexin V FITC/PI.Results:1.The morphologic characters of JAR/MTX cells disposed by Lip, SODN and ASODNAfter 24 hours, there were tight intercellular connections, polygon cells and big nucleus in the control group. The cellular characters in the Lip group and SODN group were the same as the control group besides liposome bulb in the cytoplasm.However,in the ASODN group the amount of JAR/MTX cells obviously decreased.Some cells became shrunk and distorted.Its cytoplasmic acidophily increased.Pyknosis,karyorrhexis and karyolysis could be seen.It showed that survivin-ASODN could inhibit the growth of JAR/MTX and induce apoptosis.2.Survivin-ASODN can induce the apoptosisThe apoptosis effect was the strongest in the ASODN group(P<0.05). The others had a gentle apoptosis and there was no difference among three group(sP>0.05).It showed that survivin-ASODN can induce the apoptosis of JAR/MTX,and only by inhibing the expression of survivin gene cellular apoptosis could enhance.Only survivin had this effect,and other genes inhibiting apoptosis had no this effect.For example,inhibiting the expression of bcl-2 by antisense technology can increase the cellular sensibility to apoptosis,but it has no enough ability to induce cell death. 3.Expression of survivin mRNA and protein after survivin-ASODN acted on JAR/MTX for 24 hours.Specific survivin gene strip can be seen clearly in control group,Lip group and SODN group, but was relatively weakened in ASODN group by RT-PCR.The gray scale ratio of survivin mRNA in ASODN group was relatively reduced.The cell nucleus and plasm were stained brown in control,Lip and SODN groups,while were stained light by immunocytochemistry.It showed that the mRNA and protein expression of survivin in ASODN group was significantly decreased as compared with SODN group and Lip group(P<0.05),at the same time,there were no significantly differences among control,Lip and SODN groups(P>0.05)。It showed that survivin-ASODN had a specific inhibition. Survivin has three ways to inhibit the activity of Caspase.①Survivin directly combines with the Caspase-9;②Survivin blocks SAMC to protect IAP;③Survivin enhances the functions of IAP to counteract SAMC.It was through down-regulating the gene expression of the endogenic survivin that survivin-ASODN made the above three functions defective so that the apoptosis level increased. At the same time,the above results also showed that survivin was expressed in JAR/MTX cells and related to the drug-resistant of the JAR/MTX cells. 4.Inhibition of cell growth by the survivin-ASODN.By use of the MTT assay, it can be seen that the growth-inhibitory effect of the ASODN group was stronger respectively compared with the Lip and SODN group.The defferance had a statistic significance(P<0.05).And compared between the Lip group with the SODN group,there was no statistic defferance.In 24 hours after the start of transfection,survivin antisense oligonucleotide had reduced JAR/MTX cell growth dose dependently. At the concentration of 800nmol/L, the inhibiting rate was the strongest, 58.35士3.63 % .But the variance of inhibiting rate at the concentration of between 400nmol/L and 600nmol/L was distinct.The mechanism of inhibiting cell growth :survivin,unlike to other inhibitors of apoptosis,is expressed in the G2/M phase in a cell cycle-regulated manner,and its interaction with the mitotic spindle apparatus is essential for antiapoptotic function.This could imply that the IAP survivin counteracts a default induction of apoptosis in the G2/M phase of the cell cycle.Survivin-ASODN can decrease the survivin expression of JAR/MTX cells,which made cells blocked in the G2/M phase.So abnormal cell growth was inhibited and apoptosis was induced.It implied that survivin antisense oligonucleotide could not only induce the apoptosis of JAR/MTX cells,but also inhibit the proliferation of JAR/MTX cells.However,the antisense oligonucleotides targeting other IAPs had no the ability to interfere cell proliferation.Conclusions:1.Survivin, an inhibitor of apoptosis protein, was expressed in the human choriocarcinoma cell line JAR/MTX,and related to the drug-resistant of the JAR/MTX cells. 2.The survivin antisense oligonucleotide can down-regulate the mRNA and protein expression of the JAR/MTX cells.3. The survivin antisense oligonucleotide can induce cell apoptosis. The mechanism: It was through down-regulating the gene expression of the endogenic survivin that survivin-ASODN made its functions defective so that the apoptosis level increased.4.The survivin antisense oligonucleotide had reduced JAR/MTX cell growth dose dependently.The mechanism: Survivin-ASODN could decrease the survivin expression of JAR/MTX cells,which made cells blocked in the G2/M phase.So abnormal cell growth was inhibited and apoptosis was induced.To sum up,the survivin antisense oligonucleotide could induce apoptosis and reduce cell growth dose dependently of JAR/MTX cells by down-regulating the mRNA and protein expression of survivin.Moreover,because of selective expression of survivin,the down-regulating expression of survivin would not affect on normal tissues obviously. The antisense oligonucleotide technology targeting survivin has the possibility to become a novel approach to cure drug-resistant choriocarcinoma.