Study Of The Expression And Significance Of Survivin In Pancreatic Carcinoma And The Influence On The Apoptosis And Chemosensitivity By RNAi In PANC-1

[Objective]Pancreatic carcinoma is resist to most of all chemotherapeutical drugs.Survivin,as the most potent one of inhibitor of apoptosis(IAP) families,up-regulating in many cancer tissues and cells, is thought an important factor contributed to carcinoma proliferation and chemoresistance.The main aims of this study are as follows.(1) To discuss the expression of survivin in the pancreatic carcinoma tissue and the relationship between it's expression and the clinicopathologic parameters.(2) To observe the change of the expression of survivin in the pancreatic carcinoma cell line PANC-1 induced by chemotherapeutical drug gemcitabine.(3) To investigate survivin inhibited by RNA interference(RNAi) vectors,and induce apoptosis.(4) To investigate the sensitivity to chemotherapeutical drug gemcitabine enhanced after survivin inhibited by RNAi.Lay an experimental foundation for further exploration of new therapy of pancreatic carcinoma.[Method](1) 35 samples of resected pancreatic carcinoma tissues and 10 normal tissues from the carcinoma besides examined by immunohistochemistry and the relationship of survivin expression to the clinicopathologic parameters analyzed.(2) The pancreatic carcinoma cell line PANC-1 cultured with the gemcitabine,and survivin expression examined by RT-PCR and Western blot.(3) RNA interference vectors with U6 promoter against survivin constructed and transfected into the pancreatic carcinoma cell line PANC-1,the expression of survivin examined by RT-PCR and Western blot,FCM employed to examine the apoptosis index of PANC-1 and apoptotic morphology was examined by Hoechst 33258.(4) The sensitivity of PANC-1 to chemotherapeutical drug gemcitabine further examined by MTT,FCM and Hoechst 33258.[Result](1) Survivin expressed in pancreatic carcinoma tissues, and none of carcinoma besides tissues;the survivin expression rate of carcinoma tissues(26/35,74.29%) higher than the carcinoma besides normal tissues(0/10,0%),and the difference between them was statistically significant(P＜0.05).The rate was related to the pathological grade and the clinical stage(P＜0.05),not related to the metastasis (P＞0.05).(2) 24 and 48 hours after cultured by gemcitabine with the concentration of 1μg/ml and 10μg/ml,survivin mRNA level was increased(1.34±0.12),(2.40±0.17),(3.33±0.20) and(4.41±0.18) folds,and the protein expression was enhanced(1.20±0.07),(1.48±0.19),(2.90±0.04) and(4.50±0.20) folds respectively,and the difference between them was statistically significant(P＜0.05).(3) RNAi vectors against survivin could inhibite the survivin mRNA and protein expression level of 52.67%±3.51%,75.33%±3.06%and 58.00%±3.61%,76.67%±4.73%at 24h and 48h instantaneous effectively.The apoptosis index examined by FCM is 9.33%±1.53%and 14.57%±1.50%. The typical apoptotic morphology of condensed,shrinked and reptured nucleoli of PANC-1 found by Hoechst33258 stained.(4) The sensitivity of PANC-1 to gemcitabine increased after survivin was inhibited,the apoptosis index examined by FCM apparent increased from 15.67%±1.16%to 35.67%±1.53%by the action of gemcitabine(P＜0.05), with the IC₅₀ to gemcitabine decreased greatly from 1.24±0.11μg/ml to 0.39±0.07μg/ml,and the difference between them was statistically significant(P＜0.05).[Conclusion](1) The positive rate of survivin higher than the carcinoma besides normal tissues,and which related to the differentiation and the clinical stage of pancreatic carcinoma indicate survivin is thought of an important factor contributed to carcinoma proliferation and prognosis.(2) The capacity of pancreatic carcinoma cell line chemoresistance could be enhanced by gemcitabine cultured with the survivin expression up-regulated.(3) The RNAi vectors with U6 promoter could effective suppress the expression of survivin in pancreatic carcionoma cell line PANC-1,and induce the PANC-1 apoptosis.(4) The chemosensitivity of gemcitabine in pancreatic carcinoma cell line PANC-1 could be enhanced by suppress the expression of survivin through RNAi technology.