13 5 Vol13 No5 29 1 Life Science Research Oct 29 29 HPV16- sirna CaSki 1, 1, 1, 1, 1 2*, 1 41219 2 4113 : RNA, HPV, HPV16- sirna, HPV16- sirna, HPV16- CaSki RT-PCR CaSki mrna, c, HPV16- sirna RT-PCR, HPV16- sirna HPV16- CaSki, mrna ; Western blotting Bcl-2 ; c, HPV16- sirna c, HPV16- sirna HPV16- sirna HPV16-E, HPV16-, HPV : RNA ; RNA; ; ; : R73733 : A : 17-7847(29)5-448-5 Depressant Effects of the HPV16--Targeted sirna on the CaSki Cell of Cervical Cancer LIU Li-peng 1 TIAN Zhi 1 HUANG Chun-xia 1 GUO Xiao-fang 1 SU Min 1 WANG Xiao-chun 2* 1 Changsha Medical College Changsha 41219 Hunan China 2 Xiangya School of Medicine Central South University Changsha 4113 Hunan China Abstract To study the effects of the HPV16--targeted sirna on the biological behavior of cervical cancer cells by using RNA interference techniques and the HPV-encoded oncoprotein HPV16- as a target Moreover we attempt to elucidate the clinical significance of its experiment The expression vectors of HPV16--targeted sirna were constructed and transfected into the HPV16--positive CaSki cells by in vitro transfection reagents The expression levels of the HPV16- mrna and the mrna encoded oncoproteins in cervical cancer CaSki cells were detected by RT-PCR and Western blotting respectively The expression and activities of the apoptosis-associated molecules were analyzed through cytochrome c assay in order to investigate the molecular mechanism of cell apoptosis induced by HPV16--targeted sirna RT-PCR assay demonstrated that after the expression vectors of HPV16--targeted sirna were : 28-12-18 : 29-5-27 : 7JJ358 : 1968- * 1956- Tel 731-8265279 E-mail wxclx@yahoocomcn
5 HPV16- sirna CaSki 449 transiently transfected into the HPV16--positive CaSki cells a variety of considerable changes occurred cervical cancer ACA ACG G dtdt-3 sirna-4 5 -GGU 5 CGA UGU AUG UCU UGU U dtdt-3 5 - [1] AAC AAG ACA UAC AUC GAC C dtdt-3 wwwgenomewimitedu RNA RNA Primer 5 interference RNAi [2,3] 12 the expression of protein and mrna in CaSki cells were down-regulated Western blotting showed that the expression of anti-apoptotic protein Bcl-2 was also down-regulated the cytochrome c liberation test displayed its release from mitochondria into cytoplasm thus inducing the cell apoptosis HPV16-- targeted sirna can significantly inhibit the proliferation of transplanted tumor cells and induce cell apoptosis It finds out a new approach for the functional study of this important oncoprotein HPV16-E and provides new experimental evidences for the gene-targeted treatment of HPV16--positive tumors especially exploring a new gene therapy for HPV infection and cervical cancer Key words RNAi sirna human papilloma virus gene therapy cervical cancer Life Science Research 29 13 5 448~452 UGG AAC AAC AUU AGA A dtdt-3 5 - QIAGEN UUC UAA UGU UGU UCC AUA C dtdt-3 1 11 CaSki 2 r / min 15 min 5 ml HPV16 2 ml 1 mol / L CaCl 2 6 psirna HPV16- sirna pcon 122 HPV16- sirna GeneBank NC1526 Eppendorf 1 μl 4 3 min 42 45 s 2 min sirna-1 5 -ACU GCG ACG UGA GGU AUA U dtdt-3 5 -AUA UAC CUC ACG UCG CAG U dtdt-3 sirna-2 5 -GUA RNAi 121 [4,5] -7 RNA HPV HPV16- sirna LB HPV16-1~2 mm 2~3 ml LB HPV16-3 r / min 1 ml 1 ml LB 3 r / min 2~4 sirna-3 5 -CCG UUG UGU GAU UUG UUA A dtdt-3 5 -UUA ACA AAU CAC OD 1 min 4 1 ml 1 mol / L CaCl 2 1 min 4 1% 2 μl -7 2 ng 15 ml 9 μl LB 37 24 r / min 45~9 min 1 μl 37 QIAGEN tip 5 3~5 ml 3 r/min
45 29 1 / 1 1 ml 1 ml LB 12 h 4 min 9 ml QIAGEN P1 9 ml QIAGEN P2 5 min 9 ml QIAGEN P3 1 min 1 r / min 15 min 3 ml QBT QIAGEN-tip QC 15 ml QF DNA 7 Cyt-c α- DNA 7% TE tubulin HSP6 123 Fugene 6 2 6 5%~8% 21 HPV16- sirna HPV16- PBS mrna sirna Fugene 6 HPV16- sirna 15 min CaSki 24 h RNA RT- 5% CO 2 37 4 h PCR RNA HPV16-1 ml PBS HPV16- sirna HPV16- mrna 1 124 TRIzol RNA 1 ml 7%~9% PBS 3 15 ml TRIzol 3 min Eppendorf 5 μl 12 r / min 15 min 1 2-2 1 h 12 r / min 15 min 7% 1 min 1%DEPC -7 125 PCR(RT-PCR) RNA [6] RT-PCR OligodT 12-15 RNA RNA DNase DNA 2 μg RNA MMV cdna 2 μl cdna PCR primer 3 GAPDH 5 - GACCACA GTCCATGCCATCAC -3 5 - GTCCAC 55 6 s 72 6 s 35 72 1 1 μl 126 c Fugene 6 [7] sirna 24 h Cytochrome c apoptosis assay kit BioVision Western blot assay 1 HPV16- sirna CaSki HPV16- RNA Fig1 The expression of proteins and mrna in CaSki cells were down-regulated by HPV16--targeted sirna 22 HPV16- sirna Bcl-2 HPV16- sirna [8] HPV16- CACCCTGTTGCTGTA -3 RNA HPV16- sirna PCR 94 2 min 94 3 s Bcl-2 Western Arbitrary unit 15 1 5 CaSki psirna pcon CaSki psirna pcon GAPDH
5 HPV16- sirna CaSki 451 blotting HPV16- sirna CaSki Bcl-2 HPV16- sirna Bcl-2 2 Arbitrary units 2 HPV16- sirna Bcl-2 Fig2 The expression of Bcl-2 were depresssed by HPV16--targeted sirna 23 HPV16- sirna c HPV- HPV- HPV16- sirna Bcl-2 CaSki SiHa HPV16- sirna p53 c HPV16- sirna CaSki [13,14] Western blot c HPV16- sirna c 3 [15] Cyto C α-tubulin Arbitrary units Bcl-2 α-tubulin 6 5 4 3 2 1 14 12 1 8 6 4 2 CaSKi psirna pcon CaSKi Cytosol Bcl-2 psirna pcon Mitochondrial CaSki pcon psirna CaSki pcon psirna A Cytosol cyt c Mitochondria cyt c CaSki pcon psirna B Cyto C Hsp6 3 HPV16- sirna c Fig3 HPV16- sirna can induce to release cytochrome c 3 HPV16 18 16 18 5 %~1 % 87 % HPV16 18 HPV [1] RNAi [9~12] RNAi RNA Dicer [3] 21~23 nt sirna E7 sirna -1 E7-2 E7 E7 sirna 96 h Choo [4] HPV16-E7 mrna CaSki HPV16-E7 CaSki Rb EF-1 bcl-2 Cho [8] c Caspase flow cytometry DNA 3 HPV16- sirna CaSki HPV16- sirna G / G 1 S G 2 / M HPV16- sirna HPV16- [16] G / G 1 HPV16-
452 29 HPV16- sirna CaSki Technology Natural Science 29 23 1 59-61 HPV16- sirna CaSki [6] c [M] Beijing Science Publishing Company 25 HPV16- sirna [7] Bcl-2 c [J] HPV16- sirna HPV16- Bcl-2 c HPV [17] sirna 494-499 HPV16- HPV16- (References): [1] HPV- [J] HUANG Shun-ling WANG Xiao-chun CHEN Wei-qun Detection of HPV- [13] AITAO H E XIN WANG CUIMING Zhu et al Apoptosis positive rate in cernical tissue samples[j] China Journal of of HeLa cells inhibited by human papillomavirus type 16 Modern Medicine 26 16 24 372-3722 protein interacting with hdaxx[j] Chinese-German Journal [2] 16 - of Clinical Oncology 28 7 12 728-731 sirna [J] [14] SEKARIC P CHERRY J J ANDROPHY E J Binding of WANG Xiao-chun ZHANG Yang-de CHEN Wei-qun Influence of targeted suppression HPV- by sirna to the human papillomavirus type 16 to AP is not required for activation of Htert[J] J Virol 28 82 1 71-72 tumor of cervical cancer[j] China Journal of Modern Medicine [15] HILLER T STUBENRAUCH F IFTNER T Isolation and 26 16 24 375-378 functional analysis of five HPV variants with respect to p53 [3] HPV16 E7 sirna degradation[j] J Med Virol 28 8 478-483 [J] GAO Guo-lan [16] MASSIMI P SHAI A LAMBERT P et al HPV HAN Jie NIE Chun-lian Inhibitory effect of HPV16 /E7 degradation of p53 and PDZ containing substrates in an AP sirnas on HPV16-positive cell lines[j] Chinese Journal of null background[j] Oncogene 28 27 12 18-184 Clinical Oncology 28 35 21 1236-124 [4] CHOO C K LING M T SUEN C K et al Retrovirus mediated delivery of HPV16E7 antisense RNA inhibited tumorigenicity of CaSki cells[j] Gynecol Oncol 2 78 3 293-31 [5] HPV16 [J] HU Ren-jian CAI Jia-li FAN Kai et al Expression of HPV16- gene in Escherichia coli[j] Journal of Chongqing Institute of [M] SAMBROOK J The Condensed Protocols Molecular Cloning HPV16- sirna WANG Xiao-chun ZHANG Yang-de CHEN Wei-qun The effect of silencing HPV16- oncogene by sirna interference on cervical cancer cells[j] Tumor 27 27 7 549-552 [8] CHO C W POO H CHO Y S et al HPV antisense induces apoptosis in CaSki cells via suppression of splicing[j] Exp Mol Med 22 34 2 159-166 [9] ELBASHIR S M HARBORTH J LENDECKE L W et al Dup lexes of 21 nucleotide RNAs mediate RNA interference in cultured mammalian cells[j] Nature 21 411 6836 [1] ELBASHIR S M LENDECKE L W TUSCHL T RNA interference is mediated by small nucleotide RNAs[J] Genes Dev 2 14 2 148-152 [11] PROVOST P DISHART D DOUCET J et al Ribonuclease activity and RNA binding of recombinant human Dicer [J] EMBO J 22 21 21 5864-5868 [12] NICHOLSON R H NICHOLSON A W Molecular characterization of a mouse cdna encoding Dicer a ribonuclease ortholog in volved in RNA interference[j] Mamm Genome 22 13 2 67-7 [17] HPV [J] ZHAO Si-cheng LUO Dan HPV- protein and its cellular targets in carcinogenesis[j] Chinese Journal of Biochemistry and Molecular Biology 29 25 3 225-228