Ribozyme targeted on HPV16E6 mRNA induced apoptosis on human cervical carcinoma CaSKi cells.
OBJECTIVE
Human papillomavirus is related to cervical cancer. Ribozyme is special kind of RNA that can cleave target RNA. The aim of this study was to investigate the characterization of the cultured cervical cancer cell line transfected with anti-HPV16E6-ribozyme (HRz) and the effect of ribozyme on proliferation and apoptosis of cervical cancer cell.


METHODS
Ribozyme targeted on HPV16E6 mRNA was designed using computer. With the method of lipofectin transfection, the anti-HPV16E6- ribozyme and empty eucaryotic expressing plasmids were transfected into the CaSKi cells, which named as CaSKi-R and CaSKi-P, respectively. The expression of E6 mRNA in the three kinds of cells was examined by Northern blot analysis. Cell cycle was determined using flow cytometry. Cell apoptosis rate was examined using fluorescent (Hoechst) staining and TUNEL (TDT-mediated dUTP nick end labeling). The expression of certain proteins, including HPV16E6, c-myc, bcl-2, p53, and Fas, were also determined using flow cytometry.


RESULTS
RNA dot blot analysis demonstrated that HRz mRNA expressed stably in the CaSKi-R cells. Northern blot analysis showed that the expression of E6 mRNA was much lower in the CaSKi-R cells than that in the CaSKi cells, while there was no difference of E6 mRNA levels between the CaSKi cells and the CaSKi-P cells. The apoptosis rate in the CaSKi-R cells was much higher than that in the CaSKi cells and the CaSKi-P cells. The cell cycle was arrested in G2 phase, with decrease in percentage of S phase cells. Anti-HPV16E6-ribozyme can significantly reduce the expression of E6, c-myc, and bcl-2 genes in the CaSKi-R cells, and increase the expression of p53 compared with that in Caski cells. This phenomenon was not found in the CaSKi-P cells. The expression of Fas was similar in the three kinds of cells.


CONCLUSIONS
Ribozyme targeted on HPVE6 mRNA can induce apoptosis of human cervical cancer CaSKi cells. The reason may be the decrease of expression of E6 gene, and the successive changes of expression of some genes, including c-myc, bcl-2, and p53 genes.
Zheng
Yan-Fang
YF
Oncology Center, Zhujiang Hospital, The First Military Medical University, Guangzhou, Guang dong, PR China. zyfcn@yahoo.com
Zhang
Ji-Ren
JR
D017209:Apoptosis; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D015870:Gene Expression; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D002583:Uterine Cervical Neoplasms / Q000473:pathology*
D004273:DNA, Neoplasm; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53