Ribozyme targeted on HPV16E6 mRNA induced apoptosis on human cervical carcinoma CaSKi cells
1Oncology Center, Zhujiang Hospital, The First Military Medical University, Guangzhou, Guang dong, PR China. zyfcn@yahoo.com
Background & 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.
Conclusion:
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.
Insights
This study shows that an anti-HPV16E6-ribozyme effectively targets human papillomavirus type 16 E6 mRNA in cervical cancer cells, inducing apoptosis and inhibiting proliferation. This ribozyme therapy offers a potential new strategy for treating cervical cancer.
Area of Science:
- Molecular Biology
- Cancer Research
- RNA Therapeutics
Background:
- Human papillomavirus (HPV) is a primary cause of cervical cancer.
- Ribozymes are catalytic RNA molecules capable of cleaving specific RNA targets.
- The HPV16 E6 oncoprotein plays a critical role in cervical carcinogenesis.
Purpose of the Study:
- To characterize a cultured cervical cancer cell line (CaSKi) transfected with an anti-HPV16E6-ribozyme (HRz).
- To evaluate the effect of the HRz on cervical cancer cell proliferation and apoptosis.
Main Methods:
- Computer-aided design of a ribozyme targeting HPV16E6 mRNA.
- Lipofectin-mediated transfection of CaSKi cells with HRz or an empty plasmid.
- Analysis of E6 mRNA expression using Northern blot.
- Cell cycle analysis via flow cytometry.
- Assessment of apoptosis using Hoechst staining and TUNEL assay.
- Determination of protein expression (HPV16E6, c-myc, bcl-2, p53, Fas) by flow cytometry.
Main Results:
- Stable expression of HRz mRNA was confirmed in transfected CaSKi-R cells.
- HRz significantly reduced HPV16E6 mRNA levels in CaSKi-R cells compared to controls.
- Increased apoptosis rates and G2 cell cycle arrest were observed in CaSKi-R cells.
- Downregulation of c-myc and bcl-2, and upregulation of p53 were noted in CaSKi-R cells.
Conclusions:
- Targeted ribozyme therapy against HPV16E6 mRNA can induce apoptosis in human cervical cancer cells.
- The observed apoptosis is likely mediated by reduced E6 gene expression, leading to downstream changes in c-myc, bcl-2, and p53.
- This ribozyme strategy holds promise for cervical cancer treatment.
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