Korean J Obstet Gynecol.  2010 Jan;53(1):35-42. 10.5468/kjog.2010.53.1.35.

Comparison of cell growth suppression in SiHa cervical carcinoma cell line by human papillomavirus type 16 E6/E7 siRNAs

• Park SH
• Park BJ
• Kim YW
• Ro DY
• Kim TE
• Jung JK
• Bae SM
• Ahn WS

• KMID: 2077958
• DOI: http://doi.org/10.5468/kjog.2010.53.1.35

Abstract

OBJECTIVE
Human cervical cancer is caused by the high-risk types of human papillomavirus (HPV) such as HPV16, which possess the E6 and E7 oncogenes, whose expressions are a prerequisite for cancer development. We performed this study to compare the efficacy of antitumor activity by HPV siRNA which silences only E6 or both E6/E7.
METHODS
We transfected siRNA 377 (HPV16 E6 siRNA), siRNA 3 (HPV16 E6 siRNA), and siRNA 198 (HPV16 E7 siRNA) into SiHa cell line (siRNA 377 silences only E6, and siRNA 3 and siRNA 198 silence both E6 and E7). We experimented cell counts and morphologic changes 24 and 48 hours after transfection and expressions of HPV16 E6/E7 mRNA by RT-PCR.
RESULTS
siRNA 377, siRNA 3, and siRNA 198 suppressed the cell growth. siRNA 3 and siRNA 198 were more potent than siRNA 377 in cell growth suppression. siRNA 377 knocked down the expression of E6 mRNA, and both siRNA 3 and siRNA 198 knocked down the expression of E6/E7 mRNA.
CONCLUSION
Our results suggest that simultaneous suppression of E6 and E7 was more potent than E6-specific suppression in cancer cell growth.

Keyword

Cervical cancer; Human papillomavirus; E6; E7; siRNA

MeSH Terms

Cell Count
Cell Line
Humans
Oncogenes
RNA, Messenger
RNA, Small Interfering
Transfection
Uterine Cervical Neoplasms
RNA, Messenger
RNA, Small Interfering

Figure

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  Fig. 1 Effects of siRNA on growth inhibition in SiHa cervical cancer cell line in vitro. Cells were transfected with universal siRNA, siRNA 3, siRNA 198 and siRNA 377 at 50 nM. Cells were trypsinized for direct cell counting after 24 and 48 hours. Cell growth inhibitions were compared with the universal siRNA control by t-test. *P<0.05, **P<0.01.

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  Fig. 2 siRNA-mediated morphological and numerical changes in SiHa cervical cancer cell line in vitro. Cells were transfected with universal siRNA, siRNA 3, siRNA 198 and siRNA 377 at 50 nM. Typical photographs were shown after 48 hours (×400 for all photographs).

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  Fig. 3 RT-PCR analysis for expressions of the HPV16 E6, E7 and β-actin mRNA. Cells were transfected with universal siRNA, siRNA 377, siRNA 3 and siRNA 198 at 50 and 100 nM. RNA was extracted for RT-PCR after 48 hours.

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  Fig. 4 Effects of siRNA on p53 expressions in SiHa cell line. Cells were transfected with universal siRNA, siRNA 377, siRNA 3 and siRNA 198 at 50 nM. Immunoblot analysis using specific p53 antibody was performed after 24 and 48 hours. The cells were collected and the extracts (40 µg) were run on 10% SDS-PAGE.

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