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Reduction of human chorionic gonadotropin beta subunit expression by modified U1 snRNA caused apoptosis in cervical cancer cells.

Jankowska A, Gunderson SI, Andrusiewicz M, Burczynska B, Szczerba A, Jarmolowski A, Nowak-Markwitz E, Warchol JB - Mol. Cancer (2008)

Bottom Line: This study documents the molecular presence of human chorionic gonadotropin beta subunit in uterine cervix cancer tissues and investigates a novel technique to reduce hCGbeta levels based on expression of a modified U1 snRNA as a method to study the hormone's role in biology of human cervical cancer cells cultured in vitro.Reduction of hCGbeta levels resulted in a significantly increased apoptosis rate with almost 90% of cells transfected with modified anti-hCGbeta U1 snRNAs showing morphological changes characteristic of the apoptotic process.These data suggest that human chorionic gonadotropin beta subunit may act as a tumor growth-stimulating factor.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Medical Sciences, Rokietnicka 5D, Poznan, Poland. ajanko@amp.edu.pl

ABSTRACT

Background: Secretion of human chorionic gonadotropin, especially its beta subunit by malignant trophoblastic tumors and varieties of tumors of different origin is now well documented; however the role of hCG in tumorogenesis is still unknown.

Results: This study documents the molecular presence of human chorionic gonadotropin beta subunit in uterine cervix cancer tissues and investigates a novel technique to reduce hCGbeta levels based on expression of a modified U1 snRNA as a method to study the hormone's role in biology of human cervical cancer cells cultured in vitro. The property of U1 snRNA to block the accumulation of specific RNA transcript when it binds to its donor sequence within the 3' terminal exon was used. The first 10 nucleotides of the human U1 snRNA gene, which normally binds to the 5'ss in pre-mRNA were replaced by a sequence complementary to a 10-nt segment in the terminal exon of the hCGbeta mRNA. Three different 5' end-mutated U1 snRNA expression plasmids were tested, each targeting a different sequence in the hCGbeta mRNA, and we found each one blocked the expression of hCGbeta in HeLa cells, a cervix carcinoma cell line, as shown by immunohistochemistry and qRT-PCR. Reduction of hCGbeta levels resulted in a significantly increased apoptosis rate with almost 90% of cells transfected with modified anti-hCGbeta U1 snRNAs showing morphological changes characteristic of the apoptotic process.

Conclusion: These data suggest that human chorionic gonadotropin beta subunit may act as a tumor growth-stimulating factor.

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Related in: MedlinePlus

Expression of hCGβ in HeLa cells transfected with anti-hCGβ U1 snRNA. Total RNA was isolated from cells, and cDNA was synthesized. HeLa cells transfected with plasmids 702P/767A, 702P/767B and 702P/767C expressed decreased hCGβ message relative to control cells and cells transfected with control plasmid 702P. Columns – mean of triplicate experiments; bars – SE.
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Figure 4: Expression of hCGβ in HeLa cells transfected with anti-hCGβ U1 snRNA. Total RNA was isolated from cells, and cDNA was synthesized. HeLa cells transfected with plasmids 702P/767A, 702P/767B and 702P/767C expressed decreased hCGβ message relative to control cells and cells transfected with control plasmid 702P. Columns – mean of triplicate experiments; bars – SE.

Mentions: Because inhibition levels are limited by transfection efficiency – for example a 90% transfection rate can at most give a 10-fold inhibition [11], and in case of using HeLa cell line and lipofectamine™2000 transfection reagent the efficiency estimated by GFP reporter gene expression was lower than 30% we applied quantitative RT-PCR to show the silencing of human chorionic gonadotropin beta subunit expression. hCGβ transcripts were detected in both control and transfected cells however, all 3 U1 snRNA anti-hCGβ constructs clearly reduced the hormone's transcription as was showed by real time PCR results. The highest – 3-fold inhibition was observed for 702P/767A plasmid. Constructs 702P/767B and 702P/767C decreased hCGβ mRNA amount 2-times while the expression level of cells transfected with 702P plasmid and control cells was the same (Figure 4). Using FuGENE – HD Transfection Reagent we got 60% transfection efficiency and the level of hormone inhibition was the same as when transfection efficiency was 30% (data not shown).


Reduction of human chorionic gonadotropin beta subunit expression by modified U1 snRNA caused apoptosis in cervical cancer cells.

Jankowska A, Gunderson SI, Andrusiewicz M, Burczynska B, Szczerba A, Jarmolowski A, Nowak-Markwitz E, Warchol JB - Mol. Cancer (2008)

Expression of hCGβ in HeLa cells transfected with anti-hCGβ U1 snRNA. Total RNA was isolated from cells, and cDNA was synthesized. HeLa cells transfected with plasmids 702P/767A, 702P/767B and 702P/767C expressed decreased hCGβ message relative to control cells and cells transfected with control plasmid 702P. Columns – mean of triplicate experiments; bars – SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335103&req=5

Figure 4: Expression of hCGβ in HeLa cells transfected with anti-hCGβ U1 snRNA. Total RNA was isolated from cells, and cDNA was synthesized. HeLa cells transfected with plasmids 702P/767A, 702P/767B and 702P/767C expressed decreased hCGβ message relative to control cells and cells transfected with control plasmid 702P. Columns – mean of triplicate experiments; bars – SE.
Mentions: Because inhibition levels are limited by transfection efficiency – for example a 90% transfection rate can at most give a 10-fold inhibition [11], and in case of using HeLa cell line and lipofectamine™2000 transfection reagent the efficiency estimated by GFP reporter gene expression was lower than 30% we applied quantitative RT-PCR to show the silencing of human chorionic gonadotropin beta subunit expression. hCGβ transcripts were detected in both control and transfected cells however, all 3 U1 snRNA anti-hCGβ constructs clearly reduced the hormone's transcription as was showed by real time PCR results. The highest – 3-fold inhibition was observed for 702P/767A plasmid. Constructs 702P/767B and 702P/767C decreased hCGβ mRNA amount 2-times while the expression level of cells transfected with 702P plasmid and control cells was the same (Figure 4). Using FuGENE – HD Transfection Reagent we got 60% transfection efficiency and the level of hormone inhibition was the same as when transfection efficiency was 30% (data not shown).

Bottom Line: This study documents the molecular presence of human chorionic gonadotropin beta subunit in uterine cervix cancer tissues and investigates a novel technique to reduce hCGbeta levels based on expression of a modified U1 snRNA as a method to study the hormone's role in biology of human cervical cancer cells cultured in vitro.Reduction of hCGbeta levels resulted in a significantly increased apoptosis rate with almost 90% of cells transfected with modified anti-hCGbeta U1 snRNAs showing morphological changes characteristic of the apoptotic process.These data suggest that human chorionic gonadotropin beta subunit may act as a tumor growth-stimulating factor.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Medical Sciences, Rokietnicka 5D, Poznan, Poland. ajanko@amp.edu.pl

ABSTRACT

Background: Secretion of human chorionic gonadotropin, especially its beta subunit by malignant trophoblastic tumors and varieties of tumors of different origin is now well documented; however the role of hCG in tumorogenesis is still unknown.

Results: This study documents the molecular presence of human chorionic gonadotropin beta subunit in uterine cervix cancer tissues and investigates a novel technique to reduce hCGbeta levels based on expression of a modified U1 snRNA as a method to study the hormone's role in biology of human cervical cancer cells cultured in vitro. The property of U1 snRNA to block the accumulation of specific RNA transcript when it binds to its donor sequence within the 3' terminal exon was used. The first 10 nucleotides of the human U1 snRNA gene, which normally binds to the 5'ss in pre-mRNA were replaced by a sequence complementary to a 10-nt segment in the terminal exon of the hCGbeta mRNA. Three different 5' end-mutated U1 snRNA expression plasmids were tested, each targeting a different sequence in the hCGbeta mRNA, and we found each one blocked the expression of hCGbeta in HeLa cells, a cervix carcinoma cell line, as shown by immunohistochemistry and qRT-PCR. Reduction of hCGbeta levels resulted in a significantly increased apoptosis rate with almost 90% of cells transfected with modified anti-hCGbeta U1 snRNAs showing morphological changes characteristic of the apoptotic process.

Conclusion: These data suggest that human chorionic gonadotropin beta subunit may act as a tumor growth-stimulating factor.

Show MeSH
Related in: MedlinePlus