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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 human chorionic beta subunit in uterine cervix. (A) Electrophoretic separation showing representative RT-PCR results performed for uterine cervix carcinomas. A 210 bp fragment of hCGβ was amplified for the uterine cervix carcinoma (lanes 1 – 4) and placenta (lane 5) samples. Molecular size marker is given in lane M. (B) RT-PCR analysis of total RNA from normal myometrium (lanes 1–2) and uterine cervix tissue (lanes 3–6). Lanes 1, 3 and 5 used RT-PCR primers specific to the hCGβ mRNA as was done in Figure 1A. Lanes 2, 4 and 6 are control RT-PCRs with primers specific to the β-actin mRNA (predict a 509 bp product) to confirm the integrity of the RNA. M – size marker.
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Figure 1: Expression of human chorionic beta subunit in uterine cervix. (A) Electrophoretic separation showing representative RT-PCR results performed for uterine cervix carcinomas. A 210 bp fragment of hCGβ was amplified for the uterine cervix carcinoma (lanes 1 – 4) and placenta (lane 5) samples. Molecular size marker is given in lane M. (B) RT-PCR analysis of total RNA from normal myometrium (lanes 1–2) and uterine cervix tissue (lanes 3–6). Lanes 1, 3 and 5 used RT-PCR primers specific to the hCGβ mRNA as was done in Figure 1A. Lanes 2, 4 and 6 are control RT-PCRs with primers specific to the β-actin mRNA (predict a 509 bp product) to confirm the integrity of the RNA. M – size marker.

Mentions: To verify at the molecular level the presence of hCGβ in uterine cervix carcinoma, total RNA was isolated from cancer tissue of patients, reverse transcribed, and a 210 bp fragment corresponding to hCGβ nucleotides 348–557 [GenBank: NM_033043] was amplified. In total, 15 samples of human uterine cervix carcinoma and 4 samples of placenta were analyzed. A representative example of the RT-PCR data is shown in Figure 1A where it is evident that we were able to amplify a specific fragment of hCGβ. Sequencing of the amplified fragment confirmed its identity with hCGβ.


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 human chorionic beta subunit in uterine cervix. (A) Electrophoretic separation showing representative RT-PCR results performed for uterine cervix carcinomas. A 210 bp fragment of hCGβ was amplified for the uterine cervix carcinoma (lanes 1 – 4) and placenta (lane 5) samples. Molecular size marker is given in lane M. (B) RT-PCR analysis of total RNA from normal myometrium (lanes 1–2) and uterine cervix tissue (lanes 3–6). Lanes 1, 3 and 5 used RT-PCR primers specific to the hCGβ mRNA as was done in Figure 1A. Lanes 2, 4 and 6 are control RT-PCRs with primers specific to the β-actin mRNA (predict a 509 bp product) to confirm the integrity of the RNA. M – size marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Expression of human chorionic beta subunit in uterine cervix. (A) Electrophoretic separation showing representative RT-PCR results performed for uterine cervix carcinomas. A 210 bp fragment of hCGβ was amplified for the uterine cervix carcinoma (lanes 1 – 4) and placenta (lane 5) samples. Molecular size marker is given in lane M. (B) RT-PCR analysis of total RNA from normal myometrium (lanes 1–2) and uterine cervix tissue (lanes 3–6). Lanes 1, 3 and 5 used RT-PCR primers specific to the hCGβ mRNA as was done in Figure 1A. Lanes 2, 4 and 6 are control RT-PCRs with primers specific to the β-actin mRNA (predict a 509 bp product) to confirm the integrity of the RNA. M – size marker.
Mentions: To verify at the molecular level the presence of hCGβ in uterine cervix carcinoma, total RNA was isolated from cancer tissue of patients, reverse transcribed, and a 210 bp fragment corresponding to hCGβ nucleotides 348–557 [GenBank: NM_033043] was amplified. In total, 15 samples of human uterine cervix carcinoma and 4 samples of placenta were analyzed. A representative example of the RT-PCR data is shown in Figure 1A where it is evident that we were able to amplify a specific fragment of hCGβ. Sequencing of the amplified fragment confirmed its identity with hCGβ.

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