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Published on: September 16, 2016
[Construction of adenoviral vector encoding Calponin-1 siRNA and its effect on human myometrium cells in vitro]
Yonghong Gu1, Changju Zhou, Lingyu Hu
1Department of Pathology, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Objective:
To investigate the effect of Calponin-1 suppression on human myometrium cells through adenovirus mediated siRNA.
Methods:
Human uterine smooth muscle tissues were digested with enzymes, cultured and confirmed with immunocytochemistry. Adenovirus siRNA-Calponin-1 plasmid was transfected into primary cultured uterine smooth muscle cells in vitro. The expressions of Calponin-1 mRNA and protein were analyzed by RT-PCR and Western blot, respectively.
Results:
The pAdEasy-pShuttle-U6-Calponin-1 siRNA plasmid was successfully constructed, and Calponin-1 siRNA mediated by recombinant adenovirus resulted in markedly reduced expression of Calponin-1 mRNA and protein in human myometrium cells. The gray values of Calponin-1 mRNA in the uterine smooth muscle cells in the experimental, blank control, and empty vector groups were 316.3+/-39.2, 1048.5+/-126.4 and 1027.2+/-127.5, respectively. The gray values of Calponin-1 protein were 323.3+/-43.2, 1021.5+/-143.4, and 1019.2+/-144.5, respectively. The difference between the experimental group and the blank control group as well as the empty vector group was significant (P< 0.05). There was no significant difference between the empty vector group and the blank control group (P>0.05).
Conclusion:
The pAdEasy-pShuttle-U6-Calponin-1 siRNA plasmid can inhibit the expression of Calponin-1 in human myometrium cells in vitro, which may be a useful approach to determine the role of Calponin-1 in delivery.
Insights
Calponin-1 suppression in human myometrium cells was achieved using adenovirus-mediated siRNA. This method effectively reduced Calponin-1 mRNA and protein expression, offering a new way to study its role in childbirth.
Area of Science:
- Molecular biology
- Cell biology
- Reproductive biology
Context:
- Calponin-1 is a protein found in smooth muscle cells, including the myometrium.
- Understanding the function of Calponin-1 in the myometrium is crucial for studying labor and delivery.
- Gene silencing techniques offer a way to investigate protein function in cellular models.
Purpose:
- To investigate the effect of suppressing Calponin-1 expression in human myometrium cells.
- To develop and validate an adenovirus-mediated siRNA approach for Calponin-1 knockdown.
- To confirm the reduction of Calponin-1 at both mRNA and protein levels.
Summary:
- A recombinant adenovirus carrying siRNA targeting Calponin-1 was successfully constructed.
- Transfection of this adenovirus into primary human myometrium cells significantly reduced Calponin-1 mRNA and protein expression.
- Statistical analysis confirmed the significant inhibitory effect of the siRNA (P<0.05).
Impact:
- This study demonstrates a viable method for inhibiting Calponin-1 expression in human myometrium cells in vitro.
- The developed technique provides a valuable tool for further research into the specific role of Calponin-1 in myometrial function and the process of delivery.
- Findings contribute to a deeper understanding of the molecular mechanisms regulating uterine contractility.
