Expression of Pdlim7 in Cancer Cells




Yanxi Jiang

Introduction

Pdlim7 is made of an N-terminal PDZ domain and three C-terminal LIM domains. The PDZ domain interacts with actin-binding proteins. Its LIM domains bind to proteins involved in mitogenic or insulin signaling such as receptor tyrosine kinases (Jennifer Krcmery et al. 2010).
When PDZ-LIM proteins do not function well, they can promote tumor growth and malignant transformation. In one study, Pdlim7 played a role in tumorigenesis in a mechanism by triggering mitosis and decreasing p53 antiproliferative activity (Cho-Rok Jung et al. 2010). Kaplan-Meier survival analysis showed that the expression level of Pdlim7 is related to the survival rate of breast cancer patients. Also, PDZ domain could interact with human papillomaviruses, which is related to cervical cancer (Kazunori Nagasaka et al. 2013). HPV-induced cancer cells often have viral sequences integrated into the cellular DNA.
Additionally, Pdlim7 produces multiple alternatively spliced mRNA variants, two of which include a unique terminal exon.
Additionally, Pdlim7 produces a slice variant.

Materials and Methods
In this study, 6 cell lines were used: MDA-MB_231, MDA-MB-468, MCF-7, T47-D, MDA-MB-435(breast cancer cells) and HeLa cells( cervical cancer cells).
RT-PCR was performed using specifically designed primers. One pair only amplified the splice variant with long terminal exon (arrows in Fig.1 and Fig.3). Another pair only amplified the splice variant with exon 8 (arrows in Fig.2 and Fig.3). GAPDH was used as the housekeeping control. Samples with no templates were used as negative template control.
RT-PCR products were visualized on 1.0 % agarose gels. PCR products were purified using QIAquick® PCR Purification Kit. Purified PCR products were sequenced by GENEWIZ® and were confirmed by BLAST.

Results
This study demonstrated that the Pdlim7 gene produces splice variants with the unique terminal exon that only highly expressed in HeLa cells, but not in breast cancer cells.

Discussion
In this study, the Pdlim7 gene produced a splice variant with the unique terminal exon only in HeLa cells but not in breast cancer cells. Different types of cancer might utilize specific Pdlim7 translation products as scaffolding proteins to facilitate the production different signaling complexes.

References
Aartjan J.W. te Velthuis AJ, Bagowski CP. 2007. PDZ and LIM domain-encoding genes: molecular interactions and their role in development. Scientific- WorldJournal 7: 1470–92.

Sheng M, Sala C. 2001. PDZ domains and the organization of supramo- lecular complexes. Annu Rev Neurosci 24: 1–29.
Fanning AS, Anderson JM. 1999. PDZ domains: fundamental building blocks in the organization of protein complexes at the plasma membrane. J Clin Invest 103: 767–72.
Jelen F, Oleksy A, Smietana K, et al. 2003. PDZ domains – Common players in the cell signaling. Acta Biochim Pol 50: 985–1017.

Kazunori Nagasaka, Kei Kawana et al. 2013. PDZ Domains and Viral Infection: Versatile Potentials of HPV-PDZ Interactions in relation to Malignancy. BioMed Research International Volume 2013: 9.
Bach I. 2000. The LIM domain: regulation by association. Mech Dev 91: 5–17.
Kadrmas JL, Beckerle MC. 2004. The LIM domain: from the cytoskeleton to the nucleus. Nat Rev Mol Cell Biol 5: 920–31
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Bagheri-Yarmand R, Mazumdar A, Sahin AA, et al. 2006. LIM kinase 1 increases tumor metastasis of human breast cancer cells via regulation of the urokinase-type plasminogen activator system. Int J Cancer 118: 2703– 10.
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http://www.cdc.gov/cancer/cervical/basic_info/
http://www.cdc.gov/cancer/breast/basic_info/



Figures


Figure 1-Figure 1. RT-PCR results of the amplification of the transcript with the above primer pair. L->R: MDA-MB-435, MDA-MB-468, T47-D, HeLa cells, MCF-7, MDA-MB-231 and negative template control. GAPDH was used as a ‘loading’ control. * :Sequencing result, ident%=99


Figure 2-Figure 2. RT-PCR results of the amplification of the transcript with the above primer pair. L->R: MDA-MB-435, MDA-MB-468, T47-D, HeLa cells, MCF-7, MDA-MB-231 and negative template control. GAPDH was used as a ‘loading’ control. * :Sequencing result, ident%=99


Figure 3-Figure 3. RT-PCR results of the amplification of the transcript with the above primer pair. L->R: MDA-MB-435, MDA-MB-468, T47-D, HeLa cells, MCF-7, MDA-MB-231 and negative template control. GAPDH was used as a ‘loading’ control.


Summary

Pdlim7 is a member of the LIM family of proteins and is comprised of an N-terminal PDZ domain, and three C-terminal LIM domains. Pdlim7 plays a role in tumorigenesis by promoting cancer cell invasion and metastasis. In this study, the Pdlim7 gene produced a splice variant with unique terminal exon highly expressed in HeLa cell, but not in breast cancer cells.

Full Paper

Acknowledgments

I would like to thank Dr. Wei for providing the RNA of tested cell lines. I would like to thank my classmates for helping me. I would like to thank Kate Reid and Catharina Grubaugh for going above and beyond their duties as Teaching Assistants and for their never-ending patience and support throughout this project. I would like to thank Dr. Rubin for his guidance and for making this project possible.


This document was last modified 05/14/2014.
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