Megan Lundell and Dr. Chin-Yo Lin, Microbiology and Molecular Biology
Estrogens, including estrodial (E2), are known to play a significant role in a number of developmental and physiological processes including the genesis and progression of breast cancer. The action of estrogens is mediated by estrogen receptors (ERs), which function as ligand-dependent transcription factors. Upon ligand binding, ERs interact with cis-regulatory elements of target genes either directly by binding to estrogen response elements or indirectly through interaction with transcription factor complexes and their respective binding sites.1 The primary objective of this project involved the knockdown, or reduction, of NCor, a co-repressor protein known to associate with ER in the presence of E2, in order to discover its effect on the expression of previously identified down-regulated genes in ER breast cancer cells.
To accomplish the primary objective of my research, this project was broken down into three phases. The first phase involved selecting a set of genes to investigate. My colleagues and I carefully researched 137 genes previously identified to induce ER effects in breast cancer cells. These 137 ER-regulated genes were discovered by Lin et al through an extensive microarray study, which surveyed approximately 19,000 genes.1 I was able to narrow my focus by examining the biological functions of these genes and their association with cancer and other diseases. The genes of interest I then selected for my project include HES1, MME, NFIA, PSCA, SLC35A1, and ZNRF3, which are all down-regulated by the ER.
The second phase of my project involved the knockdown of NCoR in MCF7 breast cancer cells. The first attempt to carry out this process involved the use of short hairpin RNA (shRNA), which forms a tight hairpin turn and silences gene expression via RNA interference (RNAi). This process is supposed to decrease the presence of NCoR by resulting in the degradation of NCoR mRNA molecules that are necessary for production of the NCoR protein. However, after several attempts of unsuccessful knockdown, I decided to conduct a similar RNAi experiment using short interfering RNA (siRNA). First, MCF7 cells were grown until they were 60-80% confluent. Next, NCoR siRNA was transfected into the cells. Once inside, NCoR siRNA was copied by the cell and recognized as foreign double stranded RNA. Then, dicer protein not only destroyed all foreign double stranded NCoR RNA, but also all single stranded endogenous NCoR mRNA. Following this process, cells were treated with E2 and real-time PCR was conducted on cDNA created from extracted RNA to confirm that the knockdown was successful.
The final phase of my project investigated how the knockdown of NCoR affected expression of HES 1, MME, NFIA, PSCA, SLC35A1, and ZNRF3, which are down-regulated by ER. To determine expression levels of these genes, real-time PCR was carried out using primers for my six genes of interest and PS2, which is an up-regulated gene used as a positive control. My results showed that reduction of NCoR in MCF7 cells decreased repression of PS2, HES1, MME, PSCA, and SLC35A1. This is shown in Figure 1 by the higher level of gene expression in the set of genes with NCoR knockdown (second column) than in the set of genes without the knockdown (first column). This is what I expected to see since NCoR is a co-repressor in the presence of E2; therefore, when knocked down, it should decrease repression of its associated genes. However, two of my genes, NFIA and ZNRF3, did not show decreased repression. Figure 1 shows how NFIA exhibited no change in expression and ZNRF3 showed further repression. Because chromatin immunoprecipitation (ChIP) experiments conducted by one of my colleagues has shown that both NFIA and ZNRF3 are both affected by NCoR, my results revealed that my primers for NFIA and ZNRF3 were not functioning properly, and that they need to be redesigned. Follow-up work for this project will include designing new primers for both genes and repeating real-time PCR to determine their expression with NCoR knockdown.
Overall, this research has been an invaluable experience. It has been not only intellectually enlarging, but has also strengthened my character as I have learned to problem solve with fellow colleagues, built long term time management skills, and developed the patience required for scientific research. Through this experience, I have been able to help the world at large in the battle of breast cancer because a better understanding of the mechanism by which ER regulates gene expression in ER+ breast cancer cells can lead to new prognostic indicators for drug targets in breast cancer treatments.
References
- Lin, C.-Y., Strom, A., Bega, V.B., Kong, S.L., Thomsen J.S., Yeo, A.L., Doray, B., Chan, W.C., Bangausamy, D.K., Tang, S., Chon, A., Bajic, V.B., Ramasamy, A., Vergara, L., Miller, L.D., Gustafsson, J.-A., and Liu, E.T. Discovery of estrogen receptor α target genes and response elements in breast tumor cells. Genome Biology 2004; 5(9):R66.