Integration of Bioinformatics Approaches and Experimental Validations to Understand the Role of Notch Signaling in Ovarian Cancer

Bioinformatics is a useful way to extract information from data sets. Experimentation is equally important for validation. We combine both approaches to investigate the role of notch signaling in ovarian cancer.

The main advantage of this technique is that researchers can utilize the information from the bioinformatics databases to plan their research direction prior to conducting an experiment. With accumulated bioinformatics data of human diseases, scientists are no longer blindfolded. Experimentation can be performed to validate specific findings, which can lead to improved therapies or diagnosis of diseases.

For meta-A analysis of the association of a gene of interest with a specific type of cancer, create an account with an academic-affiliated email address to access the PRECOG database and enter the email address and password associated with the account into the login terminal. Click View details and input the gene of interest into the search bar. Then use the scroll bar to obtain the survival Z-score for the specific cancer type of interest.

To investigate the association between gene expression levels and ovarian cancer stages, navigate to the CSIOVDB web page and input the name of the gene of interest into the search bar. Then click on the Survival tab. To assess gene expression data across diverse, normal, and tumor tissues, navigate to the GENT portal, and click the Search tab.

In the keyword section, select the gene symbol for the terms from the dropdown menu, input the gene's symbol of the gene of interest, and select Tissue for the type option. Click the Search button. Summary graphs of the gene expression in normal and tumor tissues of different cancer types, based on the U133A and U122 Plus 2 platforms will be displayed.

Then click the Result Data Download link to access the detailed information about the gene expression values, tissue types, and data sources. To search for genetic alterations and signaling networks, open the CBIO portal web page, and using the query on the landing page, click the organs or tissues of interest under Select Studies section, select the particular study of interest, and click Query By Gene. In the Select Genomic Profiles section, select the mutations, putative copy number alterations from GISTIC, or mRNA expression option.

Select the corresponding data from the Select Patient/Case Set menu, and enter the target gene symbols into the query box of Enter Genes. Click the Submit Query button, and open the Network tab to retrieve the desired gene network. Then click the File tab, and select Save As Image PNG for network image downloading.

To create flies with NICD or NICD and mam overexpression, prepare the appropriate fly stocks, and apply the temporal and regional gene expression targeting technique to control the spatiotemporal gene expression, according to standard protocols. Raise the flies at 18 degrees Celsius until adulthood, before switching to 29 degrees Celsius with yeast for 48 hours. At the end of the 29 degrees Celsius incubation, add three milliliters of PBS to an embryo collection dish, and anesthetize the fly culture with a carbon dioxide pad.

Use a dissecting microscope to identify an anesthetized female fly, and use a pair of dissecting forceps to carefully grab the lower thorax of the fly. Submerge the fly in the PBS within the embryo collection dish, and use a second pair of forceps to pinch the lower abdomen, pulling gently to release the internal organs. Locate and detach the pair of ovaries from the fly body, and break the muscular sheath, located at the posterior end of the ovaries, to separate the ovarials.

Then place the isolated ovaries in a 1.5 milliliter centrifuge tube, containing 500 microliters of PBS on ice. When all of the ovaries have been collected, replace the PBS with 500 microliters of 4%formaldehyde, and place the tube on a nutator for 10 minutes. At the end of the incubation, discard the fixative and wash the ovaries with three, 15-minute rinses in one milliliter of PBT per wash.

After the last wash, label the ovaries with 150 microliters of 10 micrograms per milliliter DAPI for 10-15 minutes on the nutator. At the end of the incubation, wash the ovaries one time for 10 minutes with one milliliter of PBT, followed by two 10-minute washes in PBS. After the second wash, remove all but the last 300 microliters of PBS, and use a 200 microliter pipette tip to triterate the ovaries several times to free the egg chambers.

Next, gently sediment the ovarian tissue through a quick spin in a microcentrifuge, and remove as much PBS as possible without disturbing the ovary pellet. Using a 1, 000 microliter pipette tip, transfer about three drops of mounting solution to the tube, and use a 200 microliter pipette tip with approximately 0.33 millimeters trimmed from the end to transfer the entire 120 microliters of ovary-containing mounting solution onto a glass microscope slide. Gently place a coverslip glass over the mounting solution, and seal the edges with transparent nail polish.

Then use a confocal microscope at a 10x magnification with a 0.8 aperture to acquire images of the stained and mounted ovaries. Using the PRECOG portal as demonstrated, the Z-scores of NOTCH2, NOTCH3, and MAML1 in ovarian cancer can be obtained. The negative Z-score values indicate the poor overall survival of patients with high expression levels of the three genes.

Using the CSIOVD database to confirm these findings, further indicates that a high expression of NOTCH2, NOTCH3, and MAML1 correlates with a poor overall and disease-free survival. Further permutation tests suggest that NOTCH2, NOTCH3, and MAML1 are highly expressed in tumor tissues. Based on these three core genes, a signaling network can be created to provide the 50 most frequently altered neighboring genes that are also in the same pathway with the highest mutation rates.

Of interest, the overexpression of the Drosophila-equivalent NOTCH gene, NICD, alone, does not induce tumors in Drosophila. Whereas the overexpression of NICD and mam together induces tumors in fruit flies, as evidenced by the presence of multiple epithelial layers and accumulated cells. Further experimentation validation can pave the way for the identification of potential new drug therapy targets, disease biomarkers, and personalized treatments.