Brian Powell and Dr. Allan Judd, Physiology and Developmental Biology

Before the turn of the century, biological research was taken by storm with the discovery of inhibitory RNA (RNAi). In 1995, Guo and Kemphues (Mello & Conte, 2004) stumbled into this discovery and were later awarded the Nobel Prize in science for their discovery. The discovery turned into a technique that is used to knock out genes with laser-like precision, and although the precision of RNAi has come into question recently, it is still a valued technique (Couzin, 2004). However, still in its infancy, the extent to which RNAi is used naturally in different tissues is unclear. To the extent of our knowledge, previous studies have identified RNAi in steroidogenic tissues but have yet to explore the existence or expression of RNAi in the adrenal gland. This project focused on establishing the existence and expression of RNAi in the adrenal gland. The project was successful in that the existence of RNAi was confirmed in adrenal cortical H295R cells, and these results have prompted further research by comparing endogenous levels of RNAi with levels after treatment with dbCAMP. Initial data also gives a possible mechanism for the acute regulation of StAR, and revealed many RNAi that are conserved across species. Although a success, this project was not without its obstacles.

The first obstacle to overcome was to identify the appropriate RNAi that we were going to be search for in the adrenal gland. The project originally specified that the northern blot technique was going to be used to identify each RNAi. The Sanger database (http://microrna.sanger.ac.uk/sequences/) is widely used among scientists to data mine RNAi that have been discovered, and it was used in identifying sequences of RNAi that would be searched for. Initial searches of the database began to turn up a large number of RNAi that would need to be screened for on an individual basis, which would take much more time than anticipated. Due to the lack of efficacy of screening for each RNAi on an individual basis, attention was placed on microarrays. Microarray will screen a large number of RNAi at the same time, making it much more effective. Because the equipment at Brigham Young University was not available for RNAi microarrays, several companies were looked into. Price comparison allowed us to have an RNAi microarray done by LCsciences. LCsciences also allowed 100 additional sequences of our choice to be added to the array, which were filled with non-human sequences. We carefully selected 100 sequences that were not included in the standard test. All of these sequences were selected for their importance relating to enzymes used in the steroidogenic pathways in the adrenal gland. Payne and Hales overview on steroidogenic enzymes was used as a guide to select the appropriate targets (Payne & Hales, 2004). After determining the company to use, samples were prepared.

Preparation of the samples was done in an incubator. Initially there were problems with mold growing with the samples despite stringent efforts to keep a sterile environment. New samples were prepared and grown in large flasks, which were kept free of mold. The number of cells was counted by taking a small sampling from within the flask and placing them on a grid under a light microscope. Only live cells were counted, which could be discriminated against with the use dye. Each large flask was estimated to contain on average about 10E6 cells before RNAi isolation was conducted.

To isolate the RNAi from cells, six samples were selected for use. These cells were subjected to an RNAi isolation procedure developed by Invitrogen. Comparison between RNAi isolation kits concluded that Invitrogen isolation kit would be the most efficient for the price. Before the kit was used on the samples, three of the six samples were treated with dbCAMP for 1hr 15min. RNAi was then isolated from each of the four samples and subjected to spectrophotometer analysis to asses their purity. Spectrophotometer results suggested that the samples were impure, so the isolation kit was repeated on one of the samples to determine if the RNAi could be purified further. Unfortunately repeating the purification procedure did not increase purity ratios. It was determined that the samples could not be purified further, so the spectrophotometer’s accuracy came into question and another machine was used to asses purity. Although purity did improve with the second spectrophotometer, interpretation of the results still concluded that the samples were impure. Later, it was concluded that the spectrophotometers used were not calibrated appropriately. A more accurate technique called nanodrop was used to asses the purity of the samples, and all samples were later found to be pure. Thereafter, one untreated sample and one treated sample were sent to LCsciences for the microarray assay.

The results of the microarray assay were mined for information. First, a large majority of RNAi was determined to be present in the adrenal gland. This information also included the discovery of many RNAi in human tissues that had been found in other species but never been accounted for in human tissues. Second, the data revealed that dbCAMP has several effects on the steroidogenic pathway. Assuming that RNAi has an inhibitory effect when upregulated, enzymes in the steroidogenic pathway that were found to have their RNAi upregulated were assumed to be inhibited. Enzymes that had corresponding RNAi downregulated were assumed to have had a permissive effect on the enzyme function. The changes in RNAi were matched to corresponding gene targets in the steroidogenic pathway, and a map was created (Fig 1). Figure 1 only shows the changes in the steroidogenic pathway that were the most consistent in data analysis. Green arrows represent a permissive affect, and red arrows represent an inhibitory affect. Preliminary results suggest that in response to dbCAMP, steroidogenesis is being altered toward the production of 17OH-Progesterone, which is later modified to produce cortisol. Third, looking at expression levels of RNAi, StAR has several RNAi expressed at high levels. StAR contains a phosphorylation site for PKA, and it is degraded very quickly (Kallen et al., 1998). Given the high expression levels of RNAi against StAR, a possible mechanism of regulation could include RNAi being used to degrade StAR mRNA. After StAR is degraded, its levels would not be replenished if RNAi keeping StAR mRNA in check. In this manner, StAR could be acutely regulated.

Currently research is continuing on this project. New samples have already been prepared and are being analyzed to confirm our results and to make further conclusions. Presentation of this data has been postponed until the new data is analyzed fully. It will be presented at the Endocrinology Society in the summer 07’, and hopefully published in a journal before that time. Overall, this experience has been priceless to my education, and I was able to work with one of the best mentors at Brigham Young University.

References

• Couzin, J. (2004). Molecular biology. RNAi shows cracks in its armor. Science, 306(5699), 1124-1125.
• Kallen, C. B., Arakane, F., Christenson, L. K., Watari, H., Devoto, L., & Strauss, J. F.,3rd. (1998). Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein. Molecular and cellular endocrinology, 145(1-2), 39-45.
• Mello, C. C., & Conte, D.,Jr. (2004). Revealing the world of RNA interference. Nature, 431(7006), 338-342.
• Payne, A. H., & Hales, D. B. (2004). Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine Reviews, 25(6), 947-970