MicroRNAs (miRNAs) are short 21-23 nucleotide (nt) nucleic acid species that have a seed-sequence complimentary to target RNAs which can then be negatively regulated via the RNA silencing pathway. miR-122 is the most highly expressed miRNA in the liver, accounting for 70% of the miRNA reads and is involved in hepatocyte differentiation and cholesterol/fatty-acid synthesis. Previous studies have indicated that total abrogation of miR-122 or competition of miR-122 by short hairpin RNAs (shRNAs) in mice caused an increased expression of the Dlk-Dio3 locus, which is highly expressed in development but downregulated in adults and ultimately leads to hepatocellular carcinoma (HCC). Furthermore, it is known that activation of the Dlk-Dio3 locus alone has been shown to lead to the development of HCC but the specifics of miR-122’s regulatory role have not been elucidated. To determine the effects of miR-122 on the Dlk-Dio3 locus, I am using human HCC cells that contain a 3-bp deletion down-stream from the miR-122 transcript. We predict that the 3-bp deletion prevents proper miR-122 transcription. By transfecting the cells with a miR-122 mimic transcript to introduce its expression, I can compare the levels of RNA expression in liver cells containing miR-122 to those without by using qPCR and small-RNA sequencing. Preliminary data indicates that cells transfected with the miR-122 mimic have decreased levels of expression of RNAs located in the Dlk-Dio3 locus such as Rtl1 and positive control targets such as Aldoa. To further confirm this relationship, I am planning on using CRISPR-mediated homology-directed repair to replace the 3-bp deletion in my cell line and endogenously re-express miR-122 in order to study its regulatory role at the Dlk-Dio3 locus.

Hypoxia refers to low concentrations of dissolved oxygen (DO) in a body of water, and can result in death of marine biota. Hypoxic events have increased since the 1970s in shallow coastal and estuarine areas to the point where DO has arguably changed more drastically than other environmental variables of importance to these ecosystems. Studies have found that the main drivers increasing the frequency and intensity of hypoxic events are eutrophication as a result of nutrient loading and increasing water temperatures due to climate change. Puget Sound in Washington State is particularly susceptible to hypoxia because the geological features of its basin restrict water circulation and the Sound receives a high influx of nutrients from rivers and anthropogenic activity. Studies have reported hypoxia in regions of Puget Sound, including Sisters Point, Lynch Cove, and Hood Canal. This study provides a temporal and spatial analysis of DO in Possession Sound, an inlet of Puget Sound where the Snohomish River empties, to contribute to the growing understanding of hypoxia in Puget Sound, particularly in an estuarine environment. DO data were analyzed along with water temperature data to determine if hypoxia occurred in Possession Sound and to assess where potential hypoxia is more likely to occur. These data were collected by students at the Ocean Research College Academy (ORCA) at six sites in Possession Sound from 2017 to 2020 using an EXO Sonde instrument which allows for vertical analysis of DO in the water column. Preliminary results show that potentially toxic concentrations of DO occurred at depth during the winter months with a minimum value of 4.08 mg/L. It also appears that DO concentrations vary substantially between sites, years, seasons, and depths.