Abstract
MicroRNAs (miRNAs) have shown to be key gene expression regulators in development processes, aging, cancer, and other diseases [8]. miR-23 is a miRNA that is believed to have a role in cancer development, and overexpression of miR-23 could potentially suppress tumor growth [9]. In order to achieve this, the genes regulated by miR-23 need to be established. The gene of interest in this experiment is OXSR1, which is responsible for regulating downstream kinases in response to environmental stress [6]. Reactive oxygen species (ROS) have been found to contribute to the survival, proliferation and metastasis in a variety of cancer cells, but if the oxidative stress is left uncontrolled, increased ROS over the threshold of death induces cell death in cancer cells [3,5]. The hypothesis of this experiment is that if OXSR1 is identified as a target of miR-23, then miR-23 overexpression will decrease OXSR1 expression levels. If OXSR1 is identified as a target of miR-23, it could possibly increase ROS above the threshold of death and lead to killing cancer cells. This hypothesis was tested by isolating RNA from HeLa cells transfected with mimics for either miR-scramble or miR-23 and then performing quantitative polymerase chain reaction (qPCR) using primers for the OXSR1 gene, a known target of miR-23, and a housekeeping gene. The cycle threshold (CT) was measured for each sample and the results were analyzed using the ∆∆CT method, which determined whether OXSR1 was a target of miR-23. The results of this experiment supported the hypothesis and found that OXSR1 expression is decreased by miR-23 overexpression, which could lead to further studies investigating whether this method could inhibit tumor growth in certain types of cancers.
Introduction
miRNAs were discovered in 1993 as a class of small, conserved, non-coding RNA molecules, and have since then shown to be regulators of post-transcriptional gene expression [8]. miRNAs have been found to be abnormally expressed in cancers, supporting the idea that their regulatory roles are significant [2]. Pre-cursor RNAs are ~70-120 nucleotide nucleotides long and are processed into mature miRNAs that are ~20-22 nucleotides long. These mature miRNAs can then bind to their complementary target sequences on mRNAs and
recruit a RNA Induced Silencing Complex (RISC). The RISC decreases expression of the target gene by repressing translation and degrading mRNA. Scientists have discovered that miRNAs have a significant role in multiple diseases, and to understand their effects on the organism, it is important to identify what their targets are [7]. Once the targets of miRNAs are known, they can be used to treat diseases caused by abnormal gene expression.
To further investigate miRNAs, the gene of interest that will be studied is OXSR1 (oxidative stress responsive kinase 1), which is responsible for regulating downstream kinases in response to environmental stress [6]. The purpose of this experiment is to identify whether OXSR1 is a target gene of miR-23. miR-23 is thought to have a role in the development of various kinds of cancers and an understanding of how it affects cancer progression can potentially lead to developing new therapeutic strategies to fight against cancers linked to miR-23 [9]. The significance of OXSR1 is that it has been shown to be upregulated in human cervical cancer, while interestingly, other studies have shown that miR-23 is downregulated in cervical cancer [1,4]. The genes that are affected due to the downregulation of miR-23 in cervical cancer are unknown, and the purpose of this experiment is to identify whether OXSR1 is a target gene of miR-23. The target gene was chosen using the website TargetScan, which identifies likely targets of miR-23. To amplify the gene and quantify expression levels using qPCR, primers for OXSR1 are then designed using Primer3Plus. RNA will then be isolated from HeLa cells transfected with either miR mimics for miR-23 or miR-scramble sequences, and converted into cDNA using primers, reverse transcriptase (RT), DNA polymerase, and dNTPs. qPCR will then be performed on OXSR1, a known target of miR-23, and a housekeeping gene. SYBR Green, a fluorescent dye that binds to double stranded DNA, was used to label the PCR products in qPCR, with an increase in fluorescence indicating detection of the target gene. This will determine the CT value for each sample and the results will be analyzed using the ∆∆CT method, which will determine whether OXSR1 is a target of miR-23. Beta-2 microglobulin (B2M), a MHC class I molecule, will be the housekeeping gene used to normalize RNA input levels among all samples, and MAP3K1, a known target of miR-23, will be the positive control gene used to make sure that the experimental conditions were working.
The expected results are seeing that the overexpression of miR-23 will decrease OXSR1 expression in the HeLa cells. ROS have been found to contribute to the survival, proliferation and metastasis in a variety of cancer cells, but if the oxidative stress is left uncontrolled, this can lead to cell death [5]. This finding has led to the expansion of treatment strategies aimed at increasing ROS production in cancer cells, which can lead to killing them. If OXSR1 is identified as a target gene of miR-23, a difference in cDNA will be seen between the miR-23 and scramble cells and the target gene of the miR-23 cells should result in a higher CT value than the target gene of the scramble cells, indicating lower expression. The hypothesis of this experiment is that miR-23 will target the OXRS1 gene RNA from the miR-23 transfected HeLa cells and the qPCR data will show decreased expression levels because the up-regulation of miR-23 would repress OXSR1 expression. If this hypothesis is correct, then an overexpression of miR-23 should down-regulate OXSR1 expression, decrease the cancer cells’ ability to respond to oxidative stress, and potentially lead to cancer cell death.
Results
The target gene of this experiment, OXSR1, a kinase involved in oxidative stress response, was tested to see whether it was a target of miR-23. The hypothesis of this experiment was that miR-23 will target the OXRS1 gene RNA from the miR-23 transfected HeLa cells and the qPCR results will show decreased expression levels because the up-regulation of miR-23 would repress OXSR1 expression. This was tested by isolating RNA from HeLa cells transfected with mimics for either miR-scramble or miR-23 and then performing qPCR using primers for the OXSR1 gene, a known target of miR-23, and a housekeeping gene. The CT value was measured for each sample and the results were analyzed using the ∆∆CT method, which determined whether OXSR1 was a target of miR-23. The experiment was performed twice to assure whether OXSR1 was a target gene of miR- 23. The CT values measured by qPCR for each sample in Tables 1 and 2 represent the increase in fluorescence, indicating detection of the target gene.