RNA interference (RNAi) is a process in which cells are able to regulate protein gene expression (Fire et al. 1998) using microRNAs (miRNA), which are small pieces of RNA from non-protein-coding regions. They are incorporated into complexes responsible for translation inhibition or mRNA transcript degradation to downregulate expression of a particular protein (Carrington and Ambros 2003). miRNAs bind to the 3' untranslated regions of a gene with which they must have some complementarity (Fang and Rajewsk 2011). Targets of miRNAs include tumor suppression, cancer, and apoptotic pathway genes (Kloosterman and Plasterk 2006). Irregular expression of miRNAs has been studied as a potential cause for many diseases (Calin et al. 2002). However, many miRNA targets remain unknown (Carrington and Ambros 2003). Although bioinformatics techniques exist for prediction of miRNA targets, the best method for determination of targets is through experimentation (Grantham 2017).
The miRNA used in this lab was hsa-miR-23b (miR-23b). Irregular expression of miR-23b has been linked to various cancers including kidney, bladder, and ovarian cancers. In the case of ovarian cancer, one study (Yan et al. 2016) notes that miR-23b is a crucial regulatory factor in progression of cancerous cells. In epithelial ovarian carcinomas, when compared with healthy ovarian tissues, it was found that miR-23b was significantly downregulated (Yan et al. 2016). Additionally, overexpression of miR-23b exhibited inhibitory properties on cancer cell proliferation and invasion, and induced apoptosis within cancerous cells (Yan et al. 2016). These results provide critical insight into the use of miRNAs such as miR-23b for therapeutic purposes in order to regulate expression of miRNA within cancer cells and promote cancer cell death.
The candidate gene for this lab, cation dependent mannose-6-phosphate receptor (CI-M6PR), has an important role in cell signalling/secretory pathways. CI-M6PR encodes a P-type lectin that is crucial for delivery of mannose-6-phosphate tagged lysosomal enzymes (ncbi). One study (Dangaj et al. 2011) showed mannose-receptor engagement with GPI anchored proteins has a significant effect in polarization of macrophages to tumour-associated macrophages (TAM) in ovarian cancer. Downregulation of this mannose-receptor interaction could aid in inhibition of polarization of macrophages to TAMs (Dangaj et al. 2011).
The objective of this experiment was to determine if CI-M6PR is a target for miR-23b. Thus, RNA extraction of miR-23b mimic (treated) and scramblase (control) transfected HeLa cells was conducted, followed by quantitative polymerase chain reaction (qPCR). It was hypothesized that should CI-M6PR be a target, miR-23b-treated cells would exhibit downregulated expression in comparison to the control. The miR-scr-treated cells were expected to have regular expression levels. Should CI-M6PR not be a target of this miRNA, both miR-23b-treated and scr-treated cells should present similar expression levels. If CI-M6PR is a target for miR-23b, there is potential for RNAi therapy in instances of ovarian cancer involving M6PR in the future. Furthermore, the experimental procedure could be used to identify other targets of miR-23b for treatment of various diseases.
Materials and Methods
Materials and methods were adapted from procedures outlined in the Biology 2030 Study Guide/Lab Manual (Grantham 2017).
Target Gene and RNA Primer Design
The miRNA used in this lab and tested against with the candidate gene was hsa-miR-23b. The candidate gene, cation dependent mannose-6-phosphate receptor (CI-M6PR) with ascension number NM_001207024 and length 1474, was chosen from the miRDB database. Primers were picked to be used for qPCR using Primer-BLAST. The chosen primer for the candidate gene had an amplicon length of 246 with a forward sequence of 5'-GGCGCTTCCTGTTTCCGGTT-3' and reverse sequence of 5'-TCACTGCCACAGCCAGGAGT-3'. Two additional primers were used for the qPCR, MAP3K1 and B2M.
RNA Isolation and Purity Test
Two cultures were made of HeLa cells, one of which was transfected with a miR-23b mimic (miR-23b-treated), and another with a miR-scramble mimic (miR-scr-treated). The miR-23b-treated cells were the treated group while miR-scr-treated cells were the control group. Cells from both groups were lysed using guanidine isothiocyanate and -mercaptoethanol, then were placed on ice in a 1:1 solution of lysis buffer and 70% ethanol. The cell lysates were transferred to RNA binding columns in 2mL microcentrifuge tubes and RNA was extracted from the cells of both cultures using the Aurum Total RNA Mini Kit from BioRad. The purified RNA extracts were collected in a new microcentrifuge tube. A small amount of the RNA extracts were used to test the purity of the extracts using the Nanodrop machine (Grantham 2017a).
Reverse Transcription and qPCR
The iScript cDNA Synthesis Kit from BioRad was used to obtain cDNA from the RNA extracts. To a microcentrifuge tube, 20 L of RNA extract was added, diluted with 7.92 L of RNase-free water. To each of two 0.5 mL thin-walled PCR tubes, 10 L of the diluted RNA was added. 10 L of +RT Supermix was added to one tube, and 10 L of no-RT Supermix was added to the other. The contents of the tubes were collected after centrifugation. Reverse transcription was conducted, and eight qPCR reactions were set up as instructed in the lab manual (Grantham 2017b), with three tubes each for +RT(miR-scr) and +RT(miR-23b), as well as two tubes for no-RT(miR-scr) and no-RT(miR-23b). With each of these products, a primer for one of three genes, MAP3K1 (positive control), B2M (reference gene), or the candidate, were used to conduct the qPCR (Grantham 2017b).
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