Analysis of miRNA and mRNA Environment in Transfected Cell Lines.

Abstract

The central dogma of biology is that DNA codes for mRNA, which is then used to produce proteins. This is one of the reasons that gene therapy is such a promising field, as mutated genes and their products can be manipulated. Mutations in patient’s genetic code can lead to dysfunctional proteins and serious disorders, but gene therapy can be used to correct defective proteins that cause disease by replacing the mutated DNA, or by granting a cell new means to produce a desired protein. However, the real process is not this straightforward since mRNA are regulated by various processes, including miRNA. The goal of the project was to determine the interactions between miRNAs and the therapeutic genes (transgenes) used in gene therapy treatments. Initially, the small RNA content of several human cell lines was analyzed to identify any endogenous miRNAs that might interfere with transgene expression by binding to its 3’ UTR. An additional goal was to identify any miRNAs that were differentially expressed after transgenes were introduced into the cell, which are miRNAs that play a role in the innate immune response to foreign DNA. Sequencing data were obtained for the miRNA and mRNA present in several cell lines: human embryonic kidney cells, HEK293-T, prostate cancer cells, PC-3, and a breast cancer cell line, MCF7. Bioconda, a Python script designed for bioinformatic research was used to align and analyze the data. The number of miRNA in each cell line increased by a statistically significant margin following transfection, although the majority of the miRNA aligned with non-regulatory domains of the plasmid and thus did not likely have a regulatory effect. Of the miRNA that were found, only miRNA 6724 was found in all three cell lines and was a reverse compliment of the 3’ UTR. Additionally, miRNA 6724 has been identified in some studies as having an inhibitory effect on mRNA transcripts to which it binds. Low levels of mRNA that covered the entire plasmid were present, which are likely products of transcription or from the genome, as the backbone regions of the plasmid should not be translated. mRNA that coded for GFP was present in the highest levels, which was to be expected since that is the protein that the plasmids were designed to express. The presence of high levels of the ampicillin resistance gene relative to the baseline suggests that strong bacterial promoters may be able to function in eukaryotic cells, which raises the possibility of off target effects and reactions from transfected cells if other parts of the plasmid are transcribed to mRNA and translated to proteins.