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Non Coding RNA Gene Expression Term Paper


Fibrosis refers to the connective tissue’s scarring and thickening. Due to the excess formation of connective issue, this phenomenon may lead to significant negative consequences for the human organism. For example, if it occurs in lungs, a person might suffer from pulmonary insufficiency. Because of the popularity of health conditions caused by this process, the phenomenon of fibrosis remains an object of many scholarly studies.

The main aim of the research is to study the implications of this process for long non-coding RNA expression and function. As it is known, the term “non-coding RNA”, which is often referred to as ncRNA, denotes those RNA molecules that do not encode a protein.

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Their expression and function are crucial for the human organism since they influence a number of cellular processes. Long non-coding RNAs (LncRNAs) represent a specific form of non-coding RNA molecules with a length above 200 nucleotides. LncRNAs are important for the human organism since they significantly influence gene expression, conducting both activation and inhibition of genes. It is planned to explore the expression and function of lncRNA during fibrosis. The chosen research problem is significant since it can help understand how lncRNA can help inhibit the development of fibrosis.

The research conducted by Milligan and Lipovich (2015) established the location of various lncRNAs. In addition to those molecules that are known to reside within introns or between genes, the study also found that the formation of some of them is connected with pseudogenes. The academic literature divides lncRNA into six groups in line with their direction, location, and connection with different genes (Peschansky & Wahlestedt, 2014).

The problem of long non-coding RNA expression has been also investigated by a number of different scientists. It is known that this process is developmentally regulated and may take a cell-type or a tissue-type specific form (Cao et al. 2013). LncRNAs expression may significantly vary across the spatial and time dimensions. Previous studies comparing lncRNAs to mRNA showed that the former differ from the latter in taking a more tissue-specific fashion of expression and demonstrating a higher degree of between-tissues-variation. Results of the academic literature showed that mRNA have an expression level with a magnitude above that of lncRNA.

The specifics of lncRNA expression and function during fibrosis are one of the most promising research problems in fibrosis investigations. There are some findings in the academic literature that could be valuable for the current study. For example, the study carried out by Teng and Ghoshal (2015) deduced a strong influence of non-coding RNAs on liver fibrosis.

In accordance with the research findings, lncRNAs can promote this condition via the role of a biomarker and, in contrast, repress it through targeting dysregulated RNAs. These processes may occur with the help of stellate cells of the human liver, which can be found on the figure above. These cells are important because they can deposit extracellular matrix of liver fibrosis. The research carried out by Micheletti et al. (2017) found that Wisper, one of long non-coding RNAs, may be therapeutically targeted in an attempt to reduce cardiac fibrosis following a myocardial infraction.

While there is a significant amount of information on relationships between lncRNA and specific types of fibrosis, little is known about the general drivers of these relationships across all the six groups of lncRNAs. Furthermore, most scholars also preferred analyzing specific types of fibrosis, which makes the external validity of their findings limited. For instance, Cao et al. (2013) launched a research on the expression of lncRNAs in lung fibrosis and found that significant changes in the expression profiles of many lncRNAs, such as S69206 and AJ005396, present a unique potential for reducing the development of lung fibrosis.

Zhang et al. (2018) conducted one of the few studies trying to generalize these tendencies. In line with the scientists’ findings, expression of lncRNAs is connected with the development of many different diseases, such as tumours and autoimmune diseases. In case of fibrosis, these molecules play the role of a functional regulator.

An analysis of the studies reviewed above shows that the problem chosen for this study presents a significant research interest. There is currently no universal concept that would allow formulating general regularities regarding the specifics of expression and function of lncRNAs during fibrosis (Liang et al. 2018; Li 2018). The current study will conduct a research on two lncRNAs, BCAR4 and MALAT1, in order to fill an obvious gap in the existing literature.

The main aim of this research is to investigate long non-coding RNA expression and function during fibrosis. The study’s hypotheses are that both lncRNAs can be successfully used as therapeutic targets for reducing the development of all the four types of fibrosis and that both MALAT1 and BCAR4 significantly change their expression and function during all the four fibrosis forms. In order to achieve this goal, it is necessary to complete the following research objectives:

⦁ To analyse the specifics of MALAT1 expression and function during the different types of fibrosis, including liver, lung, kidney, and myocardium fibrosis;

⦁ To analyse the specifics of BCAR4 expression and function during the different types of fibrosis, including livery, lung, kidney, and myocardium fibrosis;
⦁ To deduce the main mechanisms of using MALAT1 and BCAR4 as therapeutic targets for reducing the development of fibrosis.

This study will be conducted on the basis of the experiment with three different groups of mice in accordance with the types of fibrosis investigated in the research. Each group will contain at least ten mice. As the previous studies showed, the procedures of investigating gene expression in these animals can provide valuable findings for analysing the phenomenon of gene expression in the human organism (Uosaki & Taguchi 2016). The empirical part of this study implies using three methods: RNA isolation, reverse transcription PCR, and PCR.

It is planned to use the RNAlater® to ensure effective RNA protection. This stabilization solution is supposed to provide the maximum level of flexibility in choosing the exact time of RNA isolation. The process of isolation will be achieved by using Trizol. The quantitation of isolated RNA will be carried out in line with the principles of UV spectroscopy. Finally, isolated RNAs will be stored with the help of TE Buffer (pH 7.0, 1 mM EDTA).

For the reverse transcription PCR, it was decided to utilize the standard curve method. In the laboratory settings, it is planned to use a thermal cycler and specific optics for emission collection in order to conduct the necessary procedures. The MessageSensortmRT Kit will be utilized during this process.

The methods highlighted above will allow implementing PCR. The main outcome of this method will be to generate multiple copies of the chosen RNA segment. In accordance with the traditional guidelines, the implementation of PCR in the current study will entail passing a series of procedures, including initialization, denaturation, annealing, extension, final elongation, and final hold. The process will comprise of the three main stages: exponential amplification, levelling of stage, and plateau. In addition, it is also planned to use the statistical analysis from data in experiment.

All the procedures described in this section will be conducted on mice in laboratory settings. As explained in the previous section, research goal is to investigate the specifics of lncRNA expression and function during three different forms of liver. Therefore, it is planned to carry out an experiment on three different groups of mice. The methodological approach towards employing mice in studying the role of lncRNAs in fibrosis development was adapted from Leisegang (2018). The external validity of findings, therefore, is supposed to be significant.

Since it is not possible to obtain human samples for the analysis, the use of mice in studies on gene expression seems to be a rational decision. A number of investigations found a high level of similarity between the gene expression profiles of humans and mice. For instance, the study conducted by Uosaki and Taguchi (2016) determined that investigation of gene expression in mice can be beneficial for understanding the specifics of gene expression relevant to human cardiac maturation. These findings lead to a conclusion that the use of mouse in the current experiment is justified.
In order to maximize the validity and reliability of the experiment, it is planned to conduct a focus group that would help reflect on the experiment’s course and make appropriate changes in this process if necessary. This measure will ensure that no mistakes are made during the processes of RNA isolation and effectively analyses the experiment’s results.

Main outcomes and impact
The research will show how MALAT1 and BCAR4 expression and function change during the three forms of fibrosis. Eventually, the study is supposed to provide a comprehensive understanding of how these two lncRNAs can facilitate and inhibit the development of liver, lung, kidney, and myocardium fibrosis. These findings may be regarded as valuable both for further research and for application in practice settings. Unfortunately, the function and expression of lngRNAs remains an underdeveloped research problem. The amount of studies on the chosen problem is limited; thus; any research that explores the expression and function of lncRNAs during fibrosis could be considered a valuable contribution to the existing literature. It is important to point out that the results of this study might be used by other scientists in their researches on the role of lncRNAs in lung, kidney, and myocardium fibrosis.
In the practical domain, this research may significantly contribute to the improvement of fibrosis treatment. Findings of this research might enhance an understanding of the use of lncRNAs as therapeutic targets in treating lung, kidney, and myocardium fibrosis. However, these findings will gain significant practical applicability only after confirming them with the help of other studies that would establish the same patterns in experiment with other animals. The results of this study might be extremely helpful in treatment and drug intervention.

⦁ Milestones(10%)
⦁ Research backroad information on fibrosis and lncRNAs;
⦁ Review the existing literature on the role of lncRNA in fibrosis;
⦁ Analyse the existing methods of investigating lncRNA expression and function;
⦁ Determine alternative solutions;
⦁ Choose the most suitable methods;
⦁ Prepare the experiment;
⦁ Conduct the experiment and analyse its results;
⦁ Organize the focus group;
⦁ Make appropriate changes in the experiment and data analysis;
⦁ Finish the paper;
⦁ Hand in draft;
⦁ Revise the draft.

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Reference List
Baxter, MA et al. 2010, ‘Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening’, Stem Cell Research, vol. 5, no. 1, pp. 4-22.
Cao, Y et al. 2013, ‘Identification of differential expression genes in leaves of rice (Oryza sativa L.) in response to heat stress by cDNA-AFLP analysis. BioMed Research International, vol. 3, pp. 1-11.
Leisegang, MS 2018, ‘LET’s sponge: How the lncRNA PFL promotes cardiac fibrosis’, Theranostics, vol. 8, no. 4, pp. 874-877.
Li, X et al. 2018, LncRNA PFAL promotes lung fibrosis through CTGF by competitively binding miR-18a’, FASEB Journal, vol. 32, no. 10, pp. 5285-5297.
Liang, H et al. 2018, ‘LncRNA PFL contributes to cardiac fibrosis by acting as a competing endogenous RNA of let-7d’, Theranostics, vol. 8, no. 4, pp. 1180-1894.
Micheletti, R et al. 2017, ‘The long non-coding RNA, Wisper controls cardiac fibrosis and remodeling’, Science Translational Medicine, vol. 21, no. 9, pp. 1-37.
Milligan, MJ & Lipovich, L 2015, ‘Pseudogene-derived lncRNAs: Emerging regulators of gene expression’, Frontiers in Genetics, vol. 4, no. 5, pp. 1-7.
Peschansky, VJ & Wahlestedt, C 2014, ‘Non-coding RNAs as direct and indirect modulators of epigenetic regulation’, Epigenetics, vol. 9, no. 1, pp. 3-12.
Teng, KY & Ghoshal, K 2015, ‘Role of non-coding RNAs as biomarker and therapeutic targets for liver fibrosis’, Gene Expression, vol. 16, no. 4, pp. 155-162.
Uosaki, H & Taguchi, Y 2016, ‘Comparative gene expression analysis of mouse and human cardiac maturation’, Genomics Proteomics Bioinformatics, vol. 14, no. 4, pp. 207-215.
Zhang, J et al. 2018, ‘LeNup: Learning nucleosome positioning from DNA sequences with improved convolutional neural networks’, Bioinformatics, vol. 34, no. 10, pp. 1705-1712.

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