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The role of non-coding RNAs in HER2-positive breast cancer

Hafstað, Völundur (2019) MOBN03 20182
Degree Projects in Molecular Biology
Abstract
The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that is involved in many cellular pathways. The HER2 locus is amplified in 12-21% of all breast cancers and is associated with poor prognosis. The introns of HER2 contain two non-coding RNAs: a microRNA (miRNA) named mir-4728 and a box C/D snoRNA-like molecule thus far named snoID_0714. In this study, a list of target genes for miR-4728-3p was created and the effects of overexpressing the microRNA examined in those genes using real-time qRT-PCR. This study also aimed to show that snoID_0714 has a biological function. An assay to detect base-specific 2'-O-methylated (2OMe) RNA bases, utilizing RNase H-directed cleavage was optimized and used for one of the... (More)
The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that is involved in many cellular pathways. The HER2 locus is amplified in 12-21% of all breast cancers and is associated with poor prognosis. The introns of HER2 contain two non-coding RNAs: a microRNA (miRNA) named mir-4728 and a box C/D snoRNA-like molecule thus far named snoID_0714. In this study, a list of target genes for miR-4728-3p was created and the effects of overexpressing the microRNA examined in those genes using real-time qRT-PCR. This study also aimed to show that snoID_0714 has a biological function. An assay to detect base-specific 2'-O-methylated (2OMe) RNA bases, utilizing RNase H-directed cleavage was optimized and used for one of the putative targets snoID_0714, base 32 of the U2 spliceosomal RNA. Finally, we attempted to show an association of snoID_0714 with fibrillarin, the methyltransferase for canonical box C/D snoRNAs.
No effect on the expression of the predicted target genes of miR-4728-3p was observed when the microRNA was overexpressed. The assay to detect 2'-O-methylation works for the U2 spliceosomal RNA, with roughly a 1000-fold difference in cleavage observed between methylated and un-methylated controls. However, snoID_0714 does not appear to be methylating the predicted target site of base 32 of U2. Immunoprecipitation of fibrillarin has proven to be difficult, with much starting material needed for a substantial RNA analysis. (Less)
Popular Abstract
New therapeutic targets in breast cancer

Breast cancer is the most common form of cancer among women and is the cause of over 600.000 deaths every year. There are many different subtypes of breast cancer, one of which is “HER2 overexpressing”. HER2 is a gene that encodes for a protein which is deregulated in certain aggressive forms of cancer. The HER2 gene also contains two non-coding RNAs (ncRNAs) which are RNA strands that are not translated into proteins, but still have biological functions in the cell. HER2 deregulation leads not only to abnormal amounts of the protein, but of these two RNA molecules as well. Currently, all anti-HER2 therapies focus only on the HER2 protein, and the function of these two ncRNAs is still unclear.... (More)
New therapeutic targets in breast cancer

Breast cancer is the most common form of cancer among women and is the cause of over 600.000 deaths every year. There are many different subtypes of breast cancer, one of which is “HER2 overexpressing”. HER2 is a gene that encodes for a protein which is deregulated in certain aggressive forms of cancer. The HER2 gene also contains two non-coding RNAs (ncRNAs) which are RNA strands that are not translated into proteins, but still have biological functions in the cell. HER2 deregulation leads not only to abnormal amounts of the protein, but of these two RNA molecules as well. Currently, all anti-HER2 therapies focus only on the HER2 protein, and the function of these two ncRNAs is still unclear. The aim of this project was to determine whether these two ncRNAs have biological functions that potentially contribute to cancer development.

The first RNA molecule that was examined was a small snoRNA-like molecule. SnoRNAs are short RNA sequences whose main function is to direct modifications to other RNA molecules. These modifications can change the efficiency of protein production in the cell and are crucial for normal cellular functions. As the molecule we were examining is much shorter than normal snoRNAs, it has only recently been discovered and has not been shown to have a function yet.
An assay was developed to test whether this molecule was truly guiding modifications as has been predicted. A list of potential targets of the snoRNA was generated and one of those targets used to test the assay. The levels of the molecule were changed in different cell lines and the RNA modifications compared to that of un-treated cells. No changes were observed but the assay will be tested on other potential targets in the future.
We also examined whether this snoRNA-like molecule was associated with fibrillarin, which is the protein that actually carries out the modifications for normal snoRNAs. A slight enrichment of the molecule was observed when fibrillarin was isolated, but much optimization for this method still remains.

The second RNA molecule examined was a micro RNA (miRNA) named mir-4728. The main function of miRNAs is to pair to complementary messenger RNA (mRNA) sequences and repress them. Micro RNAs can therefore indirectly decrease the levels of proteins in the cell. In the case of mir-4728, overexpression of the HER2 protein also leads to abnormally high amounts of the miRNA. As miRNAs can have many potential targets, high levels of mir-4728 could lead to repression of many different proteins in HER2 positive tumors, which potentially contributes to cancer development.
A list of target genes for mir-4728 was generated and the miRNA overexpressed in HER2-negative cells. The activity of the target genes was examined and compared to untreated cells. No change in gene expression was observed. For future research, mir-4728 will be stably integrated into the genome of HER2-negative cells, which will hopefully give clearer gene expression results.

Gauging the role these two RNA molecules play will help us understand the mechanisms of HER2-positive breast cancer. If they are shown to have effects which contribute to cancer they could be used as new drug targets for used in anti-HER2 therapies.


Master’s Degree Project in Molecular Biology, 60 credits, 2019
Department of Biology, Lund University
Advisor: Snorri Þór Sigurðsson (Less)
Please use this url to cite or link to this publication:
author
Hafstað, Völundur
supervisor
organization
course
MOBN03 20182
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8990043
date added to LUP
2019-07-04 14:48:56
date last changed
2019-07-04 14:48:56
@misc{8990043,
  abstract     = {{The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that is involved in many cellular pathways. The HER2 locus is amplified in 12-21% of all breast cancers and is associated with poor prognosis. The introns of HER2 contain two non-coding RNAs: a microRNA (miRNA) named mir-4728 and a box C/D snoRNA-like molecule thus far named snoID_0714. In this study, a list of target genes for miR-4728-3p was created and the effects of overexpressing the microRNA examined in those genes using real-time qRT-PCR. This study also aimed to show that snoID_0714 has a biological function. An assay to detect base-specific 2'-O-methylated (2OMe) RNA bases, utilizing RNase H-directed cleavage was optimized and used for one of the putative targets snoID_0714, base 32 of the U2 spliceosomal RNA. Finally, we attempted to show an association of snoID_0714 with fibrillarin, the methyltransferase for canonical box C/D snoRNAs. 
No effect on the expression of the predicted target genes of miR-4728-3p was observed when the microRNA was overexpressed. The assay to detect 2'-O-methylation works for the U2 spliceosomal RNA, with roughly a 1000-fold difference in cleavage observed between methylated and un-methylated controls. However, snoID_0714 does not appear to be methylating the predicted target site of base 32 of U2. Immunoprecipitation of fibrillarin has proven to be difficult, with much starting material needed for a substantial RNA analysis.}},
  author       = {{Hafstað, Völundur}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{The role of non-coding RNAs in HER2-positive breast cancer}},
  year         = {{2019}},
}