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Evaluation of a miR-125a mimic using an established Luciferase assay and working towards a new model-system to study RNAi in Escherichia coli

Lockowandt, Marcus LU (2014) KEMT30 20141
Department of Chemistry
Abstract
RNA interference (RNAi) plays a major role in regulation of gene expression in most eukaryotic organisms. On a basic level it is a protein complex named RNA induced silencing complex (RISC) that binds a RNA strand, a micro RNA (miRNA) or a small interfering RNA (siRNA), which guides it to its intended target, an mRNA. The RISC then disrupts translation of the bound mRNA, either by restricting it thereby causing its degradation, or through interactions with other proteins that cause a halt in translation. Mutations in these miRNAs can therefore cause dysfunction of protein regulation. This in turn can lead to a wide range of diseases 1.
One of the most important issues with dysregulation of miRNAs is the correlation between aberrant miRNAs... (More)
RNA interference (RNAi) plays a major role in regulation of gene expression in most eukaryotic organisms. On a basic level it is a protein complex named RNA induced silencing complex (RISC) that binds a RNA strand, a micro RNA (miRNA) or a small interfering RNA (siRNA), which guides it to its intended target, an mRNA. The RISC then disrupts translation of the bound mRNA, either by restricting it thereby causing its degradation, or through interactions with other proteins that cause a halt in translation. Mutations in these miRNAs can therefore cause dysfunction of protein regulation. This in turn can lead to a wide range of diseases 1.
One of the most important issues with dysregulation of miRNAs is the correlation between aberrant miRNAs and cancer. Several different types of cancers show miRNA profiles that differ from normal cells. This can be used to determine what type of cancer a patient has, and what treatment might be the most efficient. It is also a possibility to give the patient siRNAs down regulating specific proteins that might make cancer cells more susceptible towards treatment. One such example is the breast cancer type 1 susceptibility protein (BRCA1). This protein is involved in multiple pathways that affect among other things, cell checkpoints and double-strand break repair. A low amount of, or deficient BRCA1 has an oncogenic effect on cells. However once the cancer needs to be treated, a low enough amount of BRCA1 increases the efficiency of DNA alkylating agents such as platinum-based drugs2. This means that if a cancer is not responding well to the therapy, it might be possible to supplement the platinum drugs with a miRNA down-regulating BRCA1 and thus sensitizing the cancer cells.
One miRNA that is responsible for regulating BRCA1, among others, is miR-125a-3p. In this work, the aim was to look at changes in regulation of modified miR-125a-3p, using luciferase expressed in human MCF-7 cells and constructing a simplified model system in Escherichia coli. The BRCA1 3’ UTR was attached to the luciferase coding DNA sequence (CDS) on a plasmid, thus causing a direct correlation between the down regulating efficiency of the miRNA and the amount of luciferase measured. The plasmid was then transfected into MCF-7 cells together with the miRNA that was to be tested. The cells were lysed at a specific time and the amount of luciferase in the sample was measured, thus allowing us to draw conclusions on the efficacy of the miRNA tested. These tests showed a significant down-regulation when using a miR-125a-3p that has full complementarity to its target region of BRCA1’s 3’ UTR compared to the native miR-125a. The remaining protein producation went from approximately 60% of normal expression with the native miR-125a-3p to 20% with the modified miR-125a-3p.
Initial attempts towards construction of a model system that expresses Ago2, the protein required for minimal functionality of a RISC (recognition and restriction of mRNA), together with superfolded GFP with a 3’ UTR in E. coli were also made. Electroporation was then supposed to be used to transfect the E. coli cells with the miRNAs. The effect these would have on the amount of sfGFP in the cells would then be measured in order to determine the efficiency of down-regulation. Due to problems with Gibson assembly this part of the project was never completed to the point that results confirming the function of the model system could be achieved. (Less)
Popular Abstract (Swedish)
Det finns tre grundläggande nivåer vid vilken en cell kan reglera mängden av ett visst protein. Den första nivån är i vilken omfattning den kodande delen av en gen transkriberas från DNA till RNA. Den andra delen är i vilken omfattning detta RNA, även kallat messengerRNA (mRNA), translateras till ett färdigt protein. Den tredje och sista delen är i vilken utsträckning detta protein bryts ner.
RNA-interferens (RNAi) kontrollerar mängden protein vid nivå två. Detta sker med hjälp av ett proteinkomplex samt korta RNA strängar (mikroRNA). Proteinkomplexet kallas för RNA induced silencing complex (RISC). Detta proteinkomplexet binder ett mikroRNA som fungerar som en guide för RISC och leder det till rätt mål. RISC kan sedan förhindra... (More)
Det finns tre grundläggande nivåer vid vilken en cell kan reglera mängden av ett visst protein. Den första nivån är i vilken omfattning den kodande delen av en gen transkriberas från DNA till RNA. Den andra delen är i vilken omfattning detta RNA, även kallat messengerRNA (mRNA), translateras till ett färdigt protein. Den tredje och sista delen är i vilken utsträckning detta protein bryts ner.
RNA-interferens (RNAi) kontrollerar mängden protein vid nivå två. Detta sker med hjälp av ett proteinkomplex samt korta RNA strängar (mikroRNA). Proteinkomplexet kallas för RNA induced silencing complex (RISC). Detta proteinkomplexet binder ett mikroRNA som fungerar som en guide för RISC och leder det till rätt mål. RISC kan sedan förhindra translation antingen genom att bryta ner mRNAt om mikroRNAt basparar fullkomligt med mRNAt, eller genom att binda till andra proteiner som sitter på mRNAt och på så vis förhindra att ribosomerna (protein som translaterar mRNA till ett protein) kan binda in. Beroende på vilket mikroRNA som är bundet till RISC kan alltså olika mRNA väljas ut som mål för RISC.
I denna studie har målet varit att försöka förstå vilken påverkan olika förändringar i mikroRNAt har på dess effektivitet. Som model valdes breast cancer type 1 susceptibility protein (BRCA1) som är ett mål för ett mikroRNA vid namn miR-125a. BRCA1 är intressant eftersom det som namnet antyder är relaterat till en ökad risk för vissa typer av cancer. För att kunna mäta förändringar i regleringen via olika modifikationer av miR-125a användes ett protein som heter luciferas som reporter. Luciferas får vid tillsats av ett specifikt substrat detta att börja lysa olika mycket beroende på koncentrationen av luciferas. Genom att koppla den kodande delen av genen för luciferas till den del av BRCA1 som är mål för miR-125a så är det alltså möjligt att mäta förändringar i uttryck av luciferas och koppla dessa till förändringar i miR-125a.
För att kompletera data från luciferas-mätningarna skulle ett modelsystem utvecklas i Escerichia coli. I detta modelsystem skulle ett av proteinen (Ago2) från humant RISC uttryckas i E. coli. Ago2 klarar ensamt av att binda in ett mikroRNA och om det basparar fullkomligt med mRNAt, att klyva det. Avsikten var därför att konstruera två olika plasmider dvs. ett litet, cirkulärt, icke-genomiskt DNA som tillåter uttryck av gener i bakterier; en som innehöll genen för humant Ago2 och en som var utformade för att uttrycka en annan reporter som kallas för Green fluorescent protein (GFP). GFP var kopplat till BRCA1s 3’ UTR på samma sätt som beskrevs i paragrafen ovan med luciferas. Tanken var att sedan introducera mikroRNAn i cellerna med hjälp av en elektrisk chock. Eftersom varje GFP molekyl avger en viss mängd ljus vid 509 nm efter excitation vid 395 nm, så kan vi mäta hur mycket GFP som finns i cellerna. På så sätt kan vi räkna ut hur effektivt mikroRNAt vi tillsatt varit på att begränsa translation av mRNAt för GFP till den färdiga GFP molekylen. Av detta kan sedan slutsatser dras om hur effektivt mikroRNAt är mot sitt mål. (Less)
Please use this url to cite or link to this publication:
author
Lockowandt, Marcus LU
supervisor
organization
course
KEMT30 20141
year
type
H2 - Master's Degree (Two Years)
subject
keywords
proteinvetenskap, protein science
language
English
id
4645884
date added to LUP
2014-09-12 16:24:13
date last changed
2014-09-12 16:24:13
@misc{4645884,
  abstract     = {RNA interference (RNAi) plays a major role in regulation of gene expression in most eukaryotic organisms. On a basic level it is a protein complex named RNA induced silencing complex (RISC) that binds a RNA strand, a micro RNA (miRNA) or a small interfering RNA (siRNA), which guides it to its intended target, an mRNA. The RISC then disrupts translation of the bound mRNA, either by restricting it thereby causing its degradation, or through interactions with other proteins that cause a halt in translation. Mutations in these miRNAs can therefore cause dysfunction of protein regulation. This in turn can lead to a wide range of diseases 1.
One of the most important issues with dysregulation of miRNAs is the correlation between aberrant miRNAs and cancer. Several different types of cancers show miRNA profiles that differ from normal cells. This can be used to determine what type of cancer a patient has, and what treatment might be the most efficient. It is also a possibility to give the patient siRNAs down regulating specific proteins that might make cancer cells more susceptible towards treatment. One such example is the breast cancer type 1 susceptibility protein (BRCA1). This protein is involved in multiple pathways that affect among other things, cell checkpoints and double-strand break repair. A low amount of, or deficient BRCA1 has an oncogenic effect on cells. However once the cancer needs to be treated, a low enough amount of BRCA1 increases the efficiency of DNA alkylating agents such as platinum-based drugs2. This means that if a cancer is not responding well to the therapy, it might be possible to supplement the platinum drugs with a miRNA down-regulating BRCA1 and thus sensitizing the cancer cells.
One miRNA that is responsible for regulating BRCA1, among others, is miR-125a-3p. In this work, the aim was to look at changes in regulation of modified miR-125a-3p, using luciferase expressed in human MCF-7 cells and constructing a simplified model system in Escherichia coli. The BRCA1 3’ UTR was attached to the luciferase coding DNA sequence (CDS) on a plasmid, thus causing a direct correlation between the down regulating efficiency of the miRNA and the amount of luciferase measured. The plasmid was then transfected into MCF-7 cells together with the miRNA that was to be tested. The cells were lysed at a specific time and the amount of luciferase in the sample was measured, thus allowing us to draw conclusions on the efficacy of the miRNA tested. These tests showed a significant down-regulation when using a miR-125a-3p that has full complementarity to its target region of BRCA1’s 3’ UTR compared to the native miR-125a. The remaining protein producation went from approximately 60% of normal expression with the native miR-125a-3p to 20% with the modified miR-125a-3p.
Initial attempts towards construction of a model system that expresses Ago2, the protein required for minimal functionality of a RISC (recognition and restriction of mRNA), together with superfolded GFP with a 3’ UTR in E. coli were also made. Electroporation was then supposed to be used to transfect the E. coli cells with the miRNAs. The effect these would have on the amount of sfGFP in the cells would then be measured in order to determine the efficiency of down-regulation. Due to problems with Gibson assembly this part of the project was never completed to the point that results confirming the function of the model system could be achieved.},
  author       = {Lockowandt, Marcus},
  keyword      = {proteinvetenskap,protein science},
  language     = {eng},
  note         = {Student Paper},
  title        = {Evaluation of a miR-125a mimic using an established Luciferase assay and working towards a new model-system to study RNAi in Escherichia coli},
  year         = {2014},
}