Določitev izražanja kanabinoidnih receptorjev na ravni informacijske RNA v celičnih linijah raka dojke

Mlinar, Karmen (author), Debeljak, Nataša (mentor)

Kanabinoidni receptorji (CBR) so membranski receptorji povezani z G-proteini, ki vežejo kanabinoide eksogenega in endogenega izvora in so del endokanabinoidnega sistema. Ločimo dva tipa CBR: tip 1 (CB1, gen CNR1) s prevladujočim izražanjem v živčnih celicah in tip 2 (CB2, gen CNR2) z značilnim izražanjem v celicah imunskega sistema. CB1 receptor je v treh izooblikah: CB1, CB1a in CB1b, CB2 receptor pa v dveh izooblikah: CB2A in CB2B. Rak dojke je najpogostejša oblika malignosti pri ženskah, saj predstavlja kar 30 % vseh oblik raka pri ženskah. Na podlagi izražanja hormonskih receptorjev (HR) in receptorja za človeški epidermalni rastni faktor 2 (HER2) ločimo HR-pozitivni oz. luminalni, HER2-pozitivni in trojno negativni rak dojke. V celicah raka dojke je potrjeno izražanje CBR. Številne študije kažejo, da kanabinoidi vplivajo na lastnosti raka, znani pa so tako pozitivni kot negativni vplivi. Namen naše raziskave je bil pregledati obstoječe RNA prepise genov CNR1 in CNR2 ter določiti njuno izražanje na ravni RNA v celičnih linijah raka dojke. V prvem, bioinformatskem, delu naloge smo pregledali podatke o izražanju in obstoječe RNA prepise genov CNR1 in CNR2 v zbirkah Ensembl, NCBI in UniProt. Znotraj zbirke RefSeq smo našli 11 prepisov izooblike CB1, 1 izooblike CB1b in 1 izooblike CB2B, za izoobliki CB1a in CB2A pa nismo našli nobenega prepisa. V drugem, laboratorijskem, delu naloge smo določili raven izražanja CBR na izbranih celičnih linijah raka dojke (MCF7, T-47D, SK-BR-3, MDA-MB-231, MDA-MB-361), ki se razlikujejo v izražanju HR in HER2, ter dveh kontrolnih vzorcih (SH-SY5Y in možgansko tkivo). Iz vzorcev smo izolirali RNA in jo reverzno prepisali v cDNA. Izvedli smo kvantitativni PCR s testi TaqMan, ki so pokrivali največje število različnih prepisov preiskovanih genov CNR1 in CNR2, ter referenčna gena RPLP0 in HPRT1, izbranih na podlagi sorodnih študij. Iz dobljenih rezultatov smo z metodama za relativno kvantifikacijo določili raven izražanja preiskovanih genov. Nasprotno s svojimi pričakovanji v nobeni analizirani celični liniji raka dojke nismo zaznali izražanja gena CNR1. Izražanja gena CNR2 nismo mogli določiti, ker izbrani vzorci za pozitivno kontrolo niso bili ustrezni. Raziskavo bomo v prihodnje razširili na več celičnih linij raka dojke, preverili izražanje na ravni proteinov in zagotovili ustrezne pozitivne in negativne kontrole.

• Language: Slovenian
• Keywords: kanabinoidni receptorji, RNA prepisi, rak dojke, celične linije raka dojke
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-129757
• COBISS.SI-ID: 79562499
• Publication date in RUL: 07.09.2021

Secondary language

• Language: English
• Title: Characterisation of cannabinoid receptors messenger RNA expression on breast cancer cell lines

Abstract

Cannabinoid receptors (CBR) are G-protein coupled receptors that bind cannabinoid ligands of exogenous and endogenous origin and are part of endocannabinoid system. There are two types of CBR: type 1 (CB1, gene CNR1) expressing mainly in neural cells, and type 2 (CB2, gene CNR2) expressing typically in immune cells. Three isoforms of CB1 receptor are known: CB1, CB1a, and CB1b, and two isoforms of CB2 receptor: CB2A and CB2B. Breast cancer is the most common form of malignancy in women, presenting 30 % of all forms of cancer in women. Based on the expression of the hormone receptors (HR) and human epidermal growth factor receptor 2 (HER2), three types of breast cancer are distinguished: HR-positive or luminal, HER2-positive, and triple-negative breast cancer. In breast cancer cells CBR expression has been confirmed. Several studies have shown the tumor modulating effect of cannabinoids, and both positive and negative effects are known. Our study aimed to overview known transcripts of the genes CNR1 and CNR2 and to characterise their expression in breast cancer cell lines on RNA level. In the first bioinformatic part of the thesis, we reviewed the expression data and known transcripts of the genes CNR1 and CNR2 in the Ensembl, NCBI and UniProt databases. In the RefSeq database, we found 11 transcripts of isoform CB1, 1 of isoform CB1b, 1 of isoform CB2B, and none of isoforms CB1a and CB2A. In the second laboratory part of the thesis, we determined the level of CBR expression on selected breast cancer cell lines (MCF7, T-47D, SK-BR-3, MDA-MB-231, MDA-MB-361) that differ HR and HER2 expression, and two control samples (SH-SY5Y and brain tissue). We isolated RNA from the samples and reverse transcribed it into cDNA. We performed quantitative PCR with TaqMan assays that cover the largest number of different transcripts of the genes of interest CNR1 and CNR2 and reference genes RPLP0 and HPRT1, which we selected based on related studies. From the obtained results, we determined the expression levels of genes of interest using methods for relative quantification. Contrary to our expectations, we were unable to detect CNR1 gene expression in any analysed breast cancer cell line. We could not determine the expression level of gene CNR2 due to not suitable positive control. The study will be extended to several breast cancer cell lines, characterization on protein level, and finding appropriate positive and negative controls.

• Keywords: cannabinoid receptors, RNA transcripts, breast cancer, breast cancer cell lines