Breast cancer is the most frequently diagnosed cancer today, with approximately 2.3 million new cases for the year 2020. Regardless of gender, these diagnoses represent 11.7% of all cases. For women, breast cancer falls under roughly one-third of cancer cases. Prognosis and treatment of breast cancer are assessed by various factors such as stage, grade, biomarker expression and genetic markers in the tumor. The subdivision of breast cancer is categorized by the expression of proto-oncogenes, steroids, and growth factors such as the estrogen receptor (ER), progesterone receptor (PR), human epithelial growth factor 2 receptor (HER2), epidermal growth factor receptor (EGFR), cytokeratins 5/6 and the proliferation marker KI67. The clinically determined subtypes of breast cancer are Luminal A, Luminal B, HER2 and Triple negative. The most common subtypes which represent approximately 70% of all breast cancer cases, Luminal A and Luminal B, overexpress the estrogen receptor (ER+) which is a key factor in their development and treatment. Prognosis for these patients is good though some respond better to treatment than others.
MicroRNAs (MiRNAs) are RNA molecules which do not code for protein but have a role in gene silencing post DNA-transcription. MiRNAs bind to mRNA resulting in their breakdown or block of protein translation. MiRNAs are flexible in their role since they can bind different mRNA molecules depending on the tissue and transcriptional profile.
A previous study had shown that microRNA-190b (miR-190b) was upregulated in ER+ breast cancer but few target genes had been associated with the molecule. Little was thus known of the role of miR-190b in breast cancer.
The first aim of this study was to investigate whether CpG methylation on miR-190b’s promoter had a role in its upregulation in ER+ breast cancers and whether it had an influence on disease progression. It was confirmed by qPCR in breast cancer samples that miR-190b was indeed upregulated in ER+ subtype. It was furthermore confirmed by pyrosequencing that miR-190b is promoter hypo-methylated in ER+ breast cancers in correlation with its expression. In other words, it was showed that when the tumors had lost CpG methylation on the miR-190b promoter (DNA), the molecule was upregulated. Finally, it was showed that breast cancer specific survival was significantly different in patients by whether their tumors were miR-190b promoter hypo-methylated or not.
The second aim of the study was to investigate miR-190b’s target genes in breast cancer and study whether their expression regulation had a similar influence on patient prognosis as miR-190b. With biotin-miR-190b-mimic pulldown followed by RNA sequencing analysis and western blot it was confirmed that RFWD3 is a miR-190b target. With survival analysis it was shown that patient prognosis was less favorable when RFWD3 was expressed, which was consistent with previous results for miR-190b in this project.
The last aim of the study was to investigate miR-190b in ovarian cancer and research whether its expression had a predictive value on patient survival. With data from The Cancer Genome Atlas (TCGA), it was shown that there was neither a connection between miR-190b expression and ER nor RFWD3 expression. It was additionally shown that there was no difference in miR-190b expression between stages or grades. With survival analysis it was shown however, that prognosis was worse in ovarian cancer patients with high levels of miR-190b indicating that it is clinically relevant in ovarian cancer and needs to be studied further.
Brjóstakrabbamein er algengasta gerð krabbameins sem greinist í dag, með um 2.3 milljón ný tilfelli á heimsvísu árið 2020. Óháð kyni eru þetta um 11.7% allra nýrra tilfella. Í konum er brjóstakrabbamein tæplega þriðjungur krabbameinstilfella. Horfur og meðferð á brjóstakrabbameini eru metin af mismunandi þáttum, svo sem stigun, gráðun, og tjáningu ákveðinna próteina og erfðamarka sem finnast í æxlinu. Brjóstakrabbamein eru flokkuð í undirhópa og ræðst sú flokkun af tjáningu foræxligena, hormóna og vaxtaþáttaviðtaka eins og estrogen viðtakans (ER), prógesterón viðtakans (PR), human epithelial growth factor 2 viðtakans (HER2), epidermal growth factor receptor (EGFR), cytokeratinin 5/6 og frumufjölgunar-merkisameindina KI67. Undirgerðir brjóstakrabbameins í klínísku samhengi eru Luminal A, Luminal B, HER2 og Þríneikvæð æxli. Algengustu undirgerðirnar eru Luminal A og Luminal B sem eru um 70% af öllum tilfellum. Þessi krabbamein yfirtjá estrogen viðtakann (ER+) sem er lykilþáttur í myndun þeirra og tekur meðferð mið af því. Lifun og batahorfur eru því tiltölulega góðar hjá þessum sjúklingum þótt ekki allir svari meðferð jafn vel.
MicroRNA (MiRNA) eru RNA-sameindir sem tjá ekki fyrir próteini en gegna hlutverki í genaþöggun eftir DNA umritun. MiRNA parast við mRNA-sameindir með þeim afleiðingum að ekki myndast prótein. MiRNA eru sveigjanleg í miðlun svipgerðar þar sem að þau hafa eiginleikann til að bindast mismunandi mRNA sameindum og fer það eftir vef og tjáningarmynstri hverju það binst.
Sýnt hafði verið fram á að microRNA-190b (miR-190b) er yfirtjáð í ER+ brjóstakrabbameinum og örfá markgen hafa verið bendluð við sameindina. Lítið er því vitað um áhrif miR-190b í brjóstakrabbameinum.
Fyrstu markmið verkefnisins voru að skoða hvort CpG metýlun á stýrilröð miR-190b væri ráðandi þáttur í tjáningu þess í ER+ brjóstakrabbameinum og hvort það hefði forspágildi fyrir framvindu sjúkdómsins. Í þessu verkefni var staðfest með qPCR úr brjóstakrabbameinssýnum að miR-190b væri yfirtjáð í ER+ brjóstakrabbameinum. Ennfremur var staðfest með pyroraðgreiningu að miR-190b væri undir-metýlað á stýrisvæði þess í ER+ brjóstakrabbameinum og að það væri í fylgni við tjáningu. Með öðrum orðum var sýnt að tap á DNA metýlun á stýrlisvæði miR-190b orsakaði yfirtjáningu á miR-190b sameindinni. Loks var sýnt að munur væri á brjóstakrabbameinsháðri lifun sjúklinga eftir því hvort tap hafði verið á DNA metýlun á stýrisvæði miR-190b eða ekki.
Önnur markmið verkefnisins voru að rannsaka markgen miR-190b í brjóstakrabbameini og skoða hvort stýring á þeim hefðu svipað forspágildi á horfur sjúklinga og sú sem miR-190b tjáning hafði. Með RNA raðgreiningu á biotin-miR-190b-mimic útfellingu og western blotti var staðfest að RFWD3 er markgen miR-190b. Með lifunargreiningu sást að horfur sjúklinga voru verri þegar RFWD3 er yfirtjáð sem var í samræmi við fyrri niðurstöður verkefnisins.
Síðasta markmið rannsóknarinnar var að skoða tjáningu miR190b í eggjastokkakrabbameini, og athuga áhrif þess á lifun sjúklinga. Með gögnum úr The Cancer Genome Atlas (TCGA) sást að hvorki voru tengsl milli miR-190b tjáningar og ER eða RFWD3 tjáningar. Einnig sást að tjáning miR-190b var ekki mismunandi milli stigunar eða gráðunar. Með lifunargreiningu var þó uppgötvað að lifun var verri hjá eggjastokkakrabbameinssjúklingum með háa tjáningu af miR-190b sem bendir til þess að það sé mikilvægt í klínísku samhengi og þarfnast frekari rannsóknar.
Læst til 30.03.2027