SUMMARY of dissertation on competition of scientific degree of doctor of philosophy (PhD)

specialty 6D070100 – «Biotechnology»

Berillo Olga Alexandrovna

Intronic miRNAs in diagnostics of human gastroenteric tract and breast cancer

General characteristics of the work. The work is to research intronic microRNAs(in-miRNAs) and mRNAs of genes which participate in development of gastroenteric tractcancer (GETC) and breast cancer (BC); and to search for the in-miRNAs as markers fordiagnostics.

Novelty of the research. Cancer is one of the reasons leading to death in the world.The majority of malignant tumors are revealed at the late stages of development, thereforein many cases patients are not curable. Carcinogenesis of gastroenteric tract and breast areoften revealed in the Republic of Kazakhstan according to statistics. Tumor and normal cellsconsiderably differ morphologically and biochemically, because of genetic mutations.Specific molecules synthesized only in cancer cells or molecules expressed in very lowconcentrations in normal tissues were named tumor markers. For the last ten years, scientistsintensively have been searching for tumor markers among nucleic acids circulating in blood.For example, the expression of miRNAs change at development of esophageal cancer (EC),stomach cancer (SC), small intestine cancer (SIC), colorectal cancer (CC) and BC. miRNAare short RNA sequences that do not coded the proteins and decrease the translation ofmRNA of target gene. They suppress mRNA translation of target genes. The majority ofmiRNAs have an intragenic origin, that is, they are coded in introns, exons, 3'UTR or5'UTR of protein coding host genes. Other miRNAs are coded in intergenic sequences.Active studying of miRNAs has been initiated approximately since 2000 year. The quantityof known human miRNAs increases every year and now more than 2500 miRNAs areknown. Only a few hundreds of miRNAs discovered first are well studied. The expressionof miRNAs are connected with emergence, metastasis and progressing of malignant tumors.One of the main advantages of miRNAs among other tumor biomarker is their circulation inall biological liquids. Therefore, it is possible to control changes of expression level ofmarker miRNAs by noninvasive method. It is possible to define tumor growth by suchtumor markers and to reveal decrease in expression of the target genes. At present time ithas been revealed that the expression of several tens of miRNAs is aberrant. However, it isimportant to know which mRNAs of genes are targets for miRNAs. miRNAs are recentlydiscoveried and are insufficiently studied. In this regard, the research in the field of systembiology are very actual allowing to put in order the information and to systematizenumerous separate data about participation of miRNAs in tumourigenesis. It is possible toachieve considerable success in molecular medicine, because of new molecular and geneticmethods, and computer technologies in studying of miRNAs and their human target genes.The necessity of this work is based on definition of in-miRNAs as new molecular markersof BC and GETC.

Objects of research – nucleotide sequences of 915 in-miRNAs and 1073 genesparticipating in development of GETC and BC.

Subject of research – modelling of interactions between in-miRNAs and mRNAs ofgenes participating in development of human BC and GETC.

Purpose and goals of the work. The purpose of the work is identification of in-miRNAs interacting with mRNAs of human oncogenes for application of in-miRNAs indiagnostics of human GETC and BC.

Research tasks. 1. To create the database of in-miRNAs and pre-miRNAs properties. 2. To research the interactions between the in-miRNAs with mRNAs of the genes re-

sponsible for development of EC, SC, SIC, CC and BC.3. To study the binding of in-miRNAs to mRNAs of host genes. 4. To identify in-miRNAs for diagnostics of human GETC and BC.Research methods: The search for intronic pre-miRNAs in human genome was used

by the miRNAFinder program. Pre-mRNAs were processed by the Lextractor program. Thesearch for miRNA binding sites in mRNAs was carried out by RNAHybrid, ERNAHybridand miRTarget programs. DIANAmicroT, miRanda, miRDB, miRWalk, PICTAR4,PICTAR5, PITA, RNA22 and Targetscan programs were used for searching of target genes.GeneNet Builder program was applied for visual presentation of gene network. Twodimensional (2D) structures were constructed by the UNAFold program. The authordeveloped programs for treatment miRNA data: miRAFinder and GeneAFinder programsare used to get an information about miRNAs and genes; the TmiRSO and TranslmiRNAprograms are used to get a perfect complementary miRNA binding sites and oligopeptidescoded by them; the TmiRUSite and TmiROSite programs are used to get a nucleotidefragments of mRNAs with binding sites and amino acid sequences coded by them.

Scientific novelty of research. For the first time, the created database contains the information about: 915 human in-

miRNAs; 615 host genes coding in-miRNAs; 389 host genes and 1073 not host target geneswhich are involved in development of EC, SC, SIC, CC and BC. 944, 306, 116, 99 and 935in-miRNA binding sites in mRNAs of genes which participate in development of EC, SC,SIC, CC and BC were revealed respectively. 184, 319, 20, 440 and 613 in-miRNA bindingsites in mRNAs of host genes which participate in development of EC, SC, SIC, CC and BCwere selected respectively.

It was revealed that the average value of in-miRNA binding site density in 5'UTR ofmRNAs was in some times more, than in 3'UTR, CDS and total mRNAs respectively.

Phylogenetic conservatism of miR-302f, miR-3162-5p, miR-4665-5p, miR-655 andmiR-548j binding sites was found in CDS of mRNAs of orthologous genes APC, APC2,BAD, CCS and PTPN12, respectively.

For the first time, arrangement of localization of intronic miR-619-5p, miR-1285-3p,miR-1285-5p, miR-5585-3p, miR-5585-5p, miR-5684, miR-5095, miR-5096 and miRNAsof miR-1273 family binding sites in nucleotide fragments of mRNA sequences of targetgenes was established.

For the first time, the method of identification of miRNA binding sites on the basis ofRNAhybrid and ERNAhybrid programs was developed. Due to this method, in-miR-566,miR-1273g-3p, miR-4447, miR-5095 and miR-5096 were offered as markers for diagnosticsof GETC and BC.

Theoretical importance of the work. In-miRNA binding sites in mRNAs of target genes associated with development of

EC, SC, SIC, CC and BC were defined. The developed programs and the created databaseare used as a basis for the solution of tasks connected with studying and application ofmiRNAs. The established arrangement of localization of binding sites in fragments ofmRNAs expands ideas of their evolutionary interaction. The predicted in-miRNA binding

sites in CDS of mRNAs of orthologous genes with high energy testify their conservatism.The method of identification marker in-miRNAs EC, SC, SIC, CC and BC can be used forprediction of markers of other diseases.

Practical value of work. The developed programs are used for studying of miRNAsof different organisms and for the solution of tasks connected with miRNA research. Thepredicted in-miRNA binding sites in mRNAs are a basis for carrying out experiments fortheir verification and use in diagnostics. The offered method of binding sites can be appliedto predict the target genes which translation can be suppressed by miRNAs. Obtained dataof intronic miRNAs can be used for development of molecular markers for diagnostics ofGITC and BC.

The basic statement submitted for protection:The average value of density of in-miRNA binding sites in 5'UTR of mRNA is in

several times more, than in 3'UTR, CDS and total mRNA. miR-302f, miR-3162-5p, miR-4665-5p, miR-655 and miR-548j have conservated

binding sites localized in the coding region of mRNAs of the orthologous APC, APC2,BAD, CCS and PTPN12 genes, respectively.

Intronic miR-1273g-3p, miR-5095, miR-5096, miR-566 and miR-4447 have bindingsites in mRNAs of many target genes participating in development of GETC and BC.

The binding sites of miR-619-5p, miR-1273, miR-1285, miR-5585, miR-5684, miR-5095 and miR-5096 the in-miRNA have the arranged localization in mRNA fragments ofhundreds of target genes.

Publications. The main content of the thesis has been reflected in 54 publications,including 3 articles and 1 thesis in journals with impact-factor, 1 article is in theinternational edition quoted in Scopus. These 5 publications are quoted in Thomson Reuters.18 articles were published in scientific republic journals of the Committee of control ineducation and sciences of Republic of Kazakhtan. Nine articles and 22 theses werepublished in materials of the international conferences and symposiums.

The dissertation's structure. The dissertation consist of 175 pages includingdesignations and abbreviations, introduction, review of literature, materials and methods,results and discussion, the conclusion and the list of used sources including 523 references,475 of them in English and French languages; contains 19 tables, 25 figures and threeappendices.