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Project titleNovel long non-coding RNAs as potential biomarkers for diagnostics of liver cancer

AffiliationAutonomous Non-Profit Organization for Higher Education "Skolkovo Institute of Science and Technology",

KeywordsLong non-coding RNAs, biomarkers, hepatocellular carcinoma, liver cancer

Annotation
The project aims identification and investigation of properties of previously undescribed long non-coding RNAs (lncRNAs) associated with liver cancer, particularly with hepatocellular carcinoma (HCC) and pediatric hepatoblastoma (HBL). These oncological diseases are characterized by a lack of symptoms, so they are often diagnosed at late stages, when treatment is ineffective. Used in clinical practice protein markers, such as alpha-fetoprotein (AFP), have low sensitivity and specificity. Thus, the search for fundamentally new biomarkers is a crucial task for the research. Long non-coding RNAs represent a widespread class of regulatory molecules that perform various functions in cell. Recent studies indicate an important role of lncRNAs in oncogenesis. Up to date about 40 lncRNAs, associated with liver cancer, are known, but the mechanisms of their functioning are poorly studied. However, simple detection and measurement of concentrations of lncRNAs by quantitative RT-PCR allows using them as diagnostic markers. Moreover, many lncRNAs circulate in the human body within extracellular vesicles along with biological fluids, and their detection does not involve expensive and highly invasive procedures. Previously, we have performed a preliminary bioinformatic search for lncRNAs with dysregulated expression in HCC, and chose the most promising candidates for further study. In this project we plan to verify this data and to carry out a characteristic analysis of the main lncRNA target and its potential homologue in mice; to find out whether it is involved in the proliferation of hepatocytes, and to determine its probable protein partners. For the estimation of the diagnostic potential of this lncRNA we will measure the levels of its expression in liver samples obtained from patients with HCC and HBL.

Expected results
In the course of this project we will identify and characterize a novel, previously undescribed, human lncRNA and its potential homologue in mice. According to the preliminary data, this lncRNA is actively expressed in human liver, but its concentration significantly decreases during the development of HCC. While this RNA is not detected in other tissues and organs. We plan to verify this data and to quantify the concentration of lncRNA in different cell lines (HCC and conventional normal hepatocytes) and in human and murine liver, and to determine its tissue-specificity. We expect confirmation of the dysregulated expression of lncRNA during carcinogenesis, thus we plan to conduct statistical analysis and to determine its concentration in liver samples from patients with HCC and HBL (compared to normal liver). In the accordance with the research, we will conclude, whether this molecule can be used as a diagnostic marker. Experiments in vitro for knockdown of the investigated lncRNA target and, on the contrary, its overexpression in cell lines, will allow answering the question whether it is important for the invasive and migratory activity of hepatocytes and their proliferation. Identification of the protein partners by the RNA-pull-down method will enable speculation of the probable mechanisms of the functioning of this lncRNA, and design of further research strategy. We expect, that upon an implementation of this project we will discover the novel liver-specific lncRNA, possessing potential oncosuppressing properties, and evaluate the possibility of its application as diagnostic biomarker of HCC and HBL.

Annotation of the results obtained in 2019
The project continued to study the properties and functions of the new long noncoding RNA (lncRNA) HELIS (aka "HEalthy LIver Specific"). Stable cell lines for overexpression of HELIS were obtained and their phenotypic differences from the original wild-type cells were found, in particular, a decrease of cell migration rates. We also have found that the efficiency of induced HELIS expression depends on the correct splicing of this lncRNA. A protocol for Northern blotting of HELIS in the obtained cell lines has been developed involving usage of DIG-labeled probes. Based on the predicted secondary structure of HELIS, we designed and synthesized a set of specific antisense oligonucleotides (ASO). Conditions of HELIS knockdown in HepaRG cells were optimized, and the influence of this lncRNA on the differentiation processes of this cell line was demonstrated. For the first time we determined by RNA-FISH method the cellular localization of HELIS, that was revealed as contained in subnuclear structures for both HepaRG cells and transgenic Huh7 and HepG2 cell lines with overexpression of HELIS. The diagnostic potential of HELIS was estimated on the population of 77 pairs (norm/tumor) of liver samples from patients diagnosed with liver cancer. For the first time we showed different expression levels of HELIS upon carcinogenesis of various etiology. We revealed a reliable correlation between expression of HELIS and the differentiation stage of the tumor and its size. Based on the results obtained for HELIS and three other previously described lncRNAs, we proposed a potential diagnostic panel that allows discriminating benign and malignant tumors.

Publications

1. Burenina O.Y., Lazarevich N.L., Kustova I.F., Shavochkina D.A., Moroz E.A., Kudashkin N.E., Patyutko Y.I., Metelin A.V., Kim E.F., Skvortsov D.A., Zatsepin T.S., Rubtsova M.P. , Dontsova O.A. Panel of potential lncRNA biomarkers can distinguish various types of liver malignant and benign tumors Journal of Cancer Research and Clinical Oncology, - (year - 2020)

2. Burenina O.Y., Lazarevich N.L., Kustova I.F., Shavochkina D.A., Moroz E.A., Kudashkin N.E., Patyutko Y.I., Metelin A.V., Kim E.F., Skvortsov D.A., Zatsepin T.S., Rubtsova M.P., Dontsova O.A. Panel of potential lncRNA biomarkers can distinguish various types of liver malignant and benign tumors Journal of Cancer Research and Clinical Oncology, - (year - 2020) https://doi.org/10.1007/s00432-020-03378-5

3. Burenina O.Y., Zatsepin T.S., Kim E.F., Metelin A.V., Skvortsov D.A., Rubtsova M.P., Dontsova O.A. Сравнительный анализ экспрессии генов длинных некодирующих РНК в клеточных культурах гепатоцитов и печени человека Доклады Российской Академии наук. Науки о жизни, - (year - 2020)

4. Burenina O., Lazarevich N., Zatsepin T., Rubtsova M., Dontsova O. Novel long non-coding RNA as a potential biomarker of liver cancer FEBS Open Bio, V. 9 (Suppl. 1). P. 40. (year - 2019) https://doi.org/10.1002/2211-5463.12674

5. Burenina O., Lazarevich N., Zatsepin T., Rubtsova M., Dontsova O. Новая длинная некодирующая РНК – потенциальный биомаркер рака печени Acta Naturae, Спецвыпуск. Т. 2. С. 18-19. (year - 2019)

6. Burenina O., Lazarevich N., Zatsepin T., Rubtsova M., Dontsova O. Новая длинная некодирующая РНК HELIS – потенциальный биомаркер нормальных гепатоцитов человека Гены и клетки, Т. 14. С.43-44. (year - 2019)

Annotation of the results obtained in 2018
A lot of long noncoding RNAs (lncRNAs) take an active part in the processes of carcinogenesis and therefore are considered as potential biomarkers. In this work, for the first time a novel liver-specific human lncRNA was described and its expression in a healthy donor liver was confirmed. This lncRNA was found to be synthesized in differentiated HepaRG cells mimicking healthy liver but was completely absent in HepG2 and Huh7 liver cancer cell lines. This fact allows us to use these cells to overexpress our lncRNA and further to study its biological role. At this stage of work the corresponding genetic constructs – vectors for integration into the eukaryotic genome - have been obtained. Also a bioinformatics search was conducted to reveal potential homologs of this lncRNA in different mammals. Total RNA samples isolated from liver tissues of mouse, naked mole rat and pig were analyzed. However, lncRNA of the interest wasn’t found in any of these animal models. To estimate the possibility of using this lncRNA as a potential biomarker for liver cancer diagnostics we have analyzed a number of total RNA samples extracted from human hepatocellular carcinoma and hepatoblastoma. In all cases expression levels of lncRNA significantly decreased in tumors when compared with adjacent normal tissues, that clearly demonstrates the diagnostic potential of this molecule.

Publications