MicroRNAs as Predictive and Prognostic Biomarkers inHuman Neoplasia : With Specific Focus on Colorectal Cancer, Giant Cell Tumor of Bone, and Leukemias

Recently, discovery of microRNA has provided new insights into cancer research, revealing the role of miRNAs in various biological processes, and evidence shows that their deregulation in many cancers has prognostic and predictive significance. Although specific miRNAs have been discovered in the malignancies studied in this thesis: colorectal cancer (CRC), giant cell tumor of bone (GCTB), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML), very little still is known about the association of miRNAs with progression and their response to treatment. By applying novel microarray techniques, we profiled miRNA expression in CRC, GCTB, ALL and, AML. In the study focusing on primary tumors of 60 metastatic CRC (mCRC) patients, we detected BRAF mutations in 5 and KRAS mutations in 15 cases. In addition to 46 altered miRNAs in mCRC patients with and without KRAS mutation vs. normal colon tissue, we observed an miRNA signature associated with KRAS status when we compared 15 patients with mutant KRAS with 40 patients without this mutation. Four differentially expressed miRNAs, over-expressed miR-92a, miR-127-3p, miR-486-5p, and under-expressed miR-378, were evident in the mutated KRAS group vs. wild-type KRAS group. In another study on CRC, miRNA profiling in primary tumors of 33 mCRC patients with wild-type BRAF and KRAS allowed identification of miRNAs related to their response to anti-EGFR monoclonal antibody treatment. We found up-regulated miR-31* and down-regulated miR-592 in progressive disease (PD) compared to that in disease control (DC). Evaluation of mRNA levels of SLC26A3 and ATN1, drug-related genes and of miR-31* target genes showed their lower level of expression in PD vs. DC. Moreover, correlation between overall survival and miRNA expression assessed by two approaches, cluster analysis and the Cox proportional hazard regression model, revealed two common miRNAs, miR-140-3p and miR-1224-5p, to be related to survival in both analyses. As for the study of the metastatic and non-metastatic GCTBs, we found 12 miRNAs as being differentially expressed (miR-136, miR-513a-5p, miR-494, miR-224, miR-542-5p). Expression levels of eight genes such as NFIB, TNC, and FLRT2 correlated inversely with miRNA results. The expression levels of miR-136 and its target gene, NFIB, were verified by use of qRT-PCR. The level of NFIB protein also was higher in metastatic than in non-metastatic GCTBs. Further, we tested the protein expression level of NFIB in an independent validation cohort of 74 primary archival GCTB specimens to assess the power of NFIB as a prognostic marker. Immunodetection showed a higher frequency of NFIB over-expression in primary tumors that developed metastases than in the disease-free group. Moreover, we studied the miRNA expression profiles of primary tumors of 90 bone marrow core biopsies of ALL patients, including 11 patients with paired samples at diagnosis and at relapse. We found a set of miRNAs (miR-1281, miR-1225-3p, miR-877*, miR-423-5p, miR-29c) significantly related to survival (q less than 0.05). Further validation of miR-423-5p expression by qRT-PCR confirmed microarray analysis results and showed a direct correlation with survival. In comparisons between the diagnosis-relapse pairs, expression of miR-654-5p and miR-431 between the two groups significantly differed, and these miRNAs were down-regulated in relapse samples. Comparison of miRNA profiling of 15 chemorefractory and 18 chemosensitive AML patients showed that the differentially expressed miRNAs were miR-363, miR-532-5p, and 342-3p, all of which were over-expressed in chemorefractory vs. chemosensitive patients. Verification by use of qRT-PCR of both miR-363 and miR-532-5p revealed similar results as with microarray. The miR-363 target genes RGS17 and HIPK3 both have been associated with drug response. These studies provide new information about genomic changes involved in progression and resistance to treatment in various types of cancer, and also highlight the power of applying genomic-wide array screening techniques in malignancies. The novel findings in these studies may serve as a useful resource for future studies and aid in development of novel therapeutic targets to increase the survival rates of cancer patients.Mikro.RNA:lla (miRNA) on keskeinen merkitys syövän synnyssä. miRNA-profiilit erottelevat hyvin eri syöpätyypit toisistaan ja niillä on merkitystä arvioitaessa syövän ennustetta ja annettavan hoidon vaikutusta. Omassa työssäni selvitin miRNA:aiden ennusteellista ja hoitotulosta ennakoivaa merkitystä suolistosyövässä, jättisolutuumorissa ja akuuteissa leukemioissa. Nämä syövät valittiin tutkimukseen ensinnäkin, koska mainittuja syöpiä on tutkittu monipuolisesti laboratoriossamme yhteistyössä muiden tutkijoiden kanssa ja toiseksi, koska miRNA:den kliinisestä merkityksestä näissä taudeissa oli vain rajallisesti tietoa. Suolistosyöpätutkimukseen valittiin 60 metastasoitunuttu potilasta. KRAS-mutaatiopositiivisilla potilailla todettiin negatiivisista poikkeava miRNA-profiili: miR-92a, miR-127-3p ja miR-486-5p olivat yliekspressoituneet ja miR-378 oli aliekspressoitunut KRAS-positiivisilla potilailla (osatyö I) Osatyössä II tutkittiin EGFR vasta-ainehoidolle sensitiivisiä ja resistenttejä KRAS-negatiivisia potilaita. Hoitoresistenteillä potilailla miRNA31* oli yliekspressoitunut ja miR-592 aliekspressoitunut. SLC26A3 ja ATN1, miR-31*:n säätelemät kohdegeenit ekspressoituivat käänteisesti miRNA:n ekspressioon nähden. miR-140-3p and ja -1224-5p yliekspressio liittyi huonoon ennusteeseen. Jättisolutuumorissa (osatyö III) Joukko miRNA:ta erotteli metastasoineet tuumorit ei-metastasoineista. NFIB-, TNC- ja FLRT2- geenit ekspressoituivat käänteisesti mRNA:hin nähden. NFIB-proteiinin yliekspressio osoittautui olevan laajennetulla 74 potilaan aineistolla ennusteellisesti huono merkki. Akuutissa lymfaattisessa leukemiassa (osatyö IV) todettiin miR-654-5p:n ja miR-431:n liittyvän tilastollisesti merkittävästi taudin uusiutumiseen. Joukko miRNA:ta (miR-1281, miR-1225-3p, miR-877*, miR-423-5p ja miR-29c) liittyi tilastollisesti merkittävästi taudin ennusteeseen. Viimeisessä osatyössä (V) osoitettiin, että miR-363, miR-532-5p ja 342-3p liittyivät akuutin myelooisen leukemian hoitoresistenssiin. Väitöskirjatutkimuksessani saatiin uutta tietoa miRNA:aiden osuudesta taudin ennusteeseen ja hoitoresistenssiin. Tutkimuksessa osoitettiin uusia biomarkkereita suoloistosyövässä, jättisolutuumorissa ja leukemioissa. Kuvatut molekulaariset muutokset saattavat tulevaisuudessa avata uusia kohteita kohdistetulle henkilökohtaiselle täsmähoidolle

Simultaneous Underexpression of let-7a-5p and let-7f-5p microRNAs in Plasma and Stool Samples from Early Stage Colorectal Carcinoma: Supplementary Issue: Biomarkers for Colon Cancer

Colorectal cancer (CRC) is the third most common malignancy and the second most common cause of cancer death worldwide. Early detection of CRC can improve patient survival rates; thus, the identification of noninvasive diagnostic markers is urgently needed. MicroRNAs (miRNAs) have extensive potential to diagnose several diseases, including cancer. In this study, we compared the expression pattern of miRNAs from plasma and stool samples of patients with early stages of CRC (I, II) with that of healthy subjects. We performed miRNA profiling using microarrays on plasma and stool samples of eight patients with CRC and four healthy subjects. Seven miRNAs were found to be underexpressed in both plasma and stool samples of patients with CRC versus healthy subjects. Then, we aimed to verify two out of these seven differentially expressed miRNAs (let-7a-5p and let-7f-5p) by quantitative reverse transcriptase polymerase chain reaction on a larger set of plasma and stool samples of 51 patients with CRC and 26 healthy subjects. We confirmed the results of microarray analysis since their expression was significantly lower in stool and plasma samples of patients with CRC. Moreover, receiver operating characteristic curve analysis demonstrated that fecal let-7f expression levels have significant sensitivity and specificity to distinguish between patients with CRC and healthy subjects. In conclusion, if the results are confirmed in larger series of patients, underexpressed let-7a-5p and let-7f-5p miRNAs in both plasma and stool samples of patients with CRC may serve potentially as noninvasive molecular biomarkers for the early detection of CRC.Peer reviewe

An integrated analysis of miRNA and gene copy numbers in xenografts of Ewing's sarcoma

<p>Abstract</p> <p>Background</p> <p>Xenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers.</p> <p>Method</p> <p>Microarray technology (array comparative genomic hybridization (aCGH) and micro RNA arrays) was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0) were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106) were selected for further validation by real time polymerase chain reaction (RT-PCR). Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods.</p> <p>Results</p> <p>The most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0). MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, <it>EWSR1, FLI1 </it>and their fusion gene (<it>EWS-FLI1</it>). Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers.</p> <p>Conclusion</p> <p>In the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, <it>FLI1, EWSR1</it>, and the <it>EWS-FLI1 </it>fusion genes.</p

MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro

Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated "Focal adhesion" and "ECM-receptor interaction" as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren't significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15.Peer reviewe

MicroRNA expression profiles in metastatic and non-metastatic giant cell tumor of bone

Giant cell tumor of bone (GCTB) is a skeletal neoplasm, a locally aggressive tumor that occasionally metastasizes to the lungs. To identify novel biomarkers associated with GCTB progression and metastasis, we performed a miRNA microarray on ten primary tumors of GCTB, of which five developed lung metastases and the rest remained metastasis-free. Between metastatic and non-metastatic GCTB, 12 miRNAs were differentially expressed (such as miR-136, miR-513a-5p, miR-494, miR-224, and miR-542-5p). A decreased level of miR-136 in metastatic versus nonmetastatic GCTB was significantly confirmed by the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (p=0.04). To identify potential target genes for the differentially expressed miRNAs, we used three target prediction databases. Then, to functionally validate the potential target genes of the differentially expressed miRNAs, we re-analyzed our previous gene expression data from the same ten patients. Eight genes such as NFIB, TNC, and FLRT2 were inversely expressed relative to their predicted miRNA regulators. NFIB expression correlated in metastatic GCTB with no or low expression of miR-136, and this gene was selected for further verification with qRT-PCR and immunohistochemistry. Verification of NFIB mRNA and protein by qRT-PCR showed elevated expression levels in metastatic GCTBs. Further, the protein expression level of NFIB was tested in an independent validation cohort of 74 primary archival GCTB specimens. In the primary tumors that developed metastases compared to the disease-free group, NFIB protein was moderately to strongly expressed at a higher frequency. Thus, in GCTB, miR-136 and NFIB may serve as prognostic makers

Simultaneous Underexpression of let-7a-5p and let-7f-5p microRNAs in Plasma and Stool Samples from Early Stage Colorectal Carcinoma

International audienc

Simultaneous underexpression of let-7a-5p and let-7f-5p microRNAs in plasma and stool samples from early stage colorectal carcinoma

The study was supported by Novartis, Sigrid Jusélius Foundation, and Cancer Society of Finland. The authors confirm that the funder had no influence over the study design, content of the article, or selection of this journal.Colorectal cancer (CRC) is the third most common malignancy and the second most common cause of cancer death worldwide. Early detection of CRC can improve patient survival rates; thus, the identification of noninvasive diagnostic markers is urgently needed. MicroRNAs (miRNAs) have extensive potential to diagnose several diseases, including cancer. In this study, we compared the expression pattern of miRNAs from plasma and stool samples of patients with early stages of CRC (I, II) with that of healthy subjects. We performed miRNA profiling using microarrays on plasma and stool samples of eight patients with CRC and four healthy subjects. Seven miRNAs were found to be underexpressed in both plasma and stool samples of patients with CRC versus healthy subjects. Then, we aimed to verify two out of these seven differentially expressed miRNAs (let-7a-5p and let-7f-5p) by quantitative reverse transcriptase polymerase chain reaction on a larger set of plasma and stool samples of 51 patients with CRC and 26 healthy subjects. We confirmed the results of microarray analysis since their expression was significantly lower in stool and plasma samples of patients with CRC. Moreover, receiver operating characteristic curve analysis demonstrated that fecal let-7f expression levels have significant sensitivity and specificity to distinguish between patients with CRC and healthy subjects. In conclusion, if the results are confirmed in larger series of patients, underexpressed let-7a-5p and let-7f-5p miRNAs in both plasma and stool samples of patients with CRC may serve potentially as noninvasive molecular biomarkers for the early detection of CRC

MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro

Abstract Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated “Focal adhesion” and “ECM-receptor interaction” as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren’t significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15

Hsa-miR-31-3p expression is linked to progression-free survival in patients with KRAS wild-type metastatic colorectal cancer treated with anti-EGFR therapy.

To identify microRNAs (miRNA) that predict response to anti-EGFR antibodies in patients with wild-type KRAS metastatic colorectal cancer (mCRC).SCOPUS: ar.jinfo:eu-repo/semantics/publishe