Elevated expression of miR-494-3p is associated with resistance to osimertinib in EGFR T790M-positive non-small cell lung cancer.

Background: Non-small cell lung cancer (NSCLC) harboring activating mutations in the gene encoding epidermal growth factor receptor (EGFR) is amenable for targeted therapy with tyrosine kinase inhibitors (TKIs). Eventually, resistance to TKI-therapy occurs resulting in disease progression. A substantial fraction of resistance mechanisms is unknown and may involve alterations in the RNA or protein landscape. MicroRNAs (miRNAs) have been frequently suggested to play roles in various forms of cancer including NSCLC. However, a role of miRNAs in acquired resistance to EGFR TKIs remains elusive. In this work, we aimed to investigate the potential involvement of miRNAs in acquired resistance to the third-generation EGFR TKI osimertinib in NSCLC. Methods: We combined miRNA expression profiling with miRNA-inhibitory screening to identify miRNAs involved in conferring resistance to osimertinib. Finally, we validated our top miRNA candidate by profiling longitudinal plasma exosomal RNA from patients receiving osimertinib as second-line therapy in a clinical trial. Results: Various miRNAs displayed differential expression in parental versus osimertinib-refractory NSCLC cells. miRNA-inhibitory screening revealed miR-494-3p to partially confer resistance to osimertinib in vitro. Expression of miR-494-3p was significantly elevated in plasma sampled at disease progression compared to plasma sampled at treatment baseline in a cohort of 21 EGFR T790M-mutation positive NSCLC patients receiving osimertinib. Conclusions: Our results highlight the need for further therapeutic exploration of miR-494-3p in in vivo models of EGFR-mutant NSCLC.

The Profile of MicroRNA Expression and Potential Role in the Regulation of Drug-Resistant Genes in Doxorubicin and Topotecan Resistant Ovarian Cancer Cell Lines.

Epithelial ovarian cancer has the highest mortality among all gynecological malignancies. The main reasons for high mortality are late diagnosis and development of resistance to chemotherapy. Resistance to chemotherapeutic drugs can result from altered expression of drug-resistance genes regulated by miRNA. The main goal of our study was to detect differences in miRNA expression levels in two doxorubicin (DOX)- and two topotecan (TOP)-resistant variants of the A2780 drug-sensitive ovarian cancer cell line by miRNA microarray. The next aim was to recognize miRNAs as factors responsible for the regulation of drug-resistance genes. We observed altered expression of 28 miRNA that may be related to drug resistance. The upregulation of miR-125b-5p and miR-935 and downregulation of miR-218-5p was observed in both DOX-resistant cell lines. In both TOP-resistant cell lines, we noted the overexpression of miR-99a-5p, miR-100-5p, miR-125b-5p, and miR-125b-2-3p and decreased expression of miR-551b-3p, miR-551b-5p, and miR-383-5p. Analysis of the targets suggested that expression of important drug-resistant genes such as the collagen type I alpha 2 chain (COL1A2), protein Tyrosine Phosphatase Receptor Type K (PTPRK), receptor tyrosine kinase-EPHA7, Roundabout Guidance Receptor 2 (ROBO2), myristoylated alanine-rich C-kinase substrate (MARCK), and the ATP-binding cassette subfamily G member 2 (ABCG2) can be regulated by miRNA.

The significance of interferon gamma inducible protein 16 (IFI16) expression in drug resistant ovarian cancer cell lines.

BACKGROUND: Inherent or developed during treatment drug resistance is the main reason for the low effectiveness of chemotherapy in ovarian cancer. IFI16 is a cytoplasmic/nuclear protein involved in response to virus's infection and cell cycle arrest associated with the cellular senescence. METHODS: Here we performed a detailed IFI16 expression analysis in ovarian cancer cell lines sensitive (A2780) and resistant to doxorubicin (DOX) (A2780DR1 and A2780DR2) and paclitaxel (PAC) (A2780PR1). IFI16 mRNA level, protein level in the nuclear and cytoplasmic fraction (Western blot analysis), the protein expression in cancer cells and nuclei (immunofluorescence analysis) and cancer patient lesions (immunohistochemistry) were performed in this study. RESULTS: We observed upregulation of IFI16 expression in drug resistant cell lines with dominant cytoplasmic localization in DOX-resistant cell lines and nuclear one in the PAC-resistant cell line. The most abundantly overexpressed isoforms of IFI16 were IFI16A and IFI16C. Finally, an analysis of a histological type of ovarian cancer (immunohistochemistry) showed expression in serous ovarian cancer. CONCLUSIONS: Expression of IFI16 in drug-resistant cell lines suggests its role in drug resistance development in ovarian cancer. Expression in serous ovarian cancer suggests its role in the pathogenesis of this histological type.

The Profile of MicroRNA Expression and Potential Role in the Regulation of Drug-Resistant Genes in Cisplatin- and Paclitaxel-Resistant Ovarian Cancer Cell Lines.

Ovarian cancer is the most lethal gynecological malignancy. The high mortality results from late diagnosis and the development of drug resistance. Drug resistance results from changes in the expression of different drug-resistance genes that may be regulated miRNA. The main aim of our study was to detect changes in miRNA expression levels in two cisplatin (CIS) and two paclitaxel (PAC)-resistant variants of the A2780 drug-sensitive ovarian cancer cell line-by miRNA microarray. The next goal was to identify miRNAs responsible for the regulation of drug-resistance genes. We observed changes in the expression of 46 miRNA that may be related to drug resistance. The overexpression of miR-125b-5p, miR-99a-5p, miR-296-3p, and miR-887-3p and downregulation of miR-218-5p, miR-221-3p, and miR-222-3p was observed in both CIS-resistant cell lines. In both PAC-resistant cell lines, we observed the upregulation of miR-221-3p, miR-222-3p, and miR-4485, and decreased expression of miR-551b-3p, miR-551b-5p, and miR-218-5p. Analysis of targets suggest that expression of important drug-resistant genes like protein Tyrosine Phosphatase Receptor Type K (PTPRK), receptor tyrosine kinase-EPHA7, Semaphorin 3A (SEMA3A), or the ATP-binding cassette subfamily B member 1 gene (ABCB1) can be regulated by miRNA.

Matched Analyses of Brain Metastases versus Primary Non-Small Cell Lung Cancer Reveal a Unique microRNA Signature.

Distant spreading of tumor cells to the central nervous system in non-small cell lung cancer (NSCLC) occurs frequently and poses major clinical issues due to limited treatment options. RNAs displaying differential expression in brain metastasis versus primary NSCLC may explain distant tumor growth and may potentially be used as therapeutic targets. In this study, we conducted systematic microRNA expression profiling from tissue biopsies of primary NSCLC and brain metastases from 25 patients. RNA analysis was performed using the nCounter Human v3 miRNA Expression Assay, NanoString technologies, followed by differential expression analysis and in silico target gene pathway analysis. We uncovered a panel of 11 microRNAs with differential expression and excellent diagnostic performance in brain metastasis versus primary NSCLC. Five microRNAs were upregulated in brain metastasis (miR-129-2-3p, miR-124-3p, miR-219a-2-3p, miR-219a-5p, and miR-9-5p) and six microRNAs were downregulated in brain metastasis (miR-142-3p, miR-150-5p, miR-199b-5p, miR-199a-3p, miR-199b-5p, and miR-199a-5p). The differentially expressed microRNAs were predicted to converge on distinct target gene networks originating from five to twelve core target genes. In conclusion, we uncovered a unique microRNA profile linked to two target gene networks. Our results highlight the potential of specific microRNAs as biomarkers for brain metastasis in NSCLC and indicate plausible mechanistic connections.

Construction of a Full-Length 3'UTR Reporter System for Identification of Cell-Cycle Regulating MicroRNAs.

Three prime untranslated region (3'UTR) reporter constructs are widely used by the scientific community to functionally link microRNAs (miRNAs) to suppression of mRNA expression. However, full-length 3'UTR vectors are rarely employed due to labor-intensive cloning work. Instead, 3'UTR fragments containing putative miRNA binding sites are commonly utilized to mechanistically validate miRNAs. Assaying truncated 3'UTRs may falsely validate miRNAs due to altered positioning of binding sites in respect to 3'UTR length and RNA secondary structure. Here we present a detailed protocol for the construction of full-length 3'UTR luciferase reporter constructs that was used to unveil miRNAs regulating multiple cell-cycle factors.

A MicroRNA Gene Panel Predicts the Vaginal Microbiota Composition.

The vaginal microbiota plays an essential role in vaginal health. The vaginas of many reproductive-age women are dominated by one of the Lactobacillus species. However, the vaginas of a large number of women are characterized by the colonization of several other anaerobes. Notably, some women with the non-Lactobacillus-dominated vaginal microbiota develop bacterial vaginosis, which has been correlated with sexually transmitted infections and other adverse outcomes. However, interactions and mechanisms linking the vaginal microbiota to host response are still under investigation. There are studies suggesting a link between human microRNAs and gut microbiota, but limited analysis has been carried out on the interplay of microRNAs and vaginal microbiota. In this study, we performed a microRNA expression array profiling on 67 vaginal samples from young Swedish women. MicroRNAs were clustered into distinct groups according to vaginal microbiota composition. Interestingly, 182 microRNAs were significantly elevated in their expression in the non-Lactobacillus-dominated community, suggesting an antagonistic relationship between Lactobacillus and microRNAs. Of the elevated microRNAs, 10 microRNAs displayed excellent diagnostic potential, visualized by receiver operating characteristics analysis. We further validated our findings in 34 independent samples where expression of top microRNA candidates strongly separated the Lactobacillus-dominated community from the non-Lactobacillus-dominated community in the vaginal microbiota. Notably, the Lactobacillus crispatus-dominated community showed the most profound differential microRNA expression compared with the non-Lactobacillus-dominated community. In conclusion, we demonstrate a strong relationship between the vaginal microbiota and numerous genital microRNAs, which may facilitate a deeper mechanistic interplay in this biological niche.IMPORTANCE Vaginal microbiota is correlated with women's health, where a non-Lactobacillus-dominated community predisposes women to a higher risk of disease, including human papillomavirus (HPV). However, the molecular relationship between the vaginal microbiota and host is largely unexplored. In this study, we investigated a link between the vaginal microbiota and host microRNAs in a group of young women. We uncovered an inverse correlation of the expression of microRNAs with the abundance of Lactobacillus species in the vaginal microbiota. Particularly, the expression of microRNA miR-23a-3p and miR-130a-3p, displaying significantly elevated levels in non-Lactobacillus-dominated communities, predicted the bacterial composition of the vaginal microbiota in an independent validation group. Since targeting of microRNAs is explored in the clinical setting, our results warrant investigation of whether microRNA modulation could be used for treating vaginosis recurrence and vaginosis-related diseases. Conversely, commensal bacteria could be used for treating diseases with microRNA aberrations.

The Significance of MicroRNAs Expression in Regulation of Extracellular Matrix and Other Drug Resistant Genes in Drug Resistant Ovarian Cancer Cell Lines.

Ovarian cancer rates the highest mortality among all gynecological malignancies. The main reason for high mortality is the development of drug resistance. It can be related to increased expression of drug transporters and increased expression of extracellular matrix (ECM) proteins. Our foremost aim was to exhibit alterations in the miRNA expression levels in cisplatin (CIS), paclitaxel (PAC), doxorubicin (DOX), and topotecan (TOP)-resistant variants of the W1 sensitive ovarian cancer cell line-using miRNA microarray. The second goal was to identify miRNAs responsible for the regulation of drug-resistant genes. According to our observation, alterations in the expression of 40 miRNAs were present. We could observe that, in at least one drug-resistant cell line, the expression of 21 miRNAs was upregulated and that of 19 miRNAs was downregulated. We identified target genes for 22 miRNAs. Target analysis showed that miRNA regulates key genes responsible for drug resistance. Among others, we observed regulation of the ATP-binding cassette subfamily B member 1 gene (ABCB1) in the paclitaxel-resistant cell line by miR-363 and regulation of the collagen type III alpha 1 chain gene (COL3A1) in the topotekan-resistant cell line by miR-29a.

Expression of Osteoblast-Specific Factor 2 (OSF-2, Periostin) Is Associated with Drug Resistance in Ovarian Cancer Cell Lines.

One of the main obstacles to the effective treatment of ovarian cancer patients continues to be the drug resistance of cancer cells. Osteoblast-Specific Factor 2 (OSF-2, Periostin) is a secreted extracellular matrix protein (ECM) expressed in fibroblasts during bone and teeth development. Expression of OSF-2 has been also related to the progression and drug resistance of different tumors. The present study investigated the role of OSF-2 by evaluating its expression in the primary serous ovarian cancer cell line, sensitive (W1) and resistant to doxorubicin (DOX) (W1DR) and methotrexate (MTX) (W1MR). The OSF-2 transcript (real-time PCR analysis), protein expression in cell lysates and cell culture medium (western blot), and expression of the OSF-2 protein in cell lines (immunofluorescence) were investigated in this study. Increased expression of OSF-2 mRNA was observed in drug-resistant cells and followed by increased protein expression in cell culture media of drug-resistant cell lines. A subpopulation of ALDH1A1-positive cells was noted for W1DR and W1MR cell lines; however, no direct co-expression with OSF-2 was demonstrated. Both drugs induced OSF-2 expression after a short period of exposure of the drug-sensitive cell line to DOX and MTX. The obtained results indicate that OSF-2 expression might be associated with the development of DOX and MTX resistance in the primary serous W1 ovarian cancer cell line.

PTPRK Expression Is Downregulated in Drug Resistant Ovarian Cancer Cell Lines, and Especially in ALDH1A1 Positive CSCs-Like Populations.

Background: Ovarian cancer is the 7th most common cancer and 8th most mortal cancer among woman. The standard treatment includes cytoreduction surgery followed by chemotherapy. Unfortunately, in most cases, after treatment, cancer develops drug resistance. Decreased expression and/or activity of protein phosphatases leads to increased signal transduction and development of drug resistance in cancer cells. Methods: Using sensitive (W1, A2780) and resistant ovarian cancer cell lines, the expression of Protein Tyrosine Phosphatase Receptor Type K (PTPRK) was performed at the mRNA (real-time PCR analysis) and protein level (Western blot, immunofluorescence analysis). The protein expression in ovarian cancer tissues was determined by immunohistochemistry. Results: The results showed a decreased level of PTPRK expression in ovarian cancer cell lines resistant to cisplatin (CIS), paclitaxel (PAC), doxorubicin (DOX), topotecan (TOP), vincristine (VIN) and methotrexate (MTX). Additionally, the lower PTPRK expression was observed in Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) positive cancer stem cells (CSCs) population, suggesting the role of PTPRK downregulation in primary as well as acquired resistance to cytotoxic drugs. Conclusions: These results provide important insights into the role of PTPRK in mechanism leading to drug resistance in ovarian cancer and has raised important questions about the role of imbalance in processes of phosphorylation and dephosphorylation.

Mutual Expression of ALDH1A1, LOX, and Collagens in Ovarian Cancer Cell Lines as Combined CSCs- and ECM-Related Models of Drug Resistance Development.

A major contributor leading to treatment failure of ovarian cancer patients is the drug resistance of cancer cell. CSCs- (cancer stem cells) and ECM (extracellular matrix)-related models of drug resistance are described as independently occurring in cancer cells. Lysyl oxidase (LOX) is another extracellular protein involved in collagen cross-linking and remodeling of extracellular matrix and has been correlated with tumor progression. The expression of LOX, COL1A2, COL3A1, and ALDH1A1 was performed in sensitive (A2780, W1) and resistant to paclitaxel (PAC) (A2780PR1 and W1PR2) and topotecan (TOP) (W1TR) cell lines at the mRNA (real-time PCR analysis) and protein level (Western blot and immunofluorescence analysis). The ALDH1A1 activity was measured with the ALDEFLUOR test and flow cytometry analysis. The protein expression in ovarian cancer tissues was determined by immunohistochemistry. We observed an increased expression of LOX and collagens in PAC and TOP resistant cell lines. Subpopulations of ALDH1A1 positive and negative cells were also noted for examined cell lines. Additionally, the coexpression of LOX with ALDH1A1 and COL1A2 with ALDH1A1 was observed. The expression of LOX, collagens, and ALDH1A1 was also detected in ovarian cancer lesions. In our study LOX, ALDH1A1 and collagens were found to be coordinately expressed by cells resistant to PAC (LOX, ALDH1A1, and COL1A2) or to TOP (LOX and ALDH1A1). This represents the study where molecules related with CSCs (ALDH1A1) and ECM (LOX, collagens) models of drug resistance are described as occurring simultaneously in ovarian cancer cells treated with PAC and TOP.

New and Old Genes Associated with Primary and Established Responses to Paclitaxel Treatment in Ovarian Cancer Cell Lines.

Development of drug resistance is the main reason for low chemotherapy effectiveness in treating ovarian cancer. Paclitaxel (PAC) is a chemotherapeutic drug used in the treatment of this cancer. We analysed the development of PAC resistance in two ovarian cancer cell lines. Exposure of drug-sensitive cell lines (A2780 and W1) to PAC was used to determine the primary response. An established response was determined in PAC-resistant sublines of the A2780 and W1 cell lines. qRT-PCR was performed to measure the expression levels of specific genes. We observed decreased expression of the PCDH9, NSBP1, MCTP1 and SEMA3A genes in the PAC-resistant cell lines. Short-term exposure to PAC led to increased expression of the MDR1 and BCRP genes in the A2780 and W1 cell lines. In the A2780 cell line, we also observed increased expression of the C4orf18 gene and decreased expression of the PCDH9 and SEMA3A genes after PAC treatment. In the W1 cell line, short-term treatment with PAC upregulated the expression of the ALDH1A1 gene, a marker of Cancer stem cells (CSCs). Our results suggest that downregulation of the PCDH9, NSBP1, MCTP1 and SEMA3A genes and upregulation of the MDR1, BCRP, C4orf18 and ALDH1A1 genes may be related to PAC resistance.

Morphogenesis-related gene-expression profile in porcine oocytes before and after in vitro maturation.

Mammalian oocyte maturation is achieved when oocytes reach metaphase II (MII) stage, and accumulate mRNA and proteins in the cytoplasm following fertilization. It has been shown that oocytes investigated before and after in vitro maturation (IVM) differ significantly in transcriptomic and proteomic profiles. Additionally, folliculogenesis and oogenesis is accompanied by morphogenetic changes, which significantly influence further zygote formation and embryo growth. This study aimed to determine new transcriptomic markers of porcine oocyte morphogenesis that are associated with cell maturation competence. An Affymetrix microarray assay was performed on an RNA template isolated from porcine oocytes before (n = 150) and after (n = 150) IVM. The brilliant cresyl blue (BCB) staining test was used for identification of cells with the highest developmental capacity. DAVID (Database for Annotation, Visualization, and Integrated Discovery) software was used for the extraction of the genes belonging to a cell morphogenesis Gene Ontology group. The control group consisted of freshly isolated oocytes. In total, 12,000 different transcripts were analysed, from which 379 genes were downregulated and 40 were upregulated in oocytes following IVM. We found five genes, SOX9, MAP1B, DAB2, FN1, and CXCL12, that were significantly upregulated in oocytes after IVM (in vitro group) compared with oocytes analysed before IVM (in vivo group). In conclusion, we found new transcriptomic markers of oocyte morphogenesis, which may be also recognized as significant mediators of cellular maturation capacity in pigs. Genes SOX9, MAP1B, DAB2, FN1, and CXCL12 may be involved in the regulation of the MII stage oocyte formation and several other processes that are crucial for porcine reproductive competence.