Santosh K. Patnaik

Evaluation of MicroRNA Expression Profiles That May Predict Recurrence of Localized Stage I Non–Small Cell Lung Cancer after Surgical Resection

Prognostic markers that can predict the relapse of localized non-small cell lung cancer (NSCLC) have yet to be defined. We surveyed expression profiles of microRNA (miRNA) in stage I NSCLC to identify patterns that might predict recurrence after surgical resection of this common deadly cancer. Small RNAs extracted from formalin-fixed and paraffin-embedded tissues were hybridized to locked nucleic acid probes against 752 human miRNAs (representing 82% of the miRNAs in the miRBase 13.0 database) to obtain expression profiles for 37 cases with recurrence and 40 cases without recurrence (with clinical follow-up for at least 32 months). Differential expression between the two case groups was detected for 49% of the miRNAs (Wilcoxon rank sum test; P<0.01). The performance of expression profiles at differentiating the two case groups was assessed by leave-one-out and Monte Carlo cross-validations. In leave-one-out cross-validation using support vector machines- or top-scoring gene pair classifier methods, which looked for six- or two-miRNA-based classifiers, the identified miRNA expression pattern predicted recurrence with an accuracy of 70% and 83%, and hazard ratio of 3.6 [95% confidence interval (95% CI), 1.8-7.1] and 9.0 (95% CI, 4.4-18.2), respectively. Mean accuracy in Monte Carlo cross-validation using 1,000 random 60-17 splits was 69% (95% CI, 68-70) and 72% (95% CI, 71-72), respectively. The specific miRNAs mir-200b*, mir-30c-1*, mir-510, mir-630, mir-657, and mir-146b-3p and mir-124*, mir-585, and mir-708, respectively, represented most commonly among the 1,000 classifiers identified in Monte Carlo cross-validation by the two methods. MiRNAs mir-488, mir-503, and mir-647 were identified as potential reference miRNAs for future studies, based on the stability of their expression patterns across the 77 cases and the two case-groups. Our findings reinforce efforts to profile miRNA expression patterns for better prognostication of stage I NSCLC.

BGMUT: NCBI dbRBC database of allelic variations of genes encoding antigens of blood group systems

Analogous to human leukocyte antigens, blood group antigens are surface markers on the erythrocyte cell membrane whose structures differ among individuals and which can be serologically identified. The Blood Group Antigen Gene Mutation Database (BGMUT) is an online repository of allelic variations in genes that determine the antigens of various human blood group systems. The database is manually curated with allelic information collated from scientific literature and from direct submissions from research laboratories. Currently, the database documents sequence variations of a total of 1251 alleles of all 40 gene loci that together are known to affect antigens of 30 human blood group systems. When available, information on the geographic or ethnic prevalence of an allele is also provided. The BGMUT website also has general information on the human blood group systems and the genes responsible for them. BGMUT is a part of the dbRBC resource of the National Center for Biotechnology Information, USA, and is available online at http://www.ncbi.nlm.nih.gov/projects/gv/rbc/xslcgi.fcgi?cmd=bgmut. The database should be of use to members of the transfusion medicine community, those interested in studies of genetic variation and related topics such as human migrations, and students as well as members of the general public.

APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages

The extent, regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific C>U RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins, including many that are involved in pathogenesis of viral diseases. APOBEC3A, which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons, mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of C>U RNA editing in mammals.

MicroRNA Expression Profiles of Whole Blood in Lung Adenocarcinoma

The association of lung cancer with changes in microRNAs in plasma shown in multiple studies suggests a utility for circulating microRNA biomarkers in non-invasive detection of the disease. We examined if presence of lung cancer is reflected in whole blood microRNA expression as well, possibly because of a systemic response. Locked nucleic acid microarrays were used to quantify the global expression of microRNAs in whole blood of 22 patients with lung adenocarcinoma and 23 controls, ten of whom had a radiographically detected non-cancerous lung nodule and the other 13 were at high risk for developing lung cancer because of a smoking history of >20 pack-years. Cases and controls differed significantly for age with a mean difference of 10.7 years, but not for gender, race, smoking history, blood hemoglobin, platelet count, or white blood cell count. Of 1282 quantified human microRNAs, 395 (31%) were identified as expressed in the study’s subjects, with 96 (24%) differentially expressed between cases and controls. Classification analyses of microRNA expression data were performed using linear kernel support vector machines (SVM) and top-scoring pairs (TSP) methods, and classifiers to identify presence of lung adenocarcinoma were internally cross-validated. In leave-one-out cross-validation, the TSP classifiers had sensitivity and specificity of 91% and 100%, respectively. The values with SVM were both 91%. In a Monte Carlo cross-validation, average sensitivity and specificity values were 86% and 97%, respectively, with TSP, and 88% and 89%, respectively, with SVM. MicroRNAs miR-190b, miR-630, miR-942, and miR-1284 were the most frequent constituents of the classifiers generated during the analyses. These results suggest that whole blood microRNA expression profiles can be used to distinguish lung cancer cases from clinically relevant controls. Further studies are needed to validate this observation, including in non-adenocarcinomatous lung cancers, and to clarify upon the confounding effect of age.

Detection of microRNAs in dried serum blots

We observed the preservation of microRNAs in unrefrigerated dried serum blots. Preservation was not adversely affected by drying or storing at 37, 45, or 60°C instead of room temperature, but it was harmed when blots were dried incompletely before storage. Preservation of microRNAs in serum was not diminished if, instead of being kept frozen at -80°C, it was stored as dried blots at room temperature for 5 months or at 37°C for 4 weeks. Thus, dried blots can be a convenient and safer way to save, transport, and store serum for microRNA assays.

MicroRNAs and lung cancer: Biology and applications in diagnosis and prognosis.

MicroRNAs are tiny non-coding RNA molecules which play important roles in the epigenetic control of cellular processes by preventing the translation of proteins from messenger RNAs (mRNAs). A single microRNA can target different mRNAs, and an mRNA can be targeted by multiple microRNAs. Such complex interplays underlie many molecular pathways in cells, and specific roles for many microRNAs in physiological as well as pathological phenomena have been identified. Changes in expression of microRNAs have been associated with a wide variety of disease conditions, and microRNA-based biomarkers are being developed for the identification and monitoring of such states. This review provides a general overview of the current state of knowledge about the biology of microRNAs, and specific information about microRNAs with regard to the diagnosis and prognosis of lung cancer.

Overexpression of the lung cancer-prognostic miR-146b microRNAs has a minimal and negative effect on the malignant phenotype of A549 lung cancer cells.

Introduction Expression levels of miR-146b-5p and -3p microRNAs in human non-small cell lung cancer (NSCLC) are associated with recurrence of the disease after surgery. To understand this, the effect of miR-146b overexpression was studied in A549 human lung cancer cells. Methods A549 cells, engineered with lentiviruses to overexpress the human pre-miR-146b precursor microRNA, were examined for proliferation, colony formation on plastic surface and in soft agar, migration and invasiveness in cell culture and in vivo in mice, chemosensitivity to cisplatin and doxorubicin, and global gene expression. miR-146b expressions were assessed in microdissected stroma and epithelia of human NSCLC tumors. Association of miR-146b-5p and -3p expression in early stage NSCLC with recurrence was analyzed. Principal Findings A549 pre-miR-146b-overexpressors had 3–8-fold higher levels of both miR-146b microRNAs than control cells. Overexpression did not alter cellular proliferation, chemosensitivity, migration, or invasiveness; affected only 0.3% of the mRNA transcriptome; and, reduced the ability to form colonies in vitro by 25%. In human NSCLC tumors, expression of both miR-146b microRNAs was 7–10-fold higher in stroma than in cancerous epithelia, and higher miR-146b-5p but lower -3p levels were predictive of recurrence. Conclusions Only a minimal effect of pre-miR-146b overexpression on the malignant phenotype was seen in A549 cells. This could be because of opposing effects of miR-146b-5p and -3p overexpression as suggested by the conflicting recurrence-predictive values of the two microRNAs, or because miR-146b expression changes in non-cancerous stroma and not cancerous epithelia of tumors are responsible for the prognostic value of miR-146b.

Overexpression of MicroRNA miR-30a or miR-191 in A549 Lung Cancer or BEAS-2B Normal Lung Cell Lines Does Not Alter Phenotype

Background MicroRNAs (miRNAs) are small, noncoding RNAs (ribonucleic acids) that regulate translation. Several miRNAs have been shown to be altered in whole cancer tissue compared to normal tissue when quantified by microarray. Based on previous such evidence of differential expression, we chose to study the functional significance of miRNAs miR-30a and -191 alterations in human lung cancer. Methodology/Principal Findings The functional significance of miRNAs miR-30a and -191 was studied by creating stable transfectants of the lung adenocarcinoma cell line A549 and the immortalized bronchial epithelial cell line BEAS-2B with modest overexpression of miR-30a or -191 using a lentiviral system. When compared to the corresponding controls, both cell lines overexpressing miR-30a or -191 do not demonstrate any significant changes in cell cycle distribution, cell proliferation, adherent colony formation, soft agar colony formation, xenograft formation in a subcutaneous SCID mouse model, and drug sensitivity to doxorubicin and cisplatin. There is a modest increase in cell migration in cell lines overexpressing miR-30a compared to their controls. Conclusions/Significance Overexpression of miR-30a or -191 does not lead to an alteration in cell cycle, proliferation, xenograft formation, and chemosensitivity of A549 and BEAS-2B cell lines. Using microarray data from whole tumors to select specific miRNAs for functional study may be a suboptimal strategy.

Transient overexpression of exogenous APOBEC3A causes C-to-U RNA editing of thousands of genes

ABSTRACT APOBEC3A cytidine deaminase induces site-specific C-to-U RNA editing of hundreds of genes in monocytes exposed to hypoxia and/or interferons and in pro-inflammatory macrophages. To examine the impact of APOBEC3A overexpression, we transiently expressed APOBEC3A in HEK293T cell line and performed RNA sequencing. APOBEC3A overexpression induces C-to-U editing at more than 4,200 sites in transcripts of 3,078 genes resulting in protein recoding of 1,110 genes. We validate recoding RNA editing of genes associated with breast cancer, hematologic neoplasms, amyotrophic lateral sclerosis, Alzheimer disease and primary pulmonary hypertension. These results highlight the fundamental impact of APOBEC3A overexpression on human transcriptome by widespread RNA editing.

MiR-205 and MiR-375 MicroRNA Assays to Distinguish Squamous Cell Carcinoma from Adenocarcinoma in Lung Cancer Biopsies

Introduction: Identification of adenocarcinoma (AC) and squamous cell carcinoma (SCC) histology of non–small-cell lung cancer (NSCLC) in biopsies is clinically important but can be inaccurate by routine histopathologic examination. We quantify this inaccuracy at a cancer center, and evaluate the utility of a microRNA-based method to histotype AC/SCC in biopsies. Methods: RNA was extracted from tissue sections with greater than 90% tumor content that were macro- or micro-dissected from formalin-fixed, paraffin-embedded biopsy specimens. MicroRNAs in RNA from the biopsies and from resected tumors were quantified by TaqMan reverse transcription-polymerase chain reaction assays and normalized against the RNU6B housekeeping RNA. Publicly available microRNA expression datasets were examined. Results: NSCLC subtyping of small biopsy specimens by routine histopathologic examination either failed or mistyped the histology of 21% of 190 cases. Using 77 resectates, an reverse transcription-polymerase chain reaction-based assay of microRNAs miR-21, miR-205, and miR-375 was developed to identify AC and SCC subtypes of NSCLC. This method identified the AC/SCC histotypes of 25 biopsies with an accuracy of 96%, and correctly histotyped all 12 cases for which the histology had been mistyped by routine histopathologic examination of the biopsy. Examination of publicly available datasets identified miR-205 and miR-375 as microRNAs with the best ability to histotype AC and SCC, and that levels of the two microRNAs in AC or SCC are unaffected by the pathologic stage of the tumor or the age or race of the patient. Conclusions: Histotypic microRNA assays can aid the subtyping of NSCLC biopsies as AC or SCC by standard histopathologic methods.