hnRNP B1 mRNA Research Articles

Magnetic nanoparticle-enhanced SPR on gold nanoslits for ultra-sensitive, label-free detection of nucleic acid biomarkers

We have demonstrated a detection method for the ultra-sensitive detection of an mRNA biomarker. The method utilizes functionalized magnetic nanoparticles (MNPs) for signal enhancement in conjunction with surface plasmon resonance (SPR) on gold nanoslits. The approach for detection includes double hybridization at two different specific locations in two steps. First, the biomarker target molecule is captured with MNPs, and second, MNPs carrying the target molecule are introduced to the SPR chip to hybridize with probes immobilized on the gold nanoslits. In this work, MNPs were applied for a dual purpose: to isolate the target molecule from the sample matrix to prevent non-specific binding and to enhance the SPR response. Gold nanoslits that provide SPR sensing were fabricated by nanoimprinting lithography on polycarbonate (PC) film. The film was integrated with a microliter volume microfluidic chip to form the SPR detection chip. This detection method was used to detect mRNA heterogeneous nuclear ribonucleoproteins (hnRNP B1) in two cancer cell lines, CL1-0 and CL1-5. hnRNP B1 is an mRNA biomarker that is overexpressed in lung cancer tissue in the early stage of cancer and can be found in the serum and plasma of lung cancer patients. A synthetic target molecule and extracted total RNA from the cell lines were used as samples. Without amplification and labeling of the target molecule, the SPR results demonstrate a specific and sensitive method for the detection of hnRNP B1 mRNA in extracted RNA from the two selected cell lines. The method is capable of measuring down to 30 fM of the target molecule in a 7 μl sample (corresponding to 1.26 × 10(5) molecules) without amplification and labeling of the target molecule.

Abstract 4680: DNA dependent protein kinase as a new molecular target for treatment of non-small cell lung cancer

Abstract Prognosis with non-small cell lung cancer (NSCLC) has improved with the recent introduction of molecular targeted therapy. However, it is limited to patients with epidermal growth factor receptor (EGFR) mutations, who benefit from EGFR tyrosine kinase inhibitors (EGFR-TKI). A new molecular target is needed with other NSCLC patients. In general, chemotherapeutic agents cause DNA double-strand breaks (DNA DSB), leading to apoptosis of cancer cells; DNA repair reverses their effect, resulting in resistance to chemotherapy. DNA DSB repair can be mediated via two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). More than 90% of DSBs in mammalian cells are repaired by NHEJ, which is accomplished by core protein components including Ku70/Ku86, DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, Artemis, and Cernunnos-XLF. We recently reported that NK314, a dual inhibitor of topoisomerase IIα (Top2β) and DNA-PK, evidences a potent anti-tumor effect in adult T-cell leukemia-lymphoma (ATL). NK314 induced DNA DSBs and inhibited DNA repair mediated through degradation of the DNA-PK catalytic subunit (DNA-PKcs). In the present study, we examined the effect of NK314 in NSCLC cell lines and assessed DNA-PK as a possible molecular target for NSCLC. Twenty separate NSCLC cell lines were used, with IC50 varying from 46-193 nM. Levels of DNA-PKcs protein varied among the cell lines, whereas those of Ku70 and Ku86 did not. The inhibitory effect of cell growth was closely related to level of DNA-PKcs protein, but not to levels of other DNA-damage-activated protein kinases such as ATM or ATR. Establishment of predictive markers concomitantly with the development of a novel molecular target is indispensable for clinical application. Because detection of DNA-PKcs in peripheral blood is difficult, we investigated an alternative marker for predicting DNA-PKcs inhibitor. We previously found that heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), an RNA binding protein, interacts with DNA-PK, and we established a system for detecting hnRNP B1 mRNA in peripheral blood. We therefore examined the suitability of hnRNP B1 as an alternative biomarker for DNA-PK inhibitors. Close correlation between levels of DNA-PKcs and hnRNP B1 proteins was established using Western blot analysis with NSCLC cell lines (p=0.995). These results suggest that DNA-PK is a novel molecular target for NSCLC, and detection of hnRNP B1 might serve as an alternative molecular marker for the amount of DNA-PK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4680. doi:1538-7445.AM2012-4680

The perioperative change and clinical significance of the expression level of peripheral blood hnRNP B1 mRNA in NSCLC patients

Objective The aim of this study was to investigate the perioperative expression of the peripheral blood hnRNP B1 mRNA in non-small-cell lung cancer (NSCLC) patients and its clinical significance.

Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay

BackgroundIn addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD) plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate. ResultsIn order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs). Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1's role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3' UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms.ConclusionsDespite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels.

Comparative Quantification of Plasma hnRNP B1 mRNA in Non-small Cell Lung Cancer Patients by Real-time PCR

Circulating cell-free nucleic acids are known to be a noninvasive diagnostic tool for cancer detection. Heterogeneous nuclear ribonucleoprotein (hnRNP) B1, a nuclear core complex, is overexpressed in early stage lung cancer. We intended to evaluate the usefulness of plasma hnRNP B1 mRNA in differentiating non-small cell lung cancer (NSCLC) from other benign lung diseases, especially pulmonary tuberculosis, which is highly prevalent in Korea and often difficult to distinguish from lung cancer. Plasma RNA was extracted from 30 patients with NSCLC, 30 patients with benign lung diseases including pulmonary tuberculosis, and 10 healthy controls. Plasma hnRNP B1 mRNA was measured by TaqMan Gene Expression Assay (Applied Biosystems, USA), and pre-developed beta-actin (ACTB) mRNA was used for normalization. We analyzed the relative gene expression data using the delta-delta Ct method. Plasma hnRPN B1 mRNA was measurable in 93.3% (28/30) of NSCLC patients. Normalized 2-DeltaDeltaCt of plasma hnRPN B1 mRNA was 62.2 (95%Cl, 6.4-210.1) in NSCLC patients and 2.7 (95%Cl, 0.5-13.6) in benign lung disease patients (P<0.001, Mann-Whitney U test). Plasma hnRNP B1 mRNA was significantly increased in patients with lung cancer compared with that in patients with other benign lung diseases. Plasma hnRNP B1 mRNA may be useful as a potential marker for the detection of NSCLC.

The Expression Inhibition of HnRNP B1 in Lung Cell Line A549 by Small Interfering RNA

Background: Preliminary studies indicate a relative link between the expression of heterogeneous nuclear ribonucleoproteins (hnRNPs) and cancer. Overexpression of hnRNP B1 correlated with the eventual development of lung cancer. Here we describe inhibition of HnRNP B1 replication in human lung cell line A549 by siRNAs targeted to various regions of hnRNP B1 sequences. Methods: Small interfering RNA technique was used to interfer the expression of hnRNPB1 in Lung Cell Line A549 in vitro; the RT-PCR and Western blot technique were used to detect the quantity of hnRNPB1 mRNA and its protein; flow cytometry was used to detect the apoptosis; then the cells were transfected to nude mice. Results: We demonstrated that plasmidderived siRNAs could inhibit hnRNP B1 mRNA and protein expression, and the siRNA expressed from activated plasmid in A549 cells could suppress the cell proliferation by increasing the G1 cells. Our experiment also found that siRNA could induce apoptosis. Conclusions: These results demonstrated the effectiveness of siRNA in lung cancer cells, which indicated a possible clinical therapy method for lung cancer development.

Establishment of a new method, transcription–reverse transcription concerted reaction, for detection of plasma hnRNP B1 mRNA, a biomarker of lung cancer

Development of an early detection marker is one of the most important strategies for improving overall prognosis in lung cancer patients. We previously reported that hnRNP B1--an RNA binding protein--is overexpressed in lung cancer tissue from the early stage of cancer, and found that hnRNP B1 mRNA is detectable in the plasma of lung cancer patients using real-time RT-PCR. The purpose of this study was to establish a quick and simple method for detecting plasma hnRNP B1mRNA for use in screening for lung cancer. TRC, a homogenous method for fluorescence real-time monitoring of isothermal RNA amplification using intercalation activating fluorescence DNA probe, was used to detect plasma hnRNP B1 mRNA. The detection limit of hnRNP B1 mRNA by TRC using synthetic control RNA or total RNA derived from a lung cancer cell line was 25 or 8.65 x 10(2) copies, respectively. Using total RNA extracted from 600 mul of plasma, we detected hnRNP B1 mRNA in 39.1% (9/23) of lung cancer patients, with levels ranging from 1.9 to 19,045.5 copies/100 ng RNA, and in 5.2% (5/97) of healthy volunteers. Copy numbers were not associated with age, gender, smoking status, or histological type of cancer. TRC could detect 10(3) copies of hnRNP B1 mRNA in 10 min. Detection of plasma hnRNP B1 mRNA by TRC is a quick, easy, and non-invasive method suitable for lung cancer screening.

Detection of plasma hnRNP B1 mRNA, a new cancer biomarker, in lung cancer patients by quantitative real-time polymerase chain reaction

Circulating cell-free nucleic acids are noninvasive diagnostic tools for cancer detection. Heterogeneous nuclear ribonucleoprotein (hnRNP) B1, an RNA binding protein, has been found overexpressed in the early stage of lung cancer, including bronchial dysplasia, a premalignant lesion of lung squamous cell carcinoma. To determine the utility of plasma hnRNP B1 RNA and as cancer detection markers for lung cancer, we analyzed plasma hnRNP B1 mRNA of lung cancer patients by real-time RT-PCR. Plasma RNA was extracted from plasma of 44 lung cancer patients, 7 lung neoplasm patients, 24 benign lung diseases and 25 healthy volunteers. Mean concentration of plasma hnRNP B1 mRNA in lung cancer patients was 0.99 pg/microg RNA, whereas that in healthy volunteers and in benign lung diseases was 0.23 pg/microg RNA and 0.30 pg/microg RNA, respectively (p<0.05). Twenty of 44 (45.5%) lung cancer patients showed more than 0.70 pg/microg RNA of plasma hnRNP B1 mRNA, compared with only 3 of 25 (12.0%) healthy volunteers. Looking at histological subtype, squamous cell carcinoma patients showed higher hnRNP B1 mRNA in the plasma than did adenocarcinoma patients, which is consistent with our previous immunohistochemistry results. These results indicate that plasma hnRNP B1 mRNA is a useful non-invasive markers for detection of lung cancer.

Expression of hnRNP A2/B1 proteins in small airway epithelial cells.

The overexpression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, especially of B1 has been reported informative to detect pre-clinical lung cancer for early detection. However, relatively little work has been performed on the expression of hnRNP A2/B1 in non-malignant airway cells; such information is clearly required to investigate whether overexpression of this protein could be a specific marker for early lung cancer detection. In order to evaluate whether the expression of hnRNP A2/B1 is specific for cancer cells, we investigated the expression of the protein in human small airway epithelial (HSAE) cells. Using immunostaining and Western blotting with monoclonal antibodies, 4G8 (specifically reacts A2) and 2B2 (specific to B1), expression of hnRNP A2/B1 in HSAE cells was evaluated. Northern blotting and quantitative RT-PCR were also performed. In HSAE cells, hnRNP A2 as well as B1 were expressed primarily in the nucleus excluding the nucleolus, although the expression of A2 protein was weaker than that of B1 protein. Staining pattern of hnRNP A2/B1 in HSAE cells was similar to that observed in control cancer cells. Western blotting with 4G8 and 2B2 apparently demonstrated expression of A2 and B1 in HSAE cells. In Northern blotting, both hnRNP A2 mRNA and B1 mRNA were observed in HSAE cells as shown in control cancer cells. hnRNP A2/B1 was apparently expressed in HSAE cells, although the expression in HSAE cells was weaker than that of cancer cells. More quantitative determination of A2/B1 is required to elucidate their significance in early lung cancer detection.

Expression of hnRNP A2/B1 in human lung cancer cell lines

To explore the mRNA expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 in human lung cancer cell lines. Expression of hnRNP A2/B1 and hnRNP B1 mRNA was detected by RT-PCR using specific primers. Expression of hnRNP B1 protein was examined by immunocytochemistry using specific anti-hnRNP B1 antibody. RT-PCR indicated that the hnRNP A2/B2 and hnRNP B1 mRNA existed in the lung adenocarcinoma cell lines SPC-A1 and Y-90, squamous cell carcinoma cell line A431 and small cell lung cancer cell line NCI-H446. According to immunocytochemistry results, the expression of hnRNP B1 was observed in lung cancer cell lines, but the expression in squamous cell carcinoma cell line A431 and small cell lung cancer cell line NCI-H446 was markedly higher than that in adenocarcinoma cell lines SPC-A1 and Y-90. The results suggest that the human lung cancer cell lines detected have overexpression of hnRNP A2/B1 and hnRNP B1.

Lung cancer prevention with (-)-epigallocatechin gallate using monitoring by heterogeneous nuclear ribonucleoprotein B1

Considering the problems involved in prevention of human lung cancer, growth inhibition of human lung cancer cell line A549 was studied with emphasis on two parameters: green tea polyphenols, such as (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG); and heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a new biomarker of human lung cancer which is highly expressed in the very early stages of human lung cancer. The inhibitory potencies of green tea polyphenols were compared with those of genistein as a control. EGCG or ECG and genistein as a control dose-dependently inhibited the growth of A549 cells, which strongly elevated hnRNP B1 protein, and increased G2/M phase cells associated with induction of apoptotic cells. The results were confirmed by previous evidence with human lung cancer cell line PC-9. Some larger differences in mechanisms of action between green tea polyphenols and genistein were presented. Treatment of A549 cells with EGCG, ECG or genistein significantly inhibited the expression levels of hnRNP B1 mRNA and the elevated levels of hnRNP B1 protein, both of which are constitutively elevated in cancer cells. Furthermore, both EGCG and genistein inhibited the promoter activity of hnRNP A2/B1 gene expression, with IC50 values 29 microM for EGCG and 66 microM for genistein, suggesting the interaction of EGCG or genistein with the transcriptional complex. Looking at our results here, and those of previously reported epidemiological studies with green tea, we discuss the steadily accumulating evidence that clinical trials with green tea extract would be an efficient means of lung cancer prevention.

Detection of amplifiable messenger RNA in the serum of patients with lung cancer.

Recently, in addition to the detection of circulating tumor cells in peripheral blood of patients with solid tumors, the presence of free circulating nucleic acids in the plasma and serum has also been described. We have focused on the possibility of isolating and amplifying intact extracellular, tumor-related mRNA from the plasma/serum of patients with lung cancer. For this purpose, we established several RT-PCR-based amplification systems for the detection of a panel of five different genes. The expression of these genes was either shown to be restricted to lung tissue or associated with malignancy. We examined two small groups of 18 patients with lung cancer before and during chemotherapy, respectively. The message for beta-actin (control for integrity of the RNA) was detected in all of the analyzed sera from the control group and patients with lung cancer. Analysis of CK-19 expression was positive in the majority of tumor patients, but positive results were also shown in all of the control sera. The expression of MAGE-2 and TTF-1 genes was not observed in any of the patients in either the lymphocyte preparations or serum samples. Expression of the PGP 9.5 gene was observed in the cells of all 18 patients, but mRNA in the serum was only detectable in one case. The hnRNP-B1 mRNA was detectable in 14/18 sera, and Her2/neu-specific mRNA could be amplified from the serum of 7/18 patients. Combining the last two markers, we were able to detect all patients with a malignant lung tumor.