Expression of HnRNP A1, ZEB1, and E-cadherin in Hepatocellular carcinoma and their impact on patients' prognosis and survival

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in Egypt. HCCs usually have a poor prognosis because of late diagnosis, aggressive metastasis, and early invasion. Heterogeneous ribonucleoproteins (HnRNPs) are nuclear proteins that play a variety of roles in telomere formation, DNA repair, cell signaling, and gene regulation. Zincfinger Eboxbinding homeoboxes (ZEBs) are transcription factors that have a consistent inverse correlation with Ecadherin in numerous types of cancer and associated with poor prognosis. Aim: This study aimed to verify the prognostic expression of HnRNP A1, ZEB1, and E-cadherin in HCC. Settings and Design: The retrospective study consisted of 54 formalin-fixed paraffin-embedded tissue blocks of hepatocellular carcinoma. Methods and Material: Immunohistochemical staining was performed using antibodies against HnRNP A1, ZEB1, and E-cadherin. The patients were followed at the Clinical Oncology Department from May 2018 to July 2021. Statistical Analysis: SPSS version 20 using the Chi-square test to compare data and the Kaplan–Meier plot for comparing survival. Results: HnRNP A1 high positivity was detected in 59.3% of the cases, whereas negative E-cadherin and ZEB 1 expression presented in 37% and 70.4% of the patients, respectively. A statistically significant relation was present between HnRNP A1, ZEB1, E-cadherin, and various clinicopathological variables. The mean progression-free survival and overall survival in low HnRNP A1 and negative ZEB1 expressions were longer than those exhibited in high HnRNP A1 and positive ZEB1 expressions. Conclusion: HnRNP A1 and ZEB1 expressions are poor prognostic factors of HCC. E-cadherin has an important role in the development of differentiated HCCs and favorable outcome.

LGR5 and Downstream Intracellular Signaling Proteins Play Critical Roles in the Cell Proliferation of Neuroblastoma, Meningioma and Pituitary Adenoma

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been reported to play critical roles in the proliferation of various cancer cells. However, the roles of LGR5 in brain tumors and the specific intracellular signaling proteins directly associated with it remain unknown. Expression of LGR5 was first measured in normal brain tissue, meningioma, and pituitary adenoma of humans. To identify the downstream signaling pathways of LGR5, siRNA-mediated knockdown of LGR5 was performed in SH-SY5Y neuroblastoma cells followed by proteomics analysis with 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In addition, the expression of LGR5-associated proteins was evaluated in LGR5-inhibited neuroblastoma cells and in human normal brain, meningioma, and pituitary adenoma tissue. Proteomics analysis showed 12 protein spots were significantly different in expression level (more than two-fold change) and subsequently identified by peptide mass fingerprinting. A protein association network was constructed from the 12 identified proteins altered by LGR5 knockdown. Direct and indirect interactions were identified among the 12 proteins. HSP 90-beta was one of the proteins whose expression was altered by LGR5 knockdown. Likewise, we observed decreased expression of proteins in the hnRNP subfamily following LGR5 knockdown. In addition, we have for the first time identified significantly higher hnRNP family expression in meningioma and pituitary adenoma compared to normal brain tissue. Taken together, LGR5 and its downstream signaling play critical roles in neuroblastoma and brain tumors such as meningioma and pituitary adenoma.

Screening specific recognition motif of RNA-binding proteins by SELEX in combination with next-generation sequencing technique / 生物工程学报

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.

Correlation between abnormal heterogeneous nuclear ribonucleo-protein expression and tumors / 中国肿瘤临床

Heterogeneous nuclear ribonucleoprotein (hnRNP) is a family of multifunctional nuclear RNA-binding proteins that regulate the alternative splicing of pre-mRNA and the transport, translation, and stability of mRNA. The most abundant and best charac-terized proteins of this group are hnRNP A1 and hnRNP A2, which share a high degree of sequence homology and functional similarity. HnRNP A1 and hnRNP A2 are upregulated in multiple human tumors and modulate the alternative splicing and mRNA stability of vari-ous tumor-related genes critical to tumor cell growth, apoptosis, inflammatory and immune reactions, and epithelial-to-mesenchymal transition. Therefore, hnRNP A1 and hnRNP A2 have potential diagnostic, prognostic, and therapeutic values.

Cellular Localization Analysis of Enhanced Green Fluorescent Protein Tagged hnRNP A1 Under Stress / 天津医药

Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.

The hnRNP A1 homolog Hrp36 is essential for normal development, female fecundity, omega speckle formation and stress tolerance in Drosophila melanogaster.

Hrp36/Hrb87F is one of the most abundant and well-characterized hnRNP A homolog in Drosophila and is shown to have roles in regulation of alternative splicing, heterochromatin formation, neurodegeneration, etc. Yet, hrp36 null individuals were reported to be viable and without any apparent phenotype, presumably because of overlapping functions provided by Hrp38 and related proteins. Here we show that loss of both copies of hrp36 gene slows down development with significant reduction in adult life span, decreased female fecundity and high sensitivity to starvation and thermal stresses. In the absence of Hrp36, the nucleoplasmic omega speckles are nearly completely disrupted. The levels of nuclear matrix protein Megator and the chromatin remodeller ISWI are significantly elevated in principal cells of larval Malpighian tubules, which also display additional endoreplication cycles and good polytene chromosomes. We suggest that besides the non-coding hsrω-n transcripts, the Hrp36 protein is also a core constituent of omega speckles. The heat-shock-induced association of other hnRNPs at the hsrω locus is affected in hrp36 null cells, which may be one of the reasons for their high sensitivity to cell stress. Therefore, in spite of the functional redundancy provided by Hrp38, Hrp36 is essential for normal development and for survival under conditions of stress.

Effect of Modulation of hnRNP L Levels on the Decay of bcl-2 mRNA in MCF-7 Cells

It has been shown that CA repeats in the 3'-untranslated region (UTR) of bcl-2 mRNA contribute the constitutive decay of bcl-2 mRNA and that hnRNP L (heterogenous nuclear ribonucleoprotein L) interacts with CA repeats in the 3'-UTR of bcl-2 mRNA, both in vitro and in vivo. The aim of this study was to determine whether the alteration of hnRNP L affects the stability of bcl-2 mRNA in vivo. Human breast carcinoma MCF-7 cells were transfected with hnRNP L-specific shRNA or hnRNP L-expressing vector to decrease or increase hnRNP L levels, respectively, followed by an actinomycin D chase. An RT-PCR analysis showed that the rate of degradation of endogenous bcl-2 mRNA was not affected by the decrease or increase in the hnRNP L levels. Furthermore, during apoptosis or autophagy, in which bcl-2 expression has been reported to decrease, no difference in the degradation of bcl-2 mRNA was observed between control and hnRNP L-knock down MCF-7 Cells. On the other hand, the levels of AUF-1 and nucleolin, transacting factors for ARE in the 3'UTR of bcl-2 mRNA, were not significantly affected by the decrease in hnRNP L, suggesting that a disturbance in the quantitative balance between these transacting factors is not likely to interfere with the effect of hnRNP L. Collectively, the findings indicate that the decay of bcl-2 mRNA does not appear to be directly controlled by hnRNP L in vivo.

Expression and clinical significance of c-met and HnRNP A2/B1 gene in non-small cell lung cancer / 肿瘤研究与临床

Objective To investigate the mRNA expression level of the hepatocyte growth factor receptor, c-met, and heterogeneous nuclear ribonucleoprotein, HnRNPA2/B1, in non-small cell lung cancer (NSCLC) patients and their relationships with invasion and metastasis of NSCLC. Methods The mRNA expressions of the c-met and HnRNP A2/B1 in postoperative samples of 46 patients with NSCLC and tissue samples of 30 patients with lung innocence disease as normal controls were detected by RT-PCR, and the efficacies of each marker and combining both of markers in the diagnosis of NSCLC were analyzed by Chi square test. Results The positive rates and relative expression quantity of c-met [65.2 % and (0.903±0.04)]and HnRNP A2/B1 [60.9 % and (0.162±0.04)] in NSCLC were significantly higher than those in control group [26.7 %, (0.205±0.06) and 20.0 %, (0.096±0.02), respectively] (P ＜0.05), and the positive rate of combining both was higher than that of single marker for NSCLC diagnosis(P ＜0.05) . The overexpression of two markers was also significantly correlated with the N stage and clinical stage, but not with age, gender and pathologic types. Conclusion The high expression of c-met and HnRNP A2/B1 in NSCLC may be of significance for the clinical stage. Combining test of two markers provides more sensitivity for NSCLC diagnosis. The expression change of two markers may involve in the carcinogenesis and development of NSCLC.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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 Effect of HSVⅠ Infection on the Expression of hnRNP H2 in Human Fetal Liver Cell / 中国生物工程杂志

Herpes simplex virusⅠ(HSVⅠ) regulating the pathway of transcription and translation modify in host cell is a very systematic and complicate system. A clear understanding of the concrete mechanisms of infection will greatly help to comprehend the virus replication and the interaction with the host cell. By the analysis of 2-DE, the heterogeneous nuclear ribonucleoprotein H2 in human fetal liver cell represent distinction after the HSVⅠinfection.Utilization of Northern blot and Western blot technologies verified the expression of hnRNP H2 in different stage of virus infection is varied.

Molecular phylogenetics and functional evolution of major RNA recognition domains of recently cloned and characterized autoimmune RNA -binding particle.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are spliceosomal macromolecular assemblages and thus actively participate in pre-mRNA metabolism. They are composed of evolutionarily conserved tandemly repeated motifs, where both RNA-binding and protein-protein recognition occur to achieve cellular activities. By yet unknown mechanisms these ribonucleoprotein particles are targeted by autoantibodies and hence play significant role in variety of human systemic autoimmune diseases. This feature makes them important prognostic markers in terms of molecular epidemiology and pathogenesis of autoimmunity. Since ribonucleoprotein (RNP) domain is one of the most conserved and widespread scaffold, evolutionary analyses of these RNA-binding domains can provide further clues on disease-specific epitope formation. The study presented herein represents a sequence comparison of RNA-recognition regions of recently cloned and characterized human hnRNP A3 with those of other relevant hnRNP A/B-type proteins. Their implications in human autoimmunity are particularly emphasized.

Cross reactivity and enzyme sensitivity of immunoaffinity purified Sm/RNP antigens.

Immunoaffinity purified Sm/RNP antigens from buffalo and goat liver were studied to determine the role of RNA and proteins towards the antigenicity of Sm and RNP antigens. A more direct approach using enzyme-linked immunosorbent assay on nylon beads has been utilized to look into the problem. The effect of enzyme treatment and the role of RNA and protein fractions in influencing antigenicity have been described. RNA seems to be involved in the maintenance of RNP specific polypeptides in suitable conformation so as to keep them in solution. Removal of RNA leads to insolubilization of RNP specific polypeptides. Antibodies to Sm and RNP antigens have been shown to cross react with poly A containing heterogeneous nuclear ribonucleoprotein with no cross reactivity with thymus RNA or DNA.