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.

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.

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.

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.