Journal: Frontiers in Oncology

Article Title: The hnRNPK/A1/R/U Complex Regulates Gene Transcription and Translation and is a Favorable Prognostic Biomarker for Human Colorectal Adenocarcinoma

doi: 10.3389/fonc.2022.845931

Figure Lengend Snippet: IHC staining of hnRNPs family members in 10 human colorectal adenocarcinoma specimens. By using IHC staining of 10 human colorectal adenocarcinoma specimens, we confirmed that hnRNPA1, hnRNPA2B1, hnRNPC, hnRNPK, hnRNPR, and hnRNPU were mainly localized in the nucleus and had higher expressions in colorectal adenocarcinoma tissues compared with adjacent normal tissues (ANT). * P < 0.05.

Article Snippet: The primary antibodies used were as follows: hnRNPK (Proteintech, #11426-1-AP, 1:1000), IgG (CST, #2729, 1:1000), hnRNPA1 (Proteintech, #11176-1-AP, 1:5000), hnRNPR (Proteintech, #15018-1-AP, 1:1000), hnRNPU (Proteintech, #16365-1-AP, 1:5000), Histone H3 (Abclonal, #A2348, 1:1000) and β-Actin (Proteintech, #20536-1-AP, 1:2000).

Techniques: Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: The hnRNPK/A1/R/U Complex Regulates Gene Transcription and Translation and is a Favorable Prognostic Biomarker for Human Colorectal Adenocarcinoma

doi: 10.3389/fonc.2022.845931

Figure Lengend Snippet: High expressions levels of hnRNPA1, hnRNPK, hnRNPR, and hnRNPU were significantly associated with better OS rates in colorectal adenocarcinoma patients. (A) The HPA database was used to examine the relationship between the expression levels of hnRNPs and the survival rates of 597 patients with colorectal adenocarcinoma. The Kaplan-Meier survival analysis demonstrated that high expressions of hnRNPK and hnRNPR were significantly associated with better OS rates for COAD patients. (B) High expressions of hnRNPA1, hnRNPK, hnRNPR, and hnRNPU were significantly associated with better OS rates for READ patients. (C) High expressions of hnRNPA1, hnRNPK, hnRNPR, and hnRNPU were significantly associated with better OS rates for colorectal adenocarcinoma patients. (D) The GEPIA database was selected to further examine the survival of 181 COAD patients and 181 READ patients. Similarly, high expression of hnRNPK was associated with better OS rates.

Article Snippet: The primary antibodies used were as follows: hnRNPK (Proteintech, #11426-1-AP, 1:1000), IgG (CST, #2729, 1:1000), hnRNPA1 (Proteintech, #11176-1-AP, 1:5000), hnRNPR (Proteintech, #15018-1-AP, 1:1000), hnRNPU (Proteintech, #16365-1-AP, 1:5000), Histone H3 (Abclonal, #A2348, 1:1000) and β-Actin (Proteintech, #20536-1-AP, 1:2000).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: The hnRNPK/A1/R/U Complex Regulates Gene Transcription and Translation and is a Favorable Prognostic Biomarker for Human Colorectal Adenocarcinoma

doi: 10.3389/fonc.2022.845931

Figure Lengend Snippet: The hnRNPK/A1/R/U network in colorectal adenocarcinoma modulated transcription and translation. (A) A gene co-expression network for hnRNPA1, hnRNPK, hnRNPR, and hnRNPU was constructed. The network displayed that the transcription-related and translation-related genes, such as DHX9, MRPL3, SFPQ, CPSF6, RAD23b, and NCBP1, were closely associated with the hnRNPK/A1/R/U network. (B) The GEPIA database revealed that the TPM levels of hnRNPK/A1/R/U with each other were positively correlated. (C, D, E, F) The functional predictions of hnRNPK/A1/R/U network were analyzed in the DAVID database. The GO analysis results showed that transcription, translation, and ribosomal large subunit assembly were regulated by the hnRNPK/A1/R/U. Furthermore, ribosomal subunit, nucleotide binding, structure constituent of ribosome, and poly(A) RNA-binding were also enriched. The KEGG analysis results showed that DNA replication and ribosomes were enriched, suggesting that hnRNPK/A1/R/U modulated the process of transcription and translation.

Article Snippet: The primary antibodies used were as follows: hnRNPK (Proteintech, #11426-1-AP, 1:1000), IgG (CST, #2729, 1:1000), hnRNPA1 (Proteintech, #11176-1-AP, 1:5000), hnRNPR (Proteintech, #15018-1-AP, 1:1000), hnRNPU (Proteintech, #16365-1-AP, 1:5000), Histone H3 (Abclonal, #A2348, 1:1000) and β-Actin (Proteintech, #20536-1-AP, 1:2000).

Techniques: Expressing, Construct, Functional Assay, Binding Assay, RNA Binding Assay

Journal: Frontiers in Oncology

Article Title: The hnRNPK/A1/R/U Complex Regulates Gene Transcription and Translation and is a Favorable Prognostic Biomarker for Human Colorectal Adenocarcinoma

doi: 10.3389/fonc.2022.845931

Figure Lengend Snippet: HnRNPK/A1/R/U complex was identified and validated in human colorectal adenocarcinoma cell lines. (A) Nuclear proteins of HCT116, SW480, and SW620 cells were extracted, with FHC as the control. After SDS-PAGE and silver staining, a differential protein band with a molecular weight of 50-55 kDa was observed (red rectangle). In detail, more enrichment was observed in SW620 cells and less enrichment was observed in SW480 and HCT116 cells. Importantly, the total enrichment amount of colorectal adenocarcinoma cells was elevated compared with normal cells. (B) The protein band in red rectangle was excised, trypsinized, and analyzed by MS. With the best peptide-spectrum sequences, hnRNPA1 (P09651), hnRNPK (P61798), hnRNPR (O43390), and hnRNPU (Q00839) were identified. (C) WB assays were performed to validate the expressions of hnRNPA1, hnRNPK, hnRNPR, and hnRNPU in the nucleus, with histone H3 as the control. The results showed that hnRNPA1, hnRNPK, hnRNPR, and hnRNPU in the nucleus had higher expressions in colorectal adenocarcinoma cells compared with FHC cells. (D) The interaction between hnRNPK and hnRNPA1/R/U proteins was confirmed by Co-IP in HCT116 cells. To confirm the reliability of this result, the interaction between hnRNPU and hnRNPA1/R/K proteins was further confirmed in HCT116 cells. In FHC cells, the interaction between hnRNPK and hnRNPA1/R/U proteins was observed, which revealed that the hnRNPK/A1/R/U interaction was not specific to cancer cells.

Article Snippet: The primary antibodies used were as follows: hnRNPK (Proteintech, #11426-1-AP, 1:1000), IgG (CST, #2729, 1:1000), hnRNPA1 (Proteintech, #11176-1-AP, 1:5000), hnRNPR (Proteintech, #15018-1-AP, 1:1000), hnRNPU (Proteintech, #16365-1-AP, 1:5000), Histone H3 (Abclonal, #A2348, 1:1000) and β-Actin (Proteintech, #20536-1-AP, 1:2000).

Techniques: SDS Page, Silver Staining, Molecular Weight, Co-Immunoprecipitation Assay

Journal: iScience

Article Title: Increased LDL receptor by SREBP2 or SREBP2-induced lncRNA LDLR-AS promotes triglyceride accumulation in fish

doi: 10.1016/j.isci.2022.104670

Figure Lengend Snippet: LDLR-AS interacts with hnRNPR to stabilize LDLR mRNA (A) Silver staining of proteins pulled down with biotinylated LDLR-AS RNA and a negative control with antisense sequences. Blue triangle denotes the band identified as hnRNPR by mass spectrometry. (B) Interaction profiles for LDLR-AS interaction with hnRNPR were predicted using the catRAPID website. (C) ChIP-qPCR assay was performed to measure the binding ability of hnRNPR to LDLR-AS. Data are presented as mean ± SEM, n = 3, Student’s t-test, ∗∗∗p < 0.001. (D) RT-qPCR results of hnRNPR knockdown in fish hepatocytes transfected with si-hnRNPR#1/2/3 for 24 h. Data are presented as mean ± SEM, n = 3, one-way ANOVA, ∗p < 0.05. (E) Western blot of LDLR and hnRNPR protein in fish hepatocytes transfected with si-hnRNPR#1 for 48 h. Data are presented as mean ± SEM, n = 3, one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01. (F) RT-qPCR results of LDLR-AS expression in fish hepatocytes transfected with si-hnRNPR#1/2/3 for 24 h. Data are presented as mean ± SEM, n = 3, one-way ANOVA, ns = not significant. (G) RT-qPCR analysis was used to evaluate the stability of LDLR mRNA after fish hepatocytes were transfected with si-hnRNPR#1 for 24 h, then further exposed to 5 μg/mL actinomycin D for 24 h. Data are presented as mean ± SEM, n = 3, Student’s t-test, ∗∗p < 0.01. (H) Interaction profiles for LDLR at the 5′-UTR interaction with hnRNPR were predicted using the catRAPID website. (I) The luciferase activity of 5′-UTR of LDLR mRNA was detected in HEK293T cells transfected pGL3-Basic, pGL3-5′UTR, or PCS2+hnRNPR vectors. Data are presented as mean ± SEM, n = 3, one-way ANOVA, ∗∗p < 0.01, ∗∗∗p < 0.001. (J) ChIP-qPCR assay was performed to measure the binding ability of hnRNPR to the 5′-UTR of LDLR. Data are presented as mean ± SEM, n = 3, Student’s t-test, ∗∗p < 0.01.

Article Snippet: Primary antibodies specific to LDLR (#10785-1-AP, Proteintech), SREBP2 (#28212-1-AP, Proteintech), hnRNPR (#15018-1-AP, Proteintech), PLCB1 (#DF6726, Affinity), β-actin (#ab8227, abcam), GAPDH (#ab9485, abcam), Histone 3 (#4499, Cell signaling Technology) were used.

Techniques: Silver Staining, Negative Control, Mass Spectrometry, Binding Assay, Quantitative RT-PCR, Knockdown, Transfection, Western Blot, Expressing, Luciferase, Activity Assay

Journal: iScience

Article Title: Increased LDL receptor by SREBP2 or SREBP2-induced lncRNA LDLR-AS promotes triglyceride accumulation in fish

doi: 10.1016/j.isci.2022.104670

Figure Lengend Snippet:

Article Snippet: Primary antibodies specific to LDLR (#10785-1-AP, Proteintech), SREBP2 (#28212-1-AP, Proteintech), hnRNPR (#15018-1-AP, Proteintech), PLCB1 (#DF6726, Affinity), β-actin (#ab8227, abcam), GAPDH (#ab9485, abcam), Histone 3 (#4499, Cell signaling Technology) were used.

Techniques: Virus, Recombinant, Chromatin Immunoprecipitation, In Situ Hybridization, Labeling, Silver Staining, Enzyme-linked Immunosorbent Assay, Sequencing, Software