Protein and gene expression characteristics of heterogeneous nuclear ribonucleoprotein H1 in esophageal squamous cell carcinoma

Figure 1 Expression and localization of heterogeneous nuclear ribonucleoprotein H1 in esophageal squamous cell carcinoma cells. A: Protein levels of HNRNPH1 were assessed by Western blots in seven ESCC cell lines. The β-actin protein was used as a loading control; B: Immunofluorescent visualization of HNRNPH1 in EC0156 cells; C: Subcellular protein levels of HNRNPH1 in EC0156 were assessed by western blots. Lamin B is a specific marker for nuclear proteins. AIF is a marker for intracellular membrane and nuclear proteins. Coomassie blue-stained SDS-PAGE gel was used as the loading control. AIF: Apoptosis inducing factor; HNRNPH1: Heterogeneous nuclear ribonucleoprotein H1; ESCC: Esophageal squamous cell carcinoma; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 2 Expression and clinicopathological characteristics of heterogeneous nuclear ribonucleoprotein H1 mRNA presented in the cancer genome atlas RNA sequencing dataset. A: Transcript models for HNRNPH1 in hg19 visualized in the NCBI RefSeq. Human HNRNPH1 was encoded on the reverse strand. Two transcript variants were transcribed with different 5’UTR region. The light grey represents the untranslated region, while dark grey represents the coding region; B: These two transcripts had different expression patterns in ESCC. NT, non-tumor tissues (n = 11); T, tumor tissues (n = 87); C: The correlation analysis between variant 1 and 2 in the tumor tissues of ESCC (n = 87). Pearson correlation coefficients were calculated between their mRNA levels; D and E: The clinicopathological characteristics analysis of variant 1 (D) and variant 2 (E) expression in the Asian ESCC cases (n = 40). T staging, tumor invasive depth; N staging, lymph node metastasis; AJCC staging, the 7^th edition cancer staging of American Joint Committee on Cancer. HNRNPH1: Heterogeneous nuclear ribonucleoprotein H1; ESCC:Esophageal squamous cell carcinoma; UTR: Untranslated region.

Figure 3 Kaplan-Meier curves of esophageal squamous cell carcinoma patients with low and high levels of variant 2 transcript of heterogeneous nuclear ribonucleoprotein H1 (n = 79). All of the cases were dichotomized into two groups, high level group and low level group, by the median RPKM values in tumor tissues. Log-rank test was used to compare the survival curves (P = 0.2400). HNRNPH1: Heterogeneous nuclear ribonucleoprotein H1; ESCC:Esophageal squamous cell carcinoma.

Figure 4 Western blotting analysis of heterogeneous nuclear ribonucleoprotein H1 in paired esophageal squamous cell carcinoma specimens. A: Representative western blotting images of tumor (C) and matched adjacent non-tumor esophageal mucosal tissues (N) from 30 patients with ESCC. β-actin protein levels are shown as a loading control. The patients were coded from 1 to 30. B: Densitometric analysis of 30 ESCC cases. The gray and black bars represent the relative band intensity of HNRNPH1 in non-tumor (N) or tumor (C) tissues. Each data point represents the mean ± SD derived from three independent experiments. The asterisks mark the cases that overexpressed HNRNPH1 in tumor tissues. HNRNPH1: Heterogeneous nuclear ribonucleoprotein H1.

Figure 5 Representative immunohistochemistry staining of heterogeneous nuclear ribonucleoprotein H1 in the tumor and non-tumor tissues from a esophageal squamous cell carcinoma patient. HNRNPH1 was mainly localized to the nuclei. The images are shown at high magnification (× 400), and the lower right panels are magnification × 40. HNRNPH1: Heterogeneous nuclear ribonucleoprotein H1; ESCC: Esophageal squamous cell carcinoma.

Figure 6 Expression levels of transcript variant 2 of heterogeneous nuclear ribonucleoprotein H1 were associated with the alternative splicing of proliferation- and apoptosis-related genes in esophageal squamous cell carcinoma. A: The correlation analysis between variant 2 and the two transcripts of MKI67 gene; B: The correlation analysis between variant 2 and the A-raf gene; C: The correlation analysis between variant 2 and the Bcl-X gene. HNRNPH1: Heterogeneous nuclear ribonucleoprotein H1; ESCC: Esophageal squamous cell carcinoma.