High Expression of Heterogeneous Nuclear Ribonucleoprotein A1 Facilitates Hepatocellular Carcinoma Growth

Abstract

Purpose: Hepatocellular carcinoma (HCC) represents one of the most common tumors in the world. Our study aims to explore new markers and therapeutic targets for HCC. Heterogeneous Nuclear ribonucleoprotein A1 (hnRNPA1) has recently been found to be involved in the progression of several types of cancer, but its role in HCC remains uncovered.

Methods: We performed bioinformatic analysis to preliminarily show the relationship between hnRNPA1 and liver cancer. Then the correlation of the hnRNPA1 gene expression with clinicopathological characteristics of HCC patients was verified by human liver cancer tissue microarrays. The functional role of this gene was evaluated by in vivo and vitro experiments.

Results: Results showed that the expression of hnRNPA1 was upregulated in HCC tissues and was associated with pathological stage of HCC patients. Knockdown of hnRNPA1 gene markedly inhibited tumor growth in vivo, and reversed the effects on proliferation, migration and invasion and promoted apoptosis in vitro. Furthermore, down-regulation of hnRNPA1 gene expression can inhibit the activity of the MEK/ERK pathway.

Conclusion: In our work, we combined bioinformatic analysis with in vivo and in vitro experiments to initially elucidate the function of hnRNPA1 in liver cancer, which may help to explore biomarkers and therapeutic targets for HCC patients.

Keywords: MEK/ERK; WGCNA; hepatocellular carcinoma; hnRNPA1; proliferation.

Figure 1

Co-expression network construction. (A). Clustering dendrogram of samples. (B). The scale-free fit index for soft-thresholding powers of 1–20 and the mean connectivity for 1–20 soft-thresholding powers. (C). Gene clustering dendrogram generated based on topological overlap matrix. (D). Modules clustering dendrogram based on correlations of module eigengenes.

Figure 2

Identification of the hub gene. (A). Heatmap of correlations of module eigengenes with clinical traits. Each row represented a gene module and each column represented a clinical trait, and each cell showed the correlation and P value. The table was color-coded by correlation according to the color legend. (B). Scatterplots of absolute gene significance (GS) for tumor stage vs absolute module membership (MM) in the turquoise module. (C). Top 30 gene networks of the turquoise module. The genes were selected according to the degree. The redder the color, the higher the degree. (D). The expression level of hnRNPA1 in tumor (n = 374) and non-tumor tissues (n = 50). Data are expressed as the means ± SD, ****P < 0.0001. (E). Kaplan-Meier survival analysis of hnRNPA1 gene.

Figure 3

Expression of hnRNPA1 in human hepatocellular carcinoma. (A). Representative IHC staining results of hnRNPA1 in liver cancer tissues and non-tumor tissues (scale bar = 50 μm). (B). Statistical analysis of the mean density (IOD/area) of hnRNPA1 in the corresponding tissues. Results are presented as mean ± SD from 75 pairs of samples. ****P < 0.0001. (C). Representative IHC staining results of hnRNPA1 in T1-T3 stage (scale bar = 50 μm). (D). Statistical analysis of the mean density (IOD/area) of hnRNPA1 in the corresponding T stage. Data are expressed as the means ± SD, *P < 0.05, **P < 0.01 and ****P < 0.0001.

Figure 4

Knockdown hnRNPA1 leads to a significant inhibition effect on tumorigenesis in vivo. (A and B). Gross appearance of xenograft tumors after subcutaneous injections with sh-NC and sh-hnRNPA1 group (n = 5). (C and D). Tumor volumes (C) and weights (D) of the sh-NC groups and sh-hnRNPA1 group (n = 5). (E and F) Representative images of HE (E) and IHC for Ki-67 (F), scale bar = 50 μm. (G) Histogram analysis revealed that hnRNPA1 was associated with Ki-67 expression (n = 5). Data are expressed as the means ± SD. *P < 0.05 and ***P < 0.001.

Figure 5

Reduced expression of hnRNPA1 decreases the proliferation, migration, and invasion, and promoted apoptosis in HCC cells. (A). Relative hnRNPA1 expression level in HepG2 and Huh7 cells transfected with si-RNAs targeting hnRNPA1 by qRT-PCR. (B–E) Proliferation of HepG2 and Huh7 cells after knockdown of hnRNPA1 by CCK-8 (B) and EDU assay (C–E), scale bar = 50 μm. (F–H) Representative results and statistical analysis of apoptosis in HepG2 (G) and Huh7 cells (H). (I–L). Representative results and statistical analysis of transwell assay (scale bar = 100 μm) in HCC cells after knockdown of hnRNPA1. Data are expressed as the means ± SD from three independent experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Figure 6

HnRNPA1 regulates the activity of MEK/ERK pathway in HCC. (A and B). Down regulation of hnRNPA1 reduces phosphorylation level of MEK/ERK pathway in HepG2 (A) and Huh7 (B). (C). Down regulation of hnRNPA1 reduces phosphorylation level of MEK/ERK pathway in vivo experiment (n = 5).