Overexpression of parathyroid hormone-like hormone facilitates hepatocellular carcinoma progression and correlates with adverse outcomes

doi:10.12122/j.issn.1673-4254.2025.10.10

Abstract

Abstract:

Objective To investigate the expression of parathyroid hormone-like hormone (PTHLH) in hepatocellular carcinoma (HCC) and analyze its correlation with clinical prognosis, its regulatory effects on HCC cell behaviors, and the signaling pathways mediating its effects. Methods We analyzed the differential expression of PTHLH in HCC and adjacent tissues and its association with patient prognosis based on data from TCGA and GEO databases and from 70 HCC patients treated in our hospital. The effects of PTHLH knockdown and overexpression on proliferation, migration, and invasion of cultured HCC cells were investigated using CCK-8 assay, colony formation assay, Transwell migration and invasion assays, and the signaling pathways activated by PTHLH were detected using Western blotting. Results TCGA and GEO database analysis showed significant overexpression of PTHLH mRNA in HCC tissues, which was associated with poor prognosis of the patients (P<0.05). High PTHLH mRNA expression was a probable independent prognostic risk factor for HCC (P<0.05). In the clinical samples, PTHLH mRNA and protein expressions were significantly higher in HCC tissues than in the adjacent tissues (P<0.001 or 0.01). Univariate and multivariate Cox regression analyses suggested that high PTHLH mRNA expression was an independent risk factor to affect postoperative disease-free survival of HCC patients (P<0.05). The prognostic prediction model based on PTHLH mRNA expression showed an improved accuracy for predicting the risk of postoperative recurrence in HCC patients. In cultured HCC cells, PTHLH overexpression significantly promoted cell proliferation, colony formation, migration and invasion, and caused activation of the ERK/JNK signaling pathway in Huh7 and Hep3B cells. Conclusion High PTHLH expression promotes HCC progression and is associated with poor patient prognosis. Its pro-tumor effects may be mediated by activation of the ERK/JNK signaling pathway.

Key words: hepatocellular carcinoma, parathyroid hormone-like hormone, clinical prognosis, biomarker, ERK/JNK pathway

Xiangzhuo MIAO, Pengyu ZHU, Huohui OU, Qing ZHU, Linyuan YU, Baitang GUO, Wei LIAO, Yu HUANG, Leyang XIANG, Dinghua YANG. Overexpression of parathyroid hormone-like hormone facilitates hepatocellular carcinoma progression and correlates with adverse outcomes[J]. Journal of Southern Medical University, 2025, 45(10): 2135-2145.

Figures/Tables 9

Tab.2 R packages used in the analysis

R package name	Description and Function
survival	Used for survival analysis
survminer	Used for drawing survival curves
ggplot2	Used for drawing graphs or data graphics
pheatmap	Used for drawing heatmaps
limma	Used for difference analysis between groups
ggpubr	Extension of the ggplot2 package
dplyr	Simplify data processing
stringr	String manipulation package
forestplot	Draw forest plots
reshape	Functions for changing array shapes
data.table	Extended R's data frame objects

Fig.1 Analysis of PTHLH mRNA expression levels and prognostic evaluation of HCC patients based on data from the TCGA database. A: Comparison of PTHLH mRNA expression levels between HCC and adjacent normal tissues. B: Kaplan-Meier curves for disease-free survival (DFS) stratified by the median expression level of PTHLH mRNA. C: Kaplan-Meier curves for overall survival (OS) stratified by the median expression level of PTHLH mRNA. D: Forest plot of univariate Cox regression analysis. E: Forest plot of multivariate Cox regression analysis.

Fig.2 Analysis of PTHLH mRNA expression levels and prognostic evaluation of HCC patients based on data from the GEO database. A: Comparison of PTHLH mRNA expression levels between HCC and adjacent normal tissues. B: Kaplan-Meier curves for DFS of HCC patients stratified by the median expression level of PTHLH mRNA. C: Kaplan-Meier curves for OS of HCC patients stratified by the median expression level of PTHLH mRNA. D: Forest plot of univariate Cox regression analysis. E: Forest plot of multivariate Cox regression analysis.

Fig.3 Expression and prognostic analysis of PTHLH in clinical HCC tissue samples. A, B: Relative PTHLH mRNA expression levels in 70 pairs of HCC and adjacent normal tissues. C: Kaplan-Meier curves for DFS of the patients stratified by the median expression level of PTHLH mRNA. D: Representative immunohistochemical staining of PTHLH in HCC and adjacent normal tissues (Scale bar=50 μm). **P<0.01, ***P<0.001.

Fig.4 Clinical prognostic value of PTHLH in HCC patients. A: Forest plot of univariate Cox regression analysis. B: Forest plot of multivariate Cox regression analysis. C: Nomogram for predicting 1-year, 2-year, and 3-year DFS in HCC patients. D: ROC curves comparing the predictive performance of the PTHLH-based nomogram and BCLC staging system.

Fig.5 Knockdown of PTHLH suppresses malignant phenotypes of HCC cells. A: Validation of PTHLH knockdown efficiency by qRT-PCR and Western blotting. B: CCK-8 assay showing cell proliferation. C: Transwell migration assay results (Original magnification: ×100). D: Matrigel invasion assay results (×100). E: Colony formation assay results. **P<0.01, ***P<0.001 vs control group (siNC).

Fig.6 Analysis of ERK/JNK signaling pathway in HCC cells by Western blotting following PTHLH knockdown. A: Western blotting of the ERK/JNK signaling pathway in Huh7 cells. B:Western blotting of the ERK/JNK signaling pathway in Hep3B cells. *P<0.05, **P<0.01, ***P<0.001 vs siNC.

Fig.7 Overexpression of PTHLH enhances malignant phenotypes of HCC cells. A: Validation of PTHLH overexpression efficiency by qRT-PCR and Western blotting. B: CCK-8 assay showing cell proliferation. C: Transwell migration assay results (×100). D: Matrigel invasion assay results (×100). *P<0.05, **P<0.01, ***P<0.001 vs ovNC.

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