CDKL3 is a promising biomarker for diagnosis and prognosis prediction in patients with hepatocellular carcinoma

Abstract

Cyclin-dependent kinase-like 3 (CDKL3) has been identified as an oncogene in certain types of tumors. Nonetheless, its function in hepatocellular carcinoma (HCC) is poorly understood. In this study, we conducted a comprehensive analysis of CDKL3 based on data from the HCC cohort of The Cancer Genome Atlas (TCGA). Our analysis included gene expression, diagnosis, prognosis, functional enrichment, tumor microenvironment and metabolic characteristics, tumor burden, mRNA expression-based stemness, alternative splicing, and prediction of therapy response. Additionally, we performed a cell counting kit-8 assay, TdT-mediated dUTP nick-end Labeling staining, migration assay, wound healing assay, colony formation assay, and nude mouse experiments to confirm the functional relevance of CDKL3 in HCC. Our findings showed that CDKL3 was significantly upregulated in HCC patients compared to controls. Various bioinformatic analyses suggested that CDKL3 could serve as a potential marker for HCC diagnosis and prognosis. Furthermore, CDKL3 was found to be involved in various mechanisms linked to the development of HCC, including copy number variation, tumor burden, genomic heterogeneity, cancer stemness, and alternative splicing of CDKL3. Notably, CDKL3 was also closely correlated with tumor immune cell infiltration and the expression of immune checkpoint markers. Additionally, CDKL3 was shown to independently function as a risk predictor for overall survival in HCC patients by multivariate Cox regression analysis. Furthermore, the knockdown of CDKL3 significantly inhibited cell proliferation in vitro and in vivo, indicating its role as an oncogene in HCC. Taken together, our findings suggest that CDKL3 shows promise as a biomarker for the detection and treatment outcome prediction of HCC patients.

Keywords: CDKL3; biomarker; hepatocellular carcinoma; prognosis; the cancer genome atlas.

FIGURE 1

Expression of CDKL3 in HCC. (A) The expression of CDKL3 in Pan-cancer based on TCGA data via Tumor IMmune Estimation Resource 2.0 (TIMER 2.0, http://timer.comp-genomics.org/). Comparative CDKL3 expression levels in HCC and adjacent normal tissue: plots of unpaired (B) and paired (C) data. HCC, hepatocellular carcinoma.

FIGURE 2

Diagnosis and prognosis of CDKL3 in HCC. (A) ROC curve for CDKL3 expression in HCC. (B) Kaplan-Meier overall survival curves of hCDKL3 and lCDKL3 and the survival difference was evaluated by log-rank test. (C) The dot plot and heatmap demonstrating the survival status and expression of CDKL3 in hCDKL3 and lCDKL3, respectively. (D) The expression of CDKL3 was compared between hCDKL3 and lCDKL3. (E) Forest plot showing the results of univariate Cox analyses for overall survival, disease-specific survival, progression-free interval and disease-free interval. HCC, hepatocellular carcinoma; hCDKL3, high-CDKL3 group; lCDKL3, low-CDKL3 group.

FIGURE 3

Construction of nomogram for predicting overall survival of hepatocellular carcinoma in TCGA cohort. (A) Forest plot showing the results of multivariate Cox analysis. (B) Nomogram.

FIGURE 4

Genetic features of CDKL3 in HCC. (A) The mRNAsi score was compared between hCDKL3 and lCDKL3. (B) The relationship between the CDKL3 expression and mRNAsi score. (C) The PSI values of three alternative splicings were compared between hCDKL3 and lCDKL3. (D) The relationship between CDKL3 expression and PSI values of CDKL3-73367-AT. (E) The relationship between CDKL3 expression and PSI values of CDKL3-73366-AT. (F) The hallmarks of tumor sets were enriched in hCDKL3 using GSEA. ***p < 0.001. HCC, hepatocellular carcinoma; hCDKL3, high-CDKL3 group; lCDKL3, low-CDKL3 group; PSI, percent splice-in.

FIGURE 5

The tumour microenvironment characteristics of hCDKL3 and lCDKL3. (A) ssGSEA enrichment score of 29 Fges in hCDKL3 and lCDKL3. (B) Expression levels of immunosuppression-related molecules in hCDKL3 and lCDKL3. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significantly significant. hCDKL3, high-CDKL3 group; lCDKL3, low-CDKL3 group; ssGSEA, single-sample gene set enrichment analysis.

FIGURE 6

Metabolism-related KEGG pathways were enriched, as demonstrated using GSEA. KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA: gene set enrichment analysis.

FIGURE 7

Verification of CDKL3 function in vitro. (A) qRT-PCR and (B) western blot assays were performed to evaluate the expression level of CDKL3 after transfection by shCDKL3 in hepatocellular carcinoma cell lines. (C) Cell viability was determined using CCK8 assays. (D) Colony formations assays were performed to evaluate cell proliferation ability. *p < 0.05, **p < 0.01. Error bars indicate mean ± SD. qRT-PCR, quantitative real-time polymerase chain reaction; CCK8, Cell Counting Kit-8.

FIGURE 8

TUNEL (Scale bar, 400 μm) assay was performed to detect cell apoptosis. TUNEL, TdT-mediated dUTP Nick-End Labeling.

FIGURE 9

Knowdown of CDKL3 reduced tumor proliferation in xenografted nude mice. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. Error bars indicate mean ± SD.