A cis-eQTL genetic variant in PLK4 confers high risk of hepatocellular carcinoma

Abstract

Purpose: The overexpression and knockdown of PLK4 were both reported to generate aneuploidy. Thus, we aimed to investigate whether genetic variants in PLK4 contribute to the development of hepatocellular carcinoma (HCC).

Methods: We evaluated associations of common variants in PLK4 and its promoter for the risk of HCC in our association study (1300 cases and 1344 controls). The genotype-tissue expression (GTEx) and The cancer genome atlas (TCGA) databases were used to quantify the expression of PLK4. Cell proliferation and migration affected by PLK4 in HCC were assessed in vitro. Drug susceptibility testing (DST) model was used to assess the sensibility of PLK4-activated HCC to CFI-400945, a small molecule inhibitor of PLK4.

Results: Herein, we found a significant association between rs3811741, located in the PLK4 intron, and liver cancer risk (OR = 1.26, P = 9.81 × 10^-5 ). Although PLK4 expressed at lower levels in somatic tissues compared to the testis, the risk allele A of rs3811741 was associated with increased PLK4 expression in liver cancer tissues. Additionally, PLK4 high expression was remarkably associated with shortened survival of HCC (HR = 1.97, P = .001). Furthermore, overexpression of PLK4 promoted, while knockdown of PLK4 suppressed cancer cell proliferation, migration, and invasion. DST model demonstrated that CFI-400945 can effectively suppress rampant proliferation of HCC with highly expressed PLK4.

Conclusion: Taken together, our study demonstrated that PLK4 is a susceptibility gene and plays an oncogenic role in HCC. Furthermore, we identified that PLK4 sensitives HCC to CFI-400945, which may be an ideal therapy target for HCC.

Keywords: PLK4; CFI-400945; aneuploidy; cancer-testis gene; hepatocellular carcinoma.

Figure 1

PLK4 is reactivated in hepatocellular carcinoma (HCC) and is related with poor survival. A, Rs3811741 is located on the enhancer of PLK4 modified by H3K4Me1 and H3K27Ac. B, Cis‐eQTL analysis of rs3811741 in tumor tissues of HCC based on data from the TCGA project, P value before and after the adjustment for methylation and copy number alterations were calculated. C, 50 paired HCC transcriptional data in TCGA was surveyed. P _{paired t test} < .01; N_paired = 50. D, Unpaired transcriptional data in TCGA were surveyed as well to show the differential expression of PLK4 in tumor and adjacent tissues. Data are expressed as the mean ± SEM. **P_{t test} < .01; N_tumor = 346, N_adjacent = 60. E, Kaplan‐Meier curve of overall survival using patients’ data in TCGA. N_TCGA = 346. The cutoff we used to classify “high” and “low” PLK4 expression was auto‐selected

Figure 2

PLK4 is critical for HCC cell proliferation and migration. A, mRNA expression of PLK4 in different HCC cell lines. B, mRNA and protein expression of PLK4 were knockdown in Huh7 cells. C, MTT assays were used to determine the viability after knocking down PLK4 in Huh7 cells. Data are expressed as the mean ± SEM. **P < .01. D, Clonogenicity assay in PLK4‐downregulated Huh7 cells showed the clonal formation ability. Data are expressed as the mean ± SEM. **P < .01. E, Scratch wound‐healing assay was performed to determine the migration of PLK4 knockdown Huh7 cells. Data are expressed as the mean ± SEM. **P < .01. F‐G, Cell migration and invasion ability were assessed by Transwell chambers in PLK4‐downregulated Huh7 cells. Data are expressed as the mean ± SEM. **P < .01

Figure 3

Overexpression of PLK4 promotes HCC cell proliferation and migration. A, The mRNA and protein expression of PLK4 was significantly higher in PLK4‐overexpressed Huh7 cell lines. B, MTT assays were used to determine the viability after PLK4 overexpression in Huh7 cells. Data are expressed as the mean ± SEM. *P < .01. C, Clonogenicity assay in PLK4‐overexpressed Huh7 cells showed the clonal formation ability. Data are expressed as the mean ± SEM. **P < .01. D, The migration ability of PLK4‐ overexpressed Huh7 cells was determined by scratch wound‐healing assay and cell migration assay. Data are expressed as the mean ± SEM. **P < .01. E‐F, Cell migration and invasion ability were assessed by Transwell chambers in PLK4‐ overexpressed Huh7 cells. Data are expressed as the mean ± SEM. **P < .01

Figure 4

PLK4 increases sensitivity of HCC to PLK4 inhibitors. A, The protein expression of PLK4 was significantly higher in Huh7 and BEL‐7402 cell lines. B, The protein expression of PLK4 was inhibited in CFI‐400945 treated cell line. C, Drug sensitivity assay of CFI‐400945 was performed in various cell lines with both high and low background PLK4 expression. The efficiency was evaluated using MTT assay. D, PLK4‐downregulated Huh7 cells treated with gradient concentrations of CFI‐400945, and the inhibition effect was assayed by MTT