Knockdown of POLQ interferes the development and progression of hepatocellular carcinoma through regulating cell proliferation, apoptosis and migration

Abstract

Background: DNA Polymerase Theta (POLQ) is a DNA polymerase involved in error-prone translesion DNA synthesis (TLS) and error-prone repair of DNA double-strand breaks (DSBs), whose function in hepatocellular carcinoma has not been investigated.

Methods: In the present study, both the data collected from the Cancer Genome Atlas (TCGA) and our group's results showed higher POLQ expression in HCC tissues than the para-cancerous tissues, which was associated with higher malignancy and poor prognosis. POLQ knockdown HCC cell model (shPOLQ) was constructed along with the corresponding negative control (shCtrl) through lentivirus infection for loss-of-function study.

Results: We found that, upon knockdown of POLQ, the proliferation and migration of HCC cells decreased and apoptosis percentage increased. Moreover, the percentage of cells in G2 phase significantly increased in shPOLQ group compared with shCtrl group. Xenografts in mice grafted with shPOLQ cells grew much slower than that transplanted with shCtrl cells, and expressed lower Ki67 level. Furthermore, an apoptosis-related signaling array was used to explore the involvement of downstream signaling pathways, suggesting the enhanced phosphorylation of HSP27 and JNK, and the de-activation of mTOR, PRAS40, ERK1/2 and STAT3 pathways.

Conclusions: Collectively, our study revealed that POLQ may participate in the development of HCC, depletion of which may be a promising treatment strategy for HCC.

Keywords: Cell apoptosis; Cell migration; Cell proliferation; Hepatocellular carcinoma; POLQ.

Fig. 1

POLQ is upregulated in HCC and associated with HCC development. A The expression of POLQ in HCC tissues and normal tissues was collected from TCGA database and compared. B The correlation between POLQ expression and HCC patients’ prognosis was analyzed by Kaplan–Meier survival analysis based on data of TCGA database. C The expression of POLQ in HCC tissues and normal tissues was detected by IHC. D The relationship between POLQ expression and HCC patients’ survival was analyzed by Kaplan–Meier survival analysis with log rank test

Fig. 2

The establishment of POLQ knockdown cell lines. A The endogenous expression of POLQ in various HCC cell lines was detected by qPCR. B The knockdown efficiencies of shRNAs designed for silencing POLQ were evaluated by qPCR in SK-HEP-1 cells. C The transfection efficiencies of lentivirus plasmids in BEL-7404 and SK-HEP-1 cells were observed and measured through fluorescence imaging. D, E Knockdown of POLQ in HCC cells was verified by qPCR (D) and western blotting (E), respectively. Data = mean ± SD (n ≥ 3). ** P < 0.01, *** P < 0.001

Fig. 3

POLQ knockdown inhibited cell proliferation and induced cell apoptosis. A The inhibition of cell proliferation by POLQ knockdown in HCC cell lines was proved by Celigo cell counting assay. B The promotion of cell apoptosis by POLQ knockdown in HCC cells was verified by flow cytometry. C The arrest of cell cycle in G2 phase in shPOLQ cells was shown by flow cytometry. Data = mean ± SD (n ≥ 3). *** P < 0.001

Fig. 4

POLQ knockdown suppressed cell migration of HCC cells in vitro. A The suppression of cell migration ability by POLQ knockdown in HCC cells was demonstrated by wound-healing assay. B The suppression of cell migration ability by POLQ knockdown in HCC cells was demonstrated by Transwell assay. Data = mean ± SD (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 5

Downregulation of POLQ restrained HCC progression in vivo. A Tumor size was measured throughout animal experiments for calculating tumor volume and drawing tumor growth curve, showing the slower growth of tumors in shPOLQ group. B In vivo fluorescence imaging was performed before sacrificing animal to observe the tumor burden, which is represented by the fluorescence intensity. C Photos of tumors were taken after sacrificing the mice model and removing the xenografts. D Weight of xenografts was carried out, recorded and used for comparison between shCtrl and shPOLQ groups. E The expression of Ki67 was detected by IHC in xenografts obtained from shCtrl and shPOLQ groups, respectively. Data = mean ± SD (n ≥ 3). *** P < 0.001

Fig. 6

Exploration of regulatory mechanism of POLQ on HCC. A, B A human apoptosis signaling array was used to detect the difference in the phosphorylation level of various apoptosis related signaling pathways. C The proteins with significantly differential phosphorylation level were shown for clearer demonstration. D The expression of ERK, JNK1, PRAS40, p-Stat3 and p-HSP27 was detected in both cell lines with or without POLQ knockdown. E Expression levels of several tumor-related factors were detected by western blotting. Data = mean ± SD (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001