Minichromosome maintenance 3 promotes hepatocellular carcinoma radioresistance by activating the NF-κB pathway

Abstract

Background: Hepatocellular carcinoma (HCC) is the most common tumors in the worldwide, it develops resistance to radiotherapy during treatment, understanding the regulatory mechanisms of radioresistance generation is the urgent need for HCC therapy.

Methods: qRT-PCR, western blot and immunohistochemistry were used to examine MCM3 expression. MTT assay, colony formation assay, terminal deoxynucleotidyl transferase nick end labeling assay and In vivo xenograft assay were used to determine the effect of MCM3 on radioresistance. Gene set enrichment analysis, luciferase reporter assay, western blot and qRT-PCR were used to examine the effect of MCM3 on NF-κB pathway.

Results: We found DNA replication initiation protein Minichromosome Maintenance 3 (MCM3) was upregulated in HCC tissues and cells, patients with high MCM3 expression had poor outcome, it was an independent prognostic factor for HCC. Cells with high MCM3 expression or MCM3 overexpression increased the radioresistance determined by MTT assay, colony formation assay, TUNEL assay and orthotopic transplantation mouse model, while cells with low MCM3 expression or MCM3 knockdown reduced the radioresistance. Mechanism analysis showed MCM3 activated NF-κB pathway, characterized by increasing the nuclear translocation of p65, the expression of the downstream genes NF-κB pathway and the phosphorylation of IKK-β and IκBα. Inhibition of NF-κB in MCM3 overexpressing cells using small molecular inhibitor reduced the radioresistance, suggesting MCM3 increased radioresistance through activating NF-κB pathway. Moreover, we found MCM3 expression positively correlated with NF-κB pathway in clinic.

Conclusions: Our findings revealed that MCM3 promoted radioresistance through activating NF-κB pathway, strengthening the role of MCM subunits in the tumor progression and providing a new target for HCC therapy.

Keywords: HCC; MCM3; NF-κB pathway; Radiotherapy resistance.

Fig. 1

MCM3 is an independent prognostic factor for HCC. a IHC images indicated MCM3 expression in relapse-free HCC tissues and relapse HCC tissues. b Kaplan-Meier analysis of relapse-free and overall survival curves of patients with high MCM3 expression versus low MCM3 expression. c Multivariate Cox regression analysis to investigate the importance of MCM3 in clinical prognosis

Fig. 2

MCM3 is elevated in HCC tissues and cells. a qRT-PCR and western blot investigated MCM3 expression in HCC tissues and normal liver tissues. GAPDH served as an internal control. b Analysis of MCM3 expression in TCGA tissues. c qRT-PCR and western blot investigated MCM3 expression in HCC cells and immortalized normal liver cell LO2. GAPDH served as an internal control

Fig. 3

High MCM3 expression is associated with increased radiotherapy resistance. a MTT assay of the proliferation of HCC cell treated with different dose of radiotherapy, cells with low MCM3 expression and high MCM3 expression, respectively. b Colony formation of the radiotherapy effect of HCC cells with high and low MCM3 expression. c TUNEL assay of the radiotherapy effect of HCC cells with high and low MCM3 expression.100 μM, every experiment was independently replicated in three times. Error bars

Fig. 4

MCM3 overexpression is associated with increased radiotherapy resistance. a MTT assay of the proliferation of MCM3 overexpressed or knocked down HCC cell treated with different dose of radiotherapy. b Colony formation of the radiotherapy effect of MCM3 overexpressed or knocked down HCC cells. c TUNEL assay of the radiotherapy effect of MCM3 overexpressed or knocked down HCC cells. 100 μM, every experiment was independently replicated in three times. Error bars

Fig. 5

MCM3 increased radiotherapy resistance of HCC in vivo. a Xenograft model in nude mice treated with radiotherapy, Representative bioluminescent images of xenograft tumors formed by HepG2 cells with Scramble control and MCM3 shRNA#1, respectively (Left). and representative images of tumors in the indicated group in nude mice (Right). b Kaplan-Meier analysis of overall survival curves of mice with high MCM3 knockdown versus Scramble control. c Western blot analyzed DNA-PKcs, Pdna-PKcs^T2609 and Cleaved PARP1. GAPDH was used as the loading control. Error bars, SD. *P < 0.05

Fig. 6

MCM3 increased radiotherapy resistance through activating NF-κB pathway. a GSEA revealed MCM3 expression significantly and positively correlated with TNFα induced NF-κB pathway and the upregulated target genes of NF-κB pathway. b Luciferase reporter assay of the effect of MCM3 overexpression or knockdown on NF-κB pathway activity. c Western blot analysis of p65 expression in the nuclear and cytoplasm, IKKβ and IκBα, and the phosphorylation of IKKβ and IκBα, p84 served as an internal control for nuclear proteins, GAPDH served as an internal control for total proteins. d qRT-PCR analysis of the expression of downstream genes of NF-κB pathway. e Colony formation analysis of the effect of inhibition of NF-κB pathway in MCM3 overexpression cells on radiotherapy resistance. g TUNEL analysis of the effect of inhibition of NF-κB pathway in MCM3 overexpression cells on radiotherapy resistance. Error bars, SD. *P < 0.05

Fig. 7

qRT-PCR analysis of CCND1, Bcl-XL and VEGF-C expression in 10 freshly collected HCC samples, western blot analysis of nuclear p65 and MCM3 expression in the same samples (Left). The correlation of nuclear p65 and MCM3 expression was showed in Right. Error bars, SD