Minichromosome maintenance 3 promotes hepatocellular carcinoma radioresistance by activating the NF-κB pathway
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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.
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.
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.
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.
KeywordsMCM3 HCC Radiotherapy resistance NF-κB pathway
Gene set enrichment analysis
Minichromosome Maintenance 3
HCC is the fifth most common tumors worldwide . Although the greatly improved in the last decades, its 5-year survival rate is only 15%, owing to the limitation of surgical intervention, radiotherapy and chemotherapy. It’s urgent need to identify potential biomarkers for prognosis and find new targets for designing more powerful therapeutic approach [2, 3, 4].
Eukaryotic DNA replication initiation includes helicase loading, helicase activation, replisome assembly and DNA synthesis, MCM2–7 complex assembled by six MCM subunits participates in all the events of DNA replication initiation [5, 6, 7, 8]. Some subunits have been studied in HCC, for example, MCM7 is a poor prognostic factor for HCC and promotes HCC growth through activating MAPK signaling , MCM6 is a novel serum biomarker for early HCC and promotes HCC metastasis through activating MEK/ERK pathway . MCM3 belongs to MCM2–7 complex, it is a poor prognosis marker for oral squamous cell carcinoma, melanoma, papillary thyroid carcinoma, cutaneous T-cell lymphomas, osteosarcoma, glioma, keratocystic odontogenic tumor, anaplastic astrocytoma and salivary gland epithelial tumors [11, 12, 13, 14, 15, 16, 17, 18, 19, 20]. MCM3 is upregulated in prostate cancer tissues samples with bone metastasis, mouse model showed that MCM3 is increased in mesenchymal-derived tumors . MCM3 also is upregulated in medulloblastoma and promotes cell migration and invasion . But these studies only investigate whether MCM3 could be a prognostic factor for various tumors, its role in tumor progression couldn’t be well investigated. Especially, it’s role in radioresistance of HCC. In this study, we main studied the effect of MCM3 on radioresistance of HCC and its regulatory mechanism, we found MCM3 was an independent prognostic factor for HCC and promoted radiotherapy resistance through activating NF-κB pathway.
Materials and methods
Immortalized normal liver cell LO2 and human HCC cell lines including SK-Hep1, SNU-475, HepG2, Huh7, Huh1, SNU-182 and Hep3B were purchased from the ATCC and cultured in DMEM high glucose (Hyclone) supplemented with 10% fetal bovine serum (FBS), the cells were maintained at 37 °C in 5% CO2 incubator.
Tissues samples and immunohistochemistry (IHC)
Eighteen fresh tissue specimens of HCC and three fresh tissue of non-tumor adjacent tissue, as well as 162 paraffin-embedded HCC specimens were utilized, the detailed information was shown in Additional file 1: Table S1 The criteria for determining patient recurrence is that tumors is found in the liver, lung, skeleton, lymph and other positions after complete healing. These samples were collected during surgical procedures from patients with HCC according to a protocol approved by the institutional review board of the First Affiliated Hospital of Sun Yat-sen University. All patients provided written, informed consent for participation in the study and provision of tumor samples. IHC was performed according to our previous methods [23, 24]. Anti-MCM3 antibody (ab4460, Abcam) was used. The images were captured using the AxioVision Rel.4.6 computerized image analysis system (Carl Zeiss Co Ltd., Jena, Germany).
Vectors, lentiviral infection and transfection
Human MCM3 cDNA was subcloned into the pSin-EF1α-puro lentiviral vector to generate pSin-EF1α-MCM3 vector (indicated as MCM3), the empty vector was used as the negative control (indicated as Vector). Two short hairpin RNAs (shRNAs) oligonucleotides sequences against MCM3 was cloned into the PLKO.1 lentiviral vector to generate PLKO.1-MCM3 shRNAs (indicated as shRNA#1 and shRNA#2, respectively), The sequences of shRNAs were: shRNA#1, 5′ GCCACAGATGATCCCAACTTT3’ and shRNA#2, 5′ GCAGGATGACAATCAGGTCAT3’. the scramble shRNA sequence was cloned PLKO.1 vector and used as the negative control (indicated as Scramble). These vectors were cotransfected with pM2.G and psPAX2 into 293 T using Exfect Transfection Reagent (Vazyme, Nanjing, China). The lentiviral supernatants were collected 48 h after transfection and filtered through a 0.45 μm filter. Supernatants plus polybrene (Sigma) were infected with growing HCC cells, after 12 h the supernatants were replaced by fresh medium. Puromycin (Sigma) was used to screen stably cell lines.
HCC cells were irradiated by different radioactive rays Gy (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0) from 6Mv-X-ray produced by a linear accelerator (Varian 600, Varian Medical Systems). The following day after irradiation, cells were used as MTT assy. Cells treated with 2 Gy radioactive rays were used as colony formation assay and TUNEL assay.
Cell proliferation assay
Total RNA was extracted using RNA isolater Total RNA Extraction Reagent (Vazyme), and reversely transcribed into cDNA using HiScript II 1st Strand cDNA Synthesis Kit with gDNA wiper (Vazyme). Relative gene expression levels were examined using AceQ qPCR SYBR Green Master Mix (Vazyme) on a CFX96 Touch Real-time PCR Detection system (Bio-Rad). GAPDH was used as the internal control.
Total proteins were extracted using RIPA buffer (50 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemental with protease inhibitors (Roche). KeyGEN Nuclear and Cytoplasmic Protein Extraction Kit (KGP150, KeyGEN BioTECH) was used to isolate nuclear proteins. Antibodies against MCM3 (ab4460, Abcam), p65 (ab16502, Abcam), p84 (ab487, Abcam), IKKβ (ab124957, Abcam), p-IKKβ (ab38515, Abcam), IκBα (ab32518, Abcam), p-IκBα (ab133462, Abcam), DNA PKcs (ab32566), DNA PKcs (phosphor S2056) (ab18192), CLEAVED PARP1 (ab32064) and GAPDH (G8795, Sigma).
In vivo xenograft assay
All animal experiments were performed under the protocols approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Sun Yat-sen University. Six weeks old BALB/c-nu mice were purchased from the Experimental Animal Center of the Guangzhou University of Chinese Medicine. 5◊106 HepG2 with MCM3 overexpression or knockdown were orthotopically injected into the liver parenchyma of mice (n = 6) to observe the tumor growth, tumor size was up to 7.0–8.0 mm, the mice were treated with 10Gy radioactive rays. The mice were continued to feed for 40 days, then were euthanized, tumors were excised.
SPSS 19.0 was used to perform all statistical analyses. All data from at least three independent experiments are presented as the mean ± s.d. Comparisons between different groups were analyzed using Student’s t-test, Survival curves were derived from Kaplan-Meier estimates, multivariate Cox-regression analysis was used to determine the prognostic value of MCM3 levels and other clinicopathologic characteristics. RNA-seq data from the TCGA HCC data set portal were used for the analyzing MCM3 expression, Salmon and DESeq2 were used to analyze MCM3 expression in HCC samples and normal liver samples. Gene set enrichment analysis (GSEA) were performed using GSEA 2.0.9 software http://software.broadinstitute.org/gsea/index.jsp. p < 0.05 was considered to be statistically significant.
High MCM3 expression is associated with poor outcome for HCC patients
MCM3 is upregulated in HCC cells and tissues
MCM3 is associated with poor radiotherapy effect in vivo and in vitro
MCM3 promoted HCC radioresistance through activating NF-κB pathway
In present study, we found MCM3 was upregulated in HCC tissues and cells, it’s an independent prognostic factor for HCC. MCM3 overexpression increased the radioresistance, while MCM3 knockdown inhibited the radioresistance. Mechanism analysis suggested that MCM3 promoted HCC progression through activating NF-κB pathway.
We found MCM3 overexpression increased the radioresistance, previous studies show cancer stem cells are the main reason for tumor relapse, metastasis, radiotherapy and chemotherapy resistance generation , Many cancer types have been reported to exist cancer stem cells, including HCC, EpCAM, CD13, CD133, CD90, CD24 and CD44 have used for the markers for HCC stem cells [39, 40]. We found MCM3 increased the radioresistance of HCC, suggesting MCM3 might promote the expansion of HCC stem cells, but this inference needed to be verified by further experiments.
NF-κB pathway regulates hepatic fibrosis and HCC [41, 42], In unstimulated cells, IκB interacts with NF-κB, leading the NF-κB/IκB complex sequesters in the cytoplasm, and prevents NF-κB from binding to DNA. Extracellular stimuli activate NF-κB signaling, these stimuli are recognized by receptors and transmitted into the cell, where adaptor signaling proteins initiate a signaling cascade. These signaling cascades activate IKK, IKK phosphorylates IκB in the cytoplasm, leading the degradation of IκB by the proteasome and releases NF-κB from the inhibitory complex. Then NF-κB proteins trans-locates into nucleus where they bind to their target sequences and activate gene transcription . We found MCM3 increased the nuclear translocation of p65 and the phosphorylation of IKK-β and IκBα, suggesting MCM3 activated NF-κB pathway. We also inhibited NF-κB pathway in MCM3 overexpressing cells, and found the radiotherapy resistance was reduced, suggesting MCM3 increased radioresistance through activating NF-κB pathway.
Although other subunits of MCM2–7 complex have been studied in tumors, such as MCM6 and MCM7, previous reporters only show MCM3 is a prognostic factor for various tumors, its function in tumor progression is reported rarely, especially in radioresistance generation, we first systematically studied the role of MCM3 in HCC radioresistance and the regulatory mechanisms. In summary, we found MCM3 increased the radiotherapy resistance of HCC through activating NF-κB pathway.
In conclusion, the present study demonstrates the role of MCM3 in HCC patients’ prognosis and radioresistance, we found MCM3 was an independent prognosis factor for HCC, it promoted radioresistance of HCC through activating NF-κB pathway. Thus, MCM3 could serve as a potential biomarker for HCC prognosis and a new target for HCC therapy.
JWZ, HPL and BSF: conceived the study, conducted experiments, acquired and analysed data, and wrote the manuscript; QY, BHX, QY, HT, WM, CCJ and XMZ: provided suggestions and participated in data analysis; WM, XMZ and YZ: contributed to the collection of the tissue specimens; QY, BHX, QY, HT, WM, CCJ and XMZ: contributed to data analysis; JWZ, HPL and BSF: responsible for conception and supervision of the study, and wrote the manuscript. All authors corrected draft versions and approved the final version of the manuscript.
This work was supported by the Natural Science Foundation of China (grant numbers 81602701, 81760496),the Natural Science Foundation of Guangdong Province (No. 2016A030313195, 2014A030313131, 2017A030313547 and 2018A030313176), the Key Scientific and Technological Projects of Guangdong Province (No. 2014B020228003, 2014B030301041, 2015A070710006 and 2016A020215053), the Science and Technology Planning Project of Guangzhou (No. 201400000001–3, 158100076), Medical Science and Technology Research Fund of Guangdong Province (No. A2017366) and the Science and Technology Projects Foundation of Guangzhou City (No. 201507020037 and 201607010260).
Ethics approval and consent to participate
This research was approved by the Human Research Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University, which is accredited by the National Council on Ethics in Human Research.
Consent for publication
All authors have agreed to publish this manuscript.
The authors declare that they have no competing interests.
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