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RAD18 Contributes to the Migration and Invasion of Human Cervical Cancer Cells Via the Interleukin‑1Β Pathway

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RAD18 Contributes to the Migration and Invasion of Human Cervical Cancer Cells Via the Interleukin‑1Β Pathway MOLECULAR MEDICINE REPORTS 20: 3415-3423, 2019 RAD18 contributes to the migration and invasion of human cervical cancer cells via the interleukin‑1β pathway PENGRONG LOU1*, SHITAO ZOU2*, ZENGFU SHANG3*, CHAO HE2, AIDI GAO2, SHUNYU HOU4 and JUNDONG ZHOU2 1Center of Chemoradio‑Oncology, Ningbo First Hospital, Ningbo, Zhejiang 315010; 2Suzhou Cancer Center Core Laboratory, Nanjing Medical University Affiliated Suzhou Hospital, Suzhou, Jiangsu 215001;3 School of Radiation Medicine and Protection, Medical College of Soochow University, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Suzhou, Jiangsu 215123; 4Department of Gynecology, Nanjing Medical University Affiliated Suzhou Hospital, Suzhou, Jiangsu 215001, P.R. China Received March 20, 2019; Accepted July 11, 2019 DOI: 10.3892/mmr.2019.10564 Abstract. The E3 ubiquitin ligase RAD18 has been identi- rate of patients with CC remains low, particularly for patients fied as an oncoprotein that exhibits prometastatic properties with metastasis (2). The five‑year survival rate of patients in various types of cancer; however, the role of RAD18 in decreased from 85 to 50% for patients with lymph node cervical cancer (CC) remains unclear. In the present study, it metastasis compared with patients with cervical carcinoma was revealed that increased expression of RAD18 was associ- in situ (3). The median OS of patients with bone metastasis ated with worse prognosis of patients with CC. Knockdown was only 23 months (4). Consequently, identifying the molec- of endogenous RAD18 suppressed the motility and invasive- ular events involved in the invasion and metastasis of CC is ness of CC cells, as evaluated by Transwell assays. mRNA required in order to develop novel therapeutic strategies and sequencing revealed that silencing RAD18 altered the improve CC outcomes. expression profile of proinflammatory mediators, such as Cellular DNA is continuously exposed to a range interleukin‑1β (IL‑1β). Furthermore, exogenous IL‑1β treat- of endogenous and exogenous damaging factors, which ment rescued RAD18‑mediated CC cell invasion. These contribute to genome instability and are thus associated findings indicated an underlying mechanism via which RAD18 with carcinogenesis or cell death (5). If DNA damage occurs promotes CC progression, suggesting that RAD18 may be a during replication, it will result in replication fork stalling, potential biomarker and therapeutic target for malignant CC. subsequently leading to the collapse of the replication fork and genome rearrangements (6). To avoid the occurrence of Introduction severe aberrations of the genome, cells have evolved a transle- sion DNA synthesis (TLS) system that employs a series of Cervical cancer (CC) is the fourth most common malignant specialized DNA polymerases with low fidelity to replicate tumor and a leading cause of cancer‑associated mortality damaged DNA templates and relieve the DNA replication in women, accounting for nearly 265,700 deaths globally stress (7). Various DNA polymerases (pols), including pol ι, in 2012 (1) Despite advances in early diagnosis, surgical resec- pol κ and pol η, belong to the Y‑family, whereas pol ζ is a tion, and radio‑ and chemotherapy, the overall survival (OS) B‑family polymerase (8). Due to the high error‑proneness of these TLS polymerases, their recruitment to DNA is tightly regulated via several mechanisms. For example, proliferating cell nuclear antigen (PCNA), as a scaffold for replicative polymerases, is monoubiquitylated at Lys164 in mammalian Correspondence to: Professor Shunyu Hou, Department of Gynecology, Nanjing Medical University Affiliated Suzhou Hospital, cells when exposed to various DNA lesion‑inducing agents, A16 Baita West Road, Suzhou, Jiangsu 215001, P.R. China subsequently stalling the replication fork and mediating E‑mail: [email protected] the access of TLS polymerases (9). The E3 ubiquitin ligase RAD18 forms a E2‑E3 complex with human homolog of Professor Jundong Zhou, Suzhou Cancer Center Core Laboratory, RAD6 (hHR6)A/hHR6B and facilitates PCNA monoubiqui- Nanjing Medical University Affiliated Suzhou Hospital, 16 Baita West Road, Suzhou, Jiangsu 215001, P.R. China tination in human cells (10,11). E‑mail: [email protected] The TLS system is a double‑edged sword in guarding the genome. It has been reported that low‑fidelity DNA polymer- *Contributed equally ases are involved in the accumulation of spontaneous and DNA damage‑induced genomic mutations, which are recognized as Key words: cervical cancer, RAD18, metastasis, interleukin‑1β a driving force for malignant transformation (12). As a key regulator of the TLS pathway, RAD18 has been identified as an oncoprotein that exhibits elevated expression in primary 3416 LOU et al: RAD18 PROMOTES CANCER PROGRESSION THROUGH IL‑1β and metastatic melanoma (13). Increased expression of RAD18 defined by the final score as follows: Low expression (score ≤6, is associated with worse 5‑year survival in patients with n=81) and high expression (score >6, n=45). melanoma (13). Zhou et al (14) revealed that RAD18 expres- sion was significantly increased in esophageal squamous Stable cell line generation. Short hairpin RNA (shRNA) cell cancer (ESCC) tissue compared with adjacent normal targeting RAD18 (shRAD18: 5'‑GCT GTT TAT CAC GCG tissues. A subsequent study by the same group demonstrated AAG A‑3') or non‑targeting negative control shRNA (shNC: that RAD18 expression levels were inversely associated with 5 ' ‑ T T C TCC GAA CGT GTC ACG T‑3') were purchased patient survival, and that RAD18 contributed to ESCC cell from Guangzhou RiboBio Co., Ltd. The fragments were proliferation and migration by activating the JNK‑matrix inserted into a lentiviral expression vector containing EGFP metalloproteinase (MMP) pathway (15). (pGLVH1/GFP+Puro; Shanghai GenePharma Co., Ltd.) In the present study, the role of RAD18 in CC progression and packaged into viral particles by Shanghai GenePharma was investigated. It was demonstrated that upregulated expres- Co., Ltd. A total of 2x106 TU of virus particles were mixed sion of RAD18 was associated with worse patient prognosis. with 5 µg/ml polybrene in 500 µl DMEM, then added to RNA sequencing (RNA‑Seq) was performed in CC cells 5x104 HeLa229 cells and SiHa cells in 24‑well plates. The cells following RAD18 knockdown, revealing that silencing RAD18 were then selected in medium containing 1 µg/ml Puromycin altered the expression profile of proinflammatory mediators, (Sigma‑Aldrich; Merck KGaA) for 1 week. The stable cell such as interleukin‑1β (IL‑1β). Furthermore, exogenous lines were validated via western blotting. IL‑1β treatment rescued RAD18‑mediated CC cell invasion following RAD18 knockdown. Western blot analysis. Cells were harvested and lysed in mammalian protein extraction reagent (Thermo Fisher Materials and methods Scientific, Inc.) containing protease inhibitors (Thermo Fisher Scientific, Inc.) for 10 min on ice. The protein concentra- Tissue samples and cell lines. A total of 126 archival tions were measured using a bicinchoninic acid protein paraffin‑embedded human CC tissues (29‑70 years) were used assay kits (Thermo Fisher Scientific, Inc.), and 20 µg/lane in the present study. The samples were collected at the Nanjing protein was separated via 10% SDS‑PAGE and transferred Medical University Affiliated Suzhou Hospital between to PVDF membranes (EMD Millipore). The membranes January 2010 and November 2011. Informed patient consent were blocked with 5% nonfat milk at room temperature for was obtained at the time of collection. The clinicopathological 1 h and incubated with primary antibodies targeting RAD18 features of the patients were staged according to the 8th Union (1:1,000; cat. no. ab186835; Abcam) or β‑actin (1:1,000; of International Control of Cancer (UICC) classification (16) cat. no. AA128; Beyotime Institute of Biotechnology) over- and presented in Table I. The use of the tissues for the present night at 4˚C. The membranes were washed and then incubated retrospective study was approved by the Institutional Ethics with HRP‑conjugated anti‑rabbit (1:2,000; cat. no. A0208) Committee of the Nanjing Medical University. The human or anti‑mouse secondary antibodies (1:2,000; cat. no. A0216; CC cell lines HeLa229 and SiHa were purchased from the Beyotime Institute of Biotechnology) at room temperature for Shanghai Cell Bank. Cells were cultured in DMEM (HyClone; 1 h. The protein bands were visualized using High‑sig ECL GE Healthcare Life Sciences) supplemented with 10% FBS Western Blotting Substrate (Tanon Science and Technology (HyClone; GE Healthcare Life Sciences) and antibiotics Co., Ltd.). (100 µg/ml streptomycin and 100 U/ml penicillin; Corning Inc.), and incubated in humidified atmosphere with 5% CO2 RNA extraction and reverse transcription‑quantitative at 37˚C. PCR (RT‑qPCR). Total RNA was isolated from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) Immunohistochemical analyses. The expression levels of according to the manufacturer's protocols. The concentrations RAD18 in human CC tissues were detected using immunohis- of RNA were determined using a NanoDrop 2000 (Nanodrop tochemistry. CC tissues (4‑µm sections) were deparaffinized Technologies; Thermo Fisher Scientific, Inc.). RNA (500 ng) and heat‑treated at 100˚C with citrate buffer for antigen from each sample was subjected to RT using a RevertAid retrieval, and then incubated 0.03% hydrogen peroxide for First Strand cDNA Synthesis kit (Thermo Fisher
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