POLE2 Knockdown Reduce Tumorigenesis in Esophageal Squamous Cells Yongjun Zhu, Gang Chen, Yang Song, Zhiming Chen* and Xiaofeng Chen*
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Zhu et al. Cancer Cell Int (2020) 20:388 https://doi.org/10.1186/s12935-020-01477-4 Cancer Cell International PRIMARY RESEARCH Open Access POLE2 knockdown reduce tumorigenesis in esophageal squamous cells Yongjun Zhu, Gang Chen, Yang Song, Zhiming Chen* and Xiaofeng Chen* Abstract Background: Esophageal squamous cell carcinoma (ESCC) is one of the most frequent malignant tumors originated from digestive system around the world and the treatment was limited by the unclear mechanism. DNA polymerase epsilon 2, accessory subunit (POLE2) is involved in DNA replication, repair, and cell cycle control, whose association with ESCC is still not clear. Methods: In this study, the expression level of POLE2 in ESCC tissues was detected by IHC. The POLE2 knockdown cell line was constructed, identifed by qPCR and western blot and used for detecting cellular functions and con- structing xenotransplantation mice model. MTT Assay, colony formation assay, fow cytometry, wound-healing assay and Transwell assay were used to detected cell proliferation, apoptosis and migration. Results: We frstly identifed that the expression of POLE2 was overexpressed in ESCC. Moreover, the high expres- sion of POLE2 can predict the tumor deterioration and poor prognosis of ESCC patients. Additionally, downregulation of POLE2 was involved in ESCC progression by promoting proliferation, migration, and inhibiting apoptosis in vitro. In vivo studies proved that POLE2 was positively correlated with ESCC tumor formation, which was consistent with the results in vitro. We also illuminated that POLE2 knockdown upregulated pro-apoptotic proteins (Bax, Caspase3, CD40L, FasL, IGFBP-5 and P21) and downregulated anti-apoptotic proteins (CLAP-2, IGF-I and sTNF-R2). In addition, POLE2 was involved in ESCC via targeting PI3K/Akt, Cyclin D1 signaling pathway. Conclusions: Therefore, POLE2 was proved to be involved in the development of ESCC, which may be a potential therapeutic target and bring new breakthroughs in the treatment of ESCC. Keywords: ESCC, POLE2, Cell proliferation, Cell apoptosis, Cell migration Introduction Moreover, it was reported that agents inhibiting erb-b2 Esophageal cancer (EC) is one of the most aggressive receptor tyrosine kinase 2 (ERBB2 or HER2), and vas- gastrointestinal cancers in the world [1]. Esophageal cular endothelial growth factor (VEGF), such as Tras- squamous cell carcinoma (ESCC) is one of the main tuzumab, Ramucirumab and Lapatinib, could improve subtypes of EC. Although the incidence of ESCC has survival of ESCC patients [4]. Unfortunately, the treat- shown a downward trend in recent years, the morbidity ments of ESCC could only relieve symptoms, but the and mortality of the disease remain high worldwide [2]. prognosis is still poor, especially with a 5-year survival At present, the comprehensive treatment ESCC consists rate of less than 40% [5, 6]. Tus, in attempted to improve of surgery and defnitive chemoradiotherapy, supple- outcomes of ESCC, novel therapeutic or preventive strat- mented with cisplatin and 5-fuorouracil (5-FU) (CF) [3]. egies are urgently needed. Te eukaryotic DNA polymerase epsilon was frst iso- lated from Saccharomyces in 1970, and belongs to the *Correspondence: [email protected]; [email protected] Department of Cardiothoracic Surgery, Huashan Hospital, Fudan DNA polymerase B family, which contains four subu- University, No. 12, Mid, Wulumuqi Rd, Shanghai, China nits. Te largest subunit is POLE (subunit A), the second © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhu et al. Cancer Cell Int (2020) 20:388 Page 2 of 12 largest subunit is POLE2 (subunit B) with a molecular the wetted air, using DMEM supplemented with 10% FBS weight of 59 kDa, respectively [7]. POLE2 is involved in (Hyclone, USA, Cat. # SV30087) at 37 °C. Te medium cellular functions such as DNA replication, repair, and was changed every 72 h. cell cycle control [8], as well as in the array-based pro- liferative signatures [9]. On the other hand, POLE2 was previously reported to be abnormally expressed in Target gene RNA interferes with the preparation breast, colorectal, cervical and bladder cancer [10–13]. of lentiviral vector For example, Zhou et al. reported that genomic changes Te target sequence of RNA interference (5′-CGA TTG of the 55 kDa Subunit of DNA Polymerase ε in human TTC TTG GAA TGA TA-3′) was designed using POLE2 breast cancer [10]. Daniel et al. believed that POLE2 gene as template to build target gene RNA interference mutation will afect colorectal cancer and make contribu- lentivirus vector. Te 20 µL reaction system was pre- tion to the heritable risk of colorectal cancer [11]. Yet to pared according to the T4 DNA Ligase, and the double- date, the role of POLE2 played in ESCC is still not clear. stranded DNA oligo was connected to the linearized Tus, further study is needed to evaluate and identify the carrier (100 ng/L). After the ligation products were role of POLE2 in ESCC development and progression. transformed into TOP10 E. coli competent cells (100 µL, Te results of this study indicated that POLE2 was sig- TIANGEN, Beijing, China, Cat. # CB104-03), 500 µL LB nifcantly upregulated in ESCC. Knockdown of POLE2 liquid medium without antibiotics was added, and it was could suppress ESCC development through regulating conducted in a shaking culture at 37 °C for 1 h. 150 µL cell proliferation, apoptosis and migration. Terefore, bacterial solution was evenly applied to LB solid medium this study suggested that POLE2 was involved in the containing Amp and cultured overnight in 37 °C incuba- development of ESCC and may be a potential therapeutic tor. A 20 µL PCR reaction system was prepared, and a target, providing a new therapeutic strategy for prevent- single colony was picked up as a template. Te reaction ing or delaying the progression of ESCC. conditions were: 94 °C for 3 min; 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, 22 cycles; 72 °C for 5 min. Te bac- Materials and methods teria with correct sequencing was selected. Subsequently, Immunohistochemical staining according to the kit instructions, plasmids was extracted Te 105 pairs of ESCC tissues and matched non-cancer (TIANGEN, Beijing, China, Cat. # DP117). Lentivirus normal tissues were purchased from Shanghai Outdo expressing shPOLE2 or shCtrl were constructed by Bio- Biotech Company, with detailed pathological data. Col- scienceres Co. Ltd (Shanghai, China). Te efciency of lection of samples with informed consent from patients the transfection of cells by lentivirus were evaluated by and the study were approved by the Institutional Animal the detection of fuorescence intensity in cells (green fu- Care and Use Committee of Fudan University. orescence protein tag on lentivirus). Te formalin fxed tissue samples were frst hydrated with xylene for 15 min and then with 100% ethanol for 10 min. After repairing and blocking of the citrate anti- qPCR gen, the sample and POLE2 antibody (1: 200, BIOSS, Firstly, total RNA was extracted according to Trizol USA, Cat. # BS-14356R) were incubated overnight in instructions (Invitrogen, Carlsbad, CA, USA). Nanodrop an incubator at 4 °C. After elution with PBS for 5 times, 2000/2000C spectrophotometric were used to analy- secondary antibody IgG (1: 400, Abcam, USA, Cat. # sis the quality of extracted RNA and relative levels of AB6721) was added, incubated at room temperature for the mRNAs. Reverse Transcription Kit (Vazyme, Nan- 30 min, and washed with PBS for 3 times. Tissue slices jing, China) was used to synthesize cDNAs. Te real- were frst stained with DAB, and then with hematoxylin. time reverse transcription PCR was performed by using Finally, images were collected with a photomicroscope AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, and analyzed according to the German immune response China). GAPDH was used as a reference control. Te score [14]. Te expression level of POLE2 was defned by qPCR was analyzed by 2−∆∆CT method and collected the total score of IHC staining. Notably, the high or low data. expression level was distinguished by the median of total Target name Sequence (5′ 3′) score of IHC. → POLE2 primer-F TGA GAA GCA ACC CTT GTC ATC Cell culture POLE2 primer-R TCA TCA ACA GAC TGA CTG CATTC ESCC cell lines Eca-109 and TE-1 were obtained from the GAPDH primer-F CGG ATT TGG TCG TAT TGG G Cell Bank of the Chinese Academy of Sciences (Shanghai, GAPDH primer-R GAT TTT GGA GGG ATC TCG C China). Tey were grown in 6-well plates with 5% CO2 in Zhu et al. Cancer Cell Int (2020) 20:388 Page 3 of 12 Western blot analysis Cells were incubated for 14 days to form colonies and the Te Eca-109 and TE-1 cells were collected and lysed medium was changed every 3 days. Ten the cells were with RIPA lysis bufer (Cell Signal Technology, Danvers, washed with PBS, fxed with paraformaldehyde for 1 h, MA, USA) according to the instructions.