Cancer Biol Med 2019. doi: 10.20892/j.issn.2095-3941.2019.0065 ORIGINAL ARTICLE PADI3 induces cell cycle arrest via the Sirt2/AKT/p21 pathway and acts as a tumor suppressor gene in colon cancer Xiaotian Chang1,2, Zhengbin Chai3, Jiaorui Zou1, Hongxing Wang1, Yao Wang1, Yabing Zheng1, Hui Wu1, Chunyan Liu1 1Medical Research Center, The First Affiliated Hospital of Shandong First Medical University, Jinan 250014, China; 2Medical Research Center of the Hospital Affiliated to Qingdao University, Qingdao 266000, China; 3Department of Laboratory Medicine, Jinan Infectious Disease Hospital, Shandong University, Jinan 250021, China ABSTRACT Objective: As a member of the peptidyl arginine deiminase (PAD) family, PADI3 is weakly expressed in colon cancer tissues and highly expressed in adjacent colon cancer tissues. However, the role of PADI3 in colon cancer is unclear. In this study, we investigated the function and molecular mechanism of PADI3 in colon cancer tumorigenesis. Methods: Western blot and real-time PCR were used to detect the expression levels of several genes. CCK-8, flow cytometry (FCM) and colony formation assays were used to examine cell proliferation, the cell cycle and colony formation ability. RNA- sequencing analysis was used to study the molecular mechanism of PADI3 in tumorigenesis. A truncation mutation experiment was performed to determine the key functional domain of PADI3. Results: PADI3 overexpression inhibited cell proliferation and colony formation and led to G1 phase arrest in both HCT116 (originating from primary colon cancer) and LoVo (originating from metastatic tumor nodules of colon cancer) cells. PADI3- expressing HCT116 cells had a lower tumor formation rate and produced smaller tumors than control cells. PADI3 significantly decreased Sirtuin2 (Sirt2) and Snail expression and AKT phosphorylation and increased p21 expression, and Sirt2 overexpression partly reversed the effects induced by PADI3 overexpression. Immunocytochemistry showed that PADI3 is mainly localized in the cytoplasm. Truncation mutation experiments showed that the C-domain is the key domain involved in the antitumor activity of PADI3. Conclusions: PADI3 suppresses Snail expression and AKT phosphorylation and promotes p21 expression by downregulating Sirt2 expression in the cytoplasm, and the C-domain is the key domain for its antitumor activity. KEYWORDS PADI3; Sirt2; colon cancer; cell cycle; C-domain Introduction Citrullination, a posttranslational modification, is catalyzed by a group of enzymes called peptidyl arginine Colorectal cancer (CRC) is a disease with high mortality deiminases (PADs). PADs are reported to be involved in worldwide1,2, but the pathogenesis of CRC remains unclear3. various biological processes in numerous human diseases, Surgical interventions, chemotherapy and radiotherapy are such as rheumatoid arthritis6, Alzheimer’s disease7, ulcerative the major treatments for CRC in the clinic. Despite these colitis8, and cancers9,10. The PAD family comprises five treatments, the poor prognosis associated with metastasis and members: PADI1, PADI2, PADI3, PADI4 and PADI6. Many recurrence postsurgery has not improved4,5. Further study of reports have shown that PADI1, PADI2 and PADI4 are the molecular mechanism of colon cancer, the identification involved in the tumorigenesis of various cancers10-13, and of colon cancer-suppressing genes, and methods to improve PADI6 is involved in early embryonic development14. PADI3 the survival rate of patients are issues that urgently need to be has been reported to be expressed in human keratinocytes addressed. and plays an important role in uncombable hair syndrome15,16. However, the role of PADI3 in tumorigenesis is poorly understood. Correspondence to: Chunyan Liu In the present study, we found that PADI3 is highly E-mail: [email protected] Received February 26, 2019; accepted May 6, 2019. expressed in adjacent colon tissues, but only a small amount Available at www.cancerbiomed.org of PADI3 expression can be detected in colon cancer tissues. Copyright © 2019 by Cancer Biology & Medicine We therefore further investigated the function of PADI3 in 730 Chang et al. PADI3 induces G1-arrest via Sirt2/AKT/p21 pathway the tumorigenesis of colon cancer. In vitro, the the Ethics Committee of Shandong Province Qianfoshan overexpression of PADI3 inhibited cell proliferation by Hospital, Jinan, China (Approval No. S0087). inducing G1 phase arrest. In vivo, PADI3-expressing HCT116 cells showed a lower tumor formation ratio and Primers and antibodies produced smaller tumors compared with the control group. We speculate that PADI3 plays an antitumor role in colon Full-length of PADI3 was amplified using PCR with the cancer. Further study showed that PADI3 can significantly primers PADI3-EcoRI-Fex and PADI3-AscI-Rex, the N- suppress Sirt2 expression at both the transcriptional and domain was amplified with the primers PADI3-N-OE- translational levels. However, the molecular mechanism of EcoRI-Fex and PADI3-N-OE-AscI-Rex, the M-domain was this process is still poorly understood. amplified with the primers PADI3-M-OE-EcoRI-Fex and + Sirt2 belongs to the Sirtuin family, which possesses NAD - PADI3-M-OE-AscI-Rex, the C-domain was amplified with 17 dependent protein deacetylase activity . Sirt2 participates in the primers PADI3-C-OE-EcoRI-Fex and PADI3-C-OE- a variety of cellular processes, including the cell cycle, the AscI-Rex, the NM-domain was amplified with the primers mitotic checkpoint, autophagy, apoptosis, migration and PADI3-N-OE-EcoRI-Fex and PADI3-M-OE-AscI-Rex, and 18-20 invasion . Sirt2 is highly expressed in multiple the MC-domain was amplified with the primers PADI3-M-OE- 21-23 tumors . Increasing evidence has demonstrated that Sirt2 EcoRI-Fex and PADI3-C-OE-AscI-Rex. The primer sequences plays an important role in tumorigenesis, but the function of were designed as shown in Supplementary Table S1. Sirt2 in tumorigenesis is still under debate17,21,24-26. The The following primary antibodies used in this study were opposing functions of Sirt2 in tumorigenesis may be commercially obtained: anti-PADI3 (Abcam, ab172959), dependent on the tissue distribution of it. anti-GAPDH (Abcam, ab181603), anti-AKT (Santa Cruz Inhibitors of Sirt2, such as TM, AK1, AGK-2, AC-93253, Biotechnology, sc5298), anti-Sirt2 (Santa Cruz AEM1, AEM2, and Tenovin-D3, have been proven to Biotechnology, sc28298), anti-p21 (Santa Cruz suppress tumorigenesis in a variety of tumors17,23,27-32. Biotechnology, sc6246), anti-phosphorylated AKT (p-AKT) According to a previous study, effective Sirt2 inhibitors are (CST, #4060), anti-Snail (CST, #3879), anti-His-tag (CST, considered to be the most promising cancer treatment. In 12698S), anti-histone 3 (Affinity, AF0863) and anti-red colon cancer, suppressing Sirt2 expression can induce cell fluorescent protein (RFP) (Abcam, ab28664). The following cycle arrest17, promote cell apoptosis33, and inhibit tumor secondary antibodies were used in this study: goat anti-rabbit angiogenesis34. Suppressing Sirt2 expression may be a wise choice in colon cancer therapy. Based on the above results, (Affinity, #s0001), goat anti-mouse (Affinity, #s0002) and we speculate that PADI3 has antitumor activity as a Sirt2 donkey anti-goat (Abcam, ab6881). inhibitor, but the molecular mechanism is still unclear. Further study showed that PADI3 can significantly Quantitative real-time-PCR analysis decrease Snail expression and AKT phosphorylation while increasing p21 expression via the downregulation of Sirt2 The total RNA of cells or tissues was extracted using Trizol expression. Truncation mutation and immunocytochemistry reagent (Life Technologies, USA). The extracted RNA was showed that PADI3 exerts its antitumor activity mainly in the reverse-transcribed into first-strand cDNA in a final volume cytoplasm and that the C-domain is the key functional of 10 μL using an RNA PCR Kit (Toyobo, Japan). The domain of PADI3. reverse-transcribed first-strand cDNA was used as the These findings indicate that PADI3 exerts its antitumor template for real-time PCR with the forward primer PADI3- activity by suppressing Sirt2 expression in the cytoplasm and QF and the reverse primer PADI3-QR; the primer sequences that the C-domain is essential for PADI3 to exert its are shown in Supplementary Table S1. The conditions of antitumor activity. real-time PCR were as follows: 10 s at 95°C; 45 cycles of 5 s at 60°C and 10 s at 72°C; and 30 s at 65°C. This experiment was Materials and methods performed in triplicate. Real-time PCR was performed using a 10 μL total volume that contained 1 μL of cDNA, 2 μL of Animals ddH2O, 5 μL of SYBR Green Real-time PCR Master Mix (Toyobo, Japan), 1 μL of forward primer and 1 μL of reverse BALB/c nude mice were housed in an Association for primer. GAPDH was used for quantity and quality control Assessment and Accreditation of Laboratory Animal Care using the forward primer of human GAPDH-QF and the (AAALAC)-accredited facility. The study was approved by reverse primer of human GAPDH-QR. The primer sequences Cancer Biol Med Vol 16, No 4 November 2019 731 are shown in Supplementary Table S1. Data were analyzed The absorbance was measured at 450 nm with a using the formula R = 2−[ΔCt sample − ΔCt control], where R is the spectrophotometer (SpectraMax 190, Molecular Devices, relative expression level, ΔCt sample is the difference between USA). Graphs showing growth were generated from the the Ct of the gene and the average GAPDH in the experiment average values of five wells in each group. sample, and ΔCt control is the difference between the Ct of the gene and the average GAPDH in the control sample. Cell colony formation assay Western blot analysis HCT116 cells were transfected with pCDNA3.1-PADI3-RFP plasmids to overexpress PADI3, and HCT116 cells One hundred micrograms of tumor tissue was homogenized transfected with pCDNA3.1-RFP plasmids were used as in 600 μL of Cell Lysis Solution (Sigma-Aldrich, USA) and controls. Following transfection for 48 h, the cells were centrifuged at 12,000 rpm for 20 min at 4°C.