Acta Biochim Biophys Sin, 2017, 1–10 doi: 10.1093/abbs/gmx099 Original Article Original Article Immature colon carcinoma transcript-1 promotes proliferation of gastric cancer cells Zishu Wang†,*, Gongsheng Jin†, Qiong Wu*, Rui Wang, and Yumei Li Department of Medical Oncology, The First Afﬁliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, China † These authors contributed equally to this work. *Correspondence address. Tel/Fax: +86-552-3074480; E-mail: firstname.lastname@example.org (Z.W.)/email@example.com (Q.W.) Received 22 February 2017; Editorial Decision 22 June 2017 Abstract Gastric cancer is the fourth most common malignant tumor and has been considered as one of the leading causes of cancer-related death worldwide. The identiﬁcation of the molecular mechanism during gastric cancer progression is urgently needed, which will help to develop more effective treatment strategies. As a component of the human mitoribosome, immature colon carcinoma transcript-1 (ICT1) might be involved in tumor formation and progression. However, its biological function and the corresponding mechanism in gastric cancer have been poorly characterized. To study the mechanism of ICT1 in gastric cancer, we ﬁrst investigated the mRNA levels of ICT1 in human normal and gastric cancer tissues using datasets from the publicly available Oncomine database. The results showed that ICT1 is overexpressed in gastric cancer tissues. Then in order to study the role of ICT1 in gastric cancer, two shRNAs were used to silence ICT1 in MGC80-3 and AGS cells. Functional analysis showed ICT1 knockdown signiﬁcantly inhibited the proliferation of gastric cancer cells and induced apoptosis. Further, mechanistic study demonstrated that ICT1 silencing induced cell-cycle arrest at G2/M phase via the suppression of cyclin A2 and cyclin B1. In addition, ICT1 silencing also increased cleaved caspase-3 and activated PARP in gastric cancer cells. These ﬁndings suggest that ICT1 may play a crucial role in promoting gastric cancer proliferation in vitro. Key words: Gastric cancer, ICT1, shRNA, cell proliferation, apoptosis Introduction Gastric cancer, as the fourth most common malignant tumor, has been considered as one of the leading causes of cancer-related death worldwide [1,2]. Despite the advancement of surgical techniques and instruments, it is still difﬁcult to completely cure gastric cancer patients at middle-late stage . Statistical analysis indicates that ~40%–60% gastric cancer patients will often have postoperative recurrence after undergoing gastric cancer radical operation . Recently, investigators have demonstrated that gastric carcinogenesis is closely associated with various genetic alterations, including oncogenes and tumor suppressor genes. Therefore, the identiﬁcation of the molecular mechanism underlying gastric cancer progression might provide more effective treatment strategies for this malignancy. Immature colon carcinoma transcript-1 (ICT1), as a component of the human mitoribosome, was initially identiﬁed between undifferentiated and differentiated human colon carcinoma cell lines HT29D4 and Caco-2 [5,6]. It is also an essential peptidyl-tRNA hydrolase component of the large subunit of the ribosome, indicating a strong association of ICT1 with protein synthesis. As a novel protein, ICT1 has been shown to pull down many mitochondrial ribosomal proteins using immunoprecipitation analysis [7,8]. Human mitochondria are essential for cell metabolism as ubiquitous organelles. Mitochondrial dysfunction by genetic alterations results in cell © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org 1 2 proliferation inhibition and apoptosis . As an essential mitochondrial protein, depletion of ICT1 causes a reduction of mitochondrial protein synthesis, leading to a loss of cell viability . In agreement with these facts, previous studies have recognized that ICT1 is implicated in promoting cell proliferation by affecting cell-cycle progression and apoptosis. ICT1 promotes colorectal cancer growth via the intracellular AMPK, SAPK/JNK, and PARP signaling pathways, and high ICT1 means more invasive tumors and worse prognosis . Knockdown of ICT1 in glioblastoma multiforme was found to inhibit cell proliferation by arresting cell-cycle at G2/M phase . In prostate cancer cells, ICT1 leads to cell-cycle arrest and induces apoptosis through mediating Bcl-2 family protein . These ﬁndings together implicate that ICT1 might be involved in tumor genesis, formation and The role of ICT1 in gastric cancer cells progression. Although the status of ICT1 has been well described in several types of cancer, its role in gastric cancer still remains unclear. In the present study, we aimed to investigate the biological functions of ICT1 in gastric cancer progression. The mRNA levels of ICT1 were ﬁrst investigated in human normal and gastric cancer tissues using the datasets from Oncomine database. Then lentivirus-mediated RNA interference was used to down-regulate ICT1 expression to carry out loss-of-function assays. It was shown that knockdown of ICT1 could suppress the proliferation of gastric cancer cells via inducing cell-cycle arrest, promoting apoptosis, and affecting the expression of related proteins. These ﬁndings will provide a novel insight into the development of experimental therapies for gastric cancer. Figure 1. Fold change in ICT1 expression between three types of adencarcinoma vs. gastric mucosa and gastric tissue using the Oncomine database Comparison was divided into six studies according to the source of specimen and P -values were indicated. 3 The role of ICT1 in gastric cancer cells Materials and Methods Analysis of oncomine data In order to determine the expression of ICT1 in human, we performed data mining using Oncomine database (www.oncomine.org). The gene expression of ICT1 was compared between cancer tissues with normal gastric tissues according to the standard procedures as previously described . Cell lines and cell culture The human gastric cancer MGC80-3, AGS and the human embryonic kidney 293 T (HEK293T) cell lines were obtained from the Cell Bank of Chinese Academy of Science (Shanghai, China). MGC80-3 and AGS cells were cultured at 37°C in RPMI 1640 medium (GIBCOBRL, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France). HEK293T cells were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM; GIBCO-BRL) containing 10% FBS. All cell lines were maintained in a fully humidiﬁed atmosphere of 95% O2 and 5% CO2. Construction of lentiviral vectors According to the ICT1 sequence downloaded from NCBI (NM_001545), two shRNA sequences (Genewiz, New York, USA) (S1: 5′-GCAGAATGTGAACAAAGTGAACTCGAGTTCACTTTGTTCAC ATTCTGCTTTTTT-3′ and S2: 5′-GCTGTTAATGCTTGTCTATAAC TCGAGTTATAGACAAGCATTAACAGCTTTTTT-3′) targeting ICT1 and a control shRNA sequence (5′-TTCTCCGAACGTGTCACGTCTC GAGACGTGACACGTTCGGAGAA-3′) were designed and synthesized. All the shRNA fragments were inserted into the pFH-L lentiviral vector (Shanghai Hollybio, Shanghai, China) containing green ﬂuorescence protein (GFP) gene between NheI and PacI restriction sites. The constructed plasmids were named as shICT1(S1), shICT1(S2), and shCon, respectively. Figure 2. Efﬁciency of ICT1 knockdown by lentivirus infection in the gastric cancer cells (A) Microscopic images of gastric cancer cells infected with lentivirus. (B) Western blot analysis validated the knockdown efﬁciency of ICT1 in gastric cancer cells. (C) qPCR analysis of ICT1 knockdown efﬁciency in gastric cancer cells. The mRNA expression of ICT1 was signiﬁcantly suppressed when the cells were infected with shICT1(S1) and shICT1(S2). **P < 0.01 compared with shCon. Scale bar, 10 μm. 4 Packaging of lentivirus and cell infection HEK293T cells were transfected with shICT1(S1), shICT1(S2), or shCon, together with two helper plasmids, pVSVG-I and pCMVΔR8.92 (Shanghai Hollybio) using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Three days after transfection, the culture supernatants were harvested to extract lentiviruses expressing shICT1(S1), shICT1(S2) or shCon. Then, lentiviruses were puriﬁed through ultracentrifugation and concentrated. For lentivirus infection, MGC80-3 and AGS The role of ICT1 in gastric cancer cells cells were cultured in six-well plates and lentiviruses were added at a multiplicity of infection (MOI) of 60. After infection for 96 h, the cells were collected and the infection efﬁciency was observed by ﬂuorescence microscopy (Olympus, Tokyo, Japan). RNA extraction and quantitative RT-PCR analysis Total RNA was extracted from MGC80-3 and AGS cells after 5 days of lentivirus infection using the Trizol reagent (Carlsbad, San Diego, USA), Figure 3. Knockdown of ICT1 inhibited the proliferation of gastric cancer cells (A) The growth of both shICT1(S1)-transfected and shICT1(S2)-transfected cells were much slower than shCon-transfected cells. Values are expressed as the mean ± SD. ***P < 0.001 compared with shCon group. (B) Representative images recorded under a ﬂuorescence microscope, showing the size and the number of colonies in each group. (C) The number of colonies in the shICT1(S1) and shICT1(S2) group was much smaller than that in the shCon group. Values are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the shCon group. 5 The role of ICT1 in gastric cancer cells and then reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega, Madison, USA). Primers for qRT-PCR designed for ICT1 sequence were as follows: ICT1-forward: 5′-CAGCCTGGA CAAGCTCTACC-3′, ICT1-reverse: 5′-GGAACCTGACTTCTG CCTTG-3′. Actin (forward: 5′-GTGGACATCCGCAAAGAC-3′ and reverse: 5′-AAAGGGTGTAACGCAACTA-3′) was used as an endogenous control. The gene-speciﬁc PCR ampliﬁcation was performed on the Bio-Rad Connet Real-Time PCR platform (Bio-Rad, Hercules, USA). The reaction system for qRT-PCR was as follows: 10 μl 2 × SYBR premix ex taq, 0.5 μl primers (2.5 μM), 5 μl cDNA and 4.5 μl ddH2O. The qRT-PCR reaction protocol included 1 min of initial denaturation at 95°C, and 40 cycles of denaturation at 95°C for 5 s, and annealing and extension at 60°C for 20 s. The mRNA expression levels of ICT1 were calculated using the comparative cycle using the 2−ΔΔCt method . detection, the membranes were blocked in phosphate-buffered saline (PBS) containing 0.05% Tween (PBST) and 5% nonfat milk for 1 h at room temperature. Then the blots were probed with primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated goat anti-rabbit (SC-2054, 1:5000; Santa Cruz) for 1 h at room temperature. GAPDH was used as an internal standard. The primary antibodies used in this study included: anticyclin A2 (18202-1-AP, 1:1000; Cell Signaling, Boston, USA), anticyclin B1 (K0128-3, 1:1000; MEDICAL, Nagoya, Japan), anticaspase-3 (#9661, 1:500; Cell signaling), anti-PARP (#9542, 1:1000; Cell Signaling), anti-ICT1 (AP20382b, 1:1000, Abgent, San Diego, USA), and anti-GAPDH (10494-1-AP, 1:50,000; Proteintech, Chicago, USA). Western blot analysis MTT assay After 7 days of infection, lentivirus-transfected MGC80-3 and AGS cells were lysed with ice-cold 2 × SDS sample buffer (10 mM EDTA, 100 mM Tris-HCl, pH 6.8, containing 4% SDS and 10% Glycine). Total extracts were centrifuged (13,839 g, 15 min, 4°C) and the protein concentration was determined using BCA protein assay kit (Pierce, Washington, USA). Equal amounts of protein (10 μg) were separated by 10% sodium dodecyl sulfatepolyacrylamide electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Darmstadt, German). For immune The proliferation of gastric cancer cells was determined by MTT assay (Beyotime, Nanjing, China) according to the manufacturer’s protocol. Brieﬂy, MGC80-3 and AGS cells were seeded in 96-well plates at a density of 2000 cells per well after 96 h lentiviral infection. MTT and acidic isopropanol (5% isopropanol, 10% SDS, and 10 mM HCl) were used as reagent to analyze the viable cell numbers at daily intervals (1, 2, 3, 4, and 5 days). The absorbance of each well was measured at the wavelength of 595 nm using a microplate reader (Bio-Rad, Hercules, USA). Figure 4. Knockdown of ICT1 blocked cell-cycle progression in MGC80-3 cells (A) FACS analysis of cell-cycle distribution in MGC80-3 cells after shICT1(S1) transfection. (B) Downregulation of ICT1 caused an increase of MGC80-3 cells in G2/M phase and a concomitant decrease of cells in G0/G1 phase and S phase. (C) Knockdown of ICT1 led to an increase of MGC80-3 cells in sub-G1 phase. (D) Effect of ICT1 downregulation on cell-cycle regulators. Western blot analysis of the expressions of cyclin A2 and cyclin B1 in MGC80-3 cells after shICT1(S1) transfection. Data are expressed as the mean ± SD. ***P < 0.001 compared with the shCon group. 6 Colony formation assay Cells (400 cells per well) were seeded in a six-well plate after 5 days of lentivirus infection. Cells were cultured in complete medium RPMI 1640 supplemented with 10% FBS (Biowest) for 8 days. Then cells were ﬁxed with PBS containing 4% paraformaldehyde for 30 min at room temperature, stained with crystal violet. After 20 min of incubation, cells were gently washed and air-dried. The number of colonies (>50 cells per colony) was counted under a microscope (Olympus, Tokyo, Japan). Cell-cycle and apoptosis analysis by ﬂow cytometry Cells were inoculated into 6-cm dishes at a density of 2 × 105 cells per dish for cell-cycle test and 80,000 cells per dish for apoptosis analysis after 40 h of lentivirus infection. Propidium iodide (PI) and Annexin V/7AAD were used to stain the cells according to the manufacturer’s instructions. FAC Scan ﬂow cytometry (Becton Dickinson, San Jose, USA) was then performed to measure the change of cell-cycle and the percentage of apoptotic cells. Statistical analysis All experiments were repeated at least for three times. All data were analyzed using GraphPad Prism software and expressed as the mean ± standard deviation (SD) from three independent experiments. The role of ICT1 in gastric cancer cells Statistical differences between groups were determined using Student’s t-test. P < 0.05 was deﬁned as a signiﬁcant difference. Results ICT1 is overexpressed in gastric cancer ICT1 mRNA levels in human gastric cancer tissues were investigated by using DErrico Gastric (GEO ID: 204868_at) and Cho Gastric data (GEO ID: ILMN_2182198) in Oncomine database (www.oncomine. org). A total of 69 tissues from patients, including 31 paired gastric carcinoma and adjacent normal gastric mucosa and seven unmatched gastric carcinoma samples, were analyzed in DErrico Gastric. A total of 90 tissues, 65 gastric adenocarcinoma, 19 paired surrounding normal tissue, and 6 gastrointestinal stromal tumor samples, were analyzed in ChoGastric data. As shown in Fig. 1, ICT1 mRNA expression was signiﬁcantly elevated in human gastric cancer tissues, including gastric intestinal type adenocarcinoma, diffuse gastric adenocarcinoma, gastric mixed adenocarcinoma, and microsatellite stable/instable gastric adenocarcinoma, compared with that in normal tissues, which suggested that ICT1 was highly expressed in gastric cancer in DErrico Gastric data; meanwhile, in Cho Gastric data, ICT1 had a signiﬁcant higher expression in gastric mixed adenocarcinoma than that in normal tissues. Collectively, ICT1 had a higher expression in gastric tissues than in normal tissues and had a clinical signiﬁcance in gastric cancer. Figure 5. Knockdown of ICT1 blocked cell-cycle progression in AGS cells (A) FACS analysis of cell-cycle distribution in AGS cells after shICT1(S1) transfection. (B) Downregulation of ICT1 caused an increase of AGS cells in the G2/M phase and G0/G1 phase and a concomitant decrease of cells in the S phase. (C) Knockdown of ICT1 led to an increase of AGS cells in the sub-G1 phase. (D) Effect of ICT1 downregulation on cell-cycle regulators. Western blot analysis of the expressions of cyclin A2 and cyclin B1 in AGS cells after shICT1(S1) transfection. Data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 compared with the shCon group. The role of ICT1 in gastric cancer cells Lentivirus-mediated shRNA suppressed ICT1 expression in gastric cancer cells To investigate the potential role of ICT1 in gastric cancer, the expression of ICT1 was silenced by lentivirus-mediated shRNA. As shown in Fig. 2A, more than 80% of MGC80-3 and AGS cells were GFP positive in shICT1(S1) and shICT1(S2) groups as revealed by ﬂuorescence microscopy, indicating high infection 7 efﬁciency. Moreover, RT-PCR and western blot analysis were performed to conﬁrm ICT1 expression. As shown in Fig. 2B,C, the mRNA and protein levels of ICT1 in both shICT1(S1)- and shICT1(S2)-infected cells were signiﬁcantly decreased compared that in shCon-infected cells (P < 0.01). These results showed that the designed ICT1 shRNAs could effectively down-regulate ICT1 expression. Figure 6. Knockdown of ICT1-induced cell apoptosis in gastric cancer cells (A) Cytogram of Annexin V-APC binding versus PI uptake after shICT1 (S1) transfection. (B) Downregulation of ICT1 led to an increase of apoptotic cells. (C) Knockdown of ICT1 altered the protein levels of apoptotic markers, including cleaved caspase-3 and PARP, as determined by western blot analysis. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the shCon group. 8 Knockdown of ICT1 repressed cell proliferation of gastric cancer cells To better understand the role of ICT1 in the gastric cancer tumorigenesis, we determined the effect of ICT1 knockdown on the proliferation of MGC80-3 and AGS cells by MTT assay for 5 days. As shown in Fig. 3A, the growths of MGC80-3 and AGS cells were signiﬁcantly decreased in the shICT1(S1) and shICT1(S2) infection groups in a time-dependent manner when compared with that in the shCon group. In addition, we evaluated the colony formation capacity in MGC80-3 cells with three different treatments: shCon, shICT1(S1), and shICT1(S2), and the colony formation capacity in AGS cells with two different groups: shCon and shICT1(S1). Consistent with results of MTT analysis, downregulation of ICT1 signiﬁcantly suppressed colony formation capability of MGC80-3 cells. It was found that there was smaller colonies and fewer colony numbers in shICT1(S1) (P < 0.01) and shICT1(S2) (P < 0.05) than those in shCon (Fig. 3B,C). A similar tendency was also found in AGS cells infected with shICT1(S1) (Fig. 3B,C, P < 0.001). Collectively, knockdown of ICT1 by RNAi could markedly suppress the proliferation and colony formation ability of MGC80-3 and AGS cells. Knockdown of ICT1-induced cell-cycle arrest in gastric cancer cells To investigate the potential mechanism of cell growth inhibition in MGC80-3 and AGS cells by ICT1 knockdown, ﬂow cytometry was performed to determine the cell-cycle distribution (Figs. 4 and 5). Obviously, the percentage of cells at G2/M phase was signiﬁcantly increased in the shICT1(S1) group in MGC80-3 cells, with 20.2% in the shCon group and 50.4% in the shICT1(S1) group, which suggested that MGC80-3 cells were arrested in the G2/M phase (Fig. 4B, P < 0.001). AGS cells were also arrested at G2/M phase after ICT1 silencing (Fig. 5B, P < 0.01). To further reveal the mechanism underlying the cell-cycle arrest, the expressions of some cellcycle markers were detected. As shown in Figs. 4D and 5D, the The role of ICT1 in gastric cancer cells expression levels of cyclin A2 and cyclin B1 which are associated with G2-M transition were decreased in the shICT1(S1) group of both MGC80-3 cells and AGS cells. Collectively, knockdown of ICT1 may mediate cell-cycle arrest most probably through a cyclindependent manner in gastric cancer cells. In addition, more cells were accumulated in the sub-G1 phase, which represents apoptotic cells, after ICT1 knockdown in both MGC80-3 cells (Fig. 4C, P < 0.001) and AGS cells (Fig. 5C, P < 0.01). Knockdown of ICT1-induced apoptosis in gastric cancer cells To further investigate whether ICT1 silencing causes any apoptosis in gastric cancer cells, Annexin V/7AAD double staining was applied to determine apoptosis. As shown in Fig. 6A,B, knockdown of ICT1 with shICT1(S1) signiﬁcantly increased apoptosis compared with the shCon group in AGS cells (P < 0.05 for early apoptosis) and in MGC80-3 cells (P < 0.001 for late apoptosis). Furthermore, the expressions of several apoptotic markers were determined by western blot analysis. As shown in Fig. 6C, the expression levels of cleaved caspase-3 and PARP were up-regulated in gastric cancer cells after ICT1 silencing. Thus, knockdown of ICT1 could signiﬁcantly induce apoptotic effect via activating PARP in gastric cancer cells. Discussion Recently ICT1 has been proved to be involved in various kinds of tumor progression. However, its role in gastric cancer has been poorly characterized. In the present study, we ﬁrst identiﬁed ICT1 as a novel molecule that can drive gastric cancer progression by Oncomine data analysis. Then we designed two different ICT1 shRNAs to speciﬁcally block its endogenous expression in human gastric cancer cell lines MGC80-3 and AGS. The pathway by which ICT1 mediates cell-cycle arrest and apoptosis can be summarized as Fig. 7. Knockdown of ICT1 can arrest cell-cycle at G2/M Figure 7. The proposed mechanism by which ICT1 mediates cell-cycle arrest and apoptosis The role of ICT1 in gastric cancer cells via cyclin-dependent pathway and promote apoptosis through activating PARP in human gastric cancer cells. This result is similar to Wang’s study conducted in prostate cancer cell lines . This phenomenon demonstrated that ICT1 may affect the proliferation of different cancers in the same mode. Different from Wang’s study, our study focused on the effect of ICT1 in patient tissues, which provided a more convincible proof to demonstrate the effect of ICT1 on gastric cancer cell proliferation. Previous studies have indicated that depletion of ICT1 inhibits cell proliferation in HeLa cells, which is ascribed to cell-cycle arrest and cell apoptosis . In our cell-cycle analysis results, ICT1- 9 silenced cells were mostly arrested in the G2/M phase compared to controls. This effect may be associated with alterations in the expression of cyclins, such as cyclin A2 and cyclin B1. Cyclins are activators of speciﬁc serine/threonine protein kinases and can promote cell-cycle process. Cyclin A2 is overexpressed in many cancers  and its overexpression could promote G2/M phase transition  to enhance cell proliferation . Cyclin B1 is an M phase promoting factor and has been demonstrated to be essential for the initiation of mitosis [20,21]. Western blot analysis showed that the expression levels of cyclin A2 and cyclin B1 were decreased in ICT1-silenced cells. Thus, it is reasonable to infer that knockdown Figure 8. High ICTI expression level is correlated with poor survival in different cancer patients (A) Breast cancer data from GSE1456_U133A database. (B) Breast cancer data from NKI database. (C) Renal cancer data from TCGA-KIRC database (HR: 1.52). (D) Renal cancer data from TCGA-KIRC database (HR: 2.1). (E) Lung cancer data from GES4573 database. (F) Skin cancer data from GES19234 database. 10 of ICT1 may induce cell-cycle arrest at G2/M phase most probably through the suppression of cyclin A2 and cyclin B1. To further explore the mechanisms involved in inhibition of proliferation by ICT1 silencing, ﬂow cytometry was used to investigate the apoptosis after ICT1 knockdown. Annexin V/7AAD double staining showed knockdown of ICT1 signiﬁcantly promoted apoptosis in human gastric cancer cells. Moreover, the expressions of cleaved caspase-3 and PARP were obviously increased in ICT1silenced cells. Apoptosis is a process of programmed cell death, which plays a crucial role in keeping balance of cell proliferation . Caspase-cascade, as a central part of cell apoptosis, could be regulated by various kinds of molecules and activated through mitochondrion-dependent pathways [22,23]. In addition, ICT1 has been identiﬁed as a mitochondrial release factor family member. Downregulation of ICT1 may impair mitochondrial function via caspase-cascade cell apoptosis pathway. These results are supported by previous studies, which demonstrated that knockdown of ICT1 in prostate cancer cells and HeLa cells could inhibit cell proliferation due to apoptotic cell death [10,13]. It is widely accepted that aberrant expression of oncogene and antioncogene may affect the clinical outcome of patients. Hence, it is necessary to study whether ICT1 level is linked to the prognosis of gastric cancer patients. Unfortunately, we are unable to acquire enough clinical cases and database concerning the effect of ICT1 on the prognosis of gastric cancer patients in the Oncomine database. However, data on the relationship between ICT1 and prognosis in breast cancer patients, lung cancer patients, renal cancer patients, and skin cancer patients are available in the Oncomine database. According to these data, we performed a Kaplan–Meier survival analysis. The results revealed that high ICT1 mRNA levels are signiﬁcantly correlated with poor prognosis (Fig. 8). Based on these results, it is reasonable for us to propose that ICT1 expression level may affect clinical outcome of gastric cancer patients. An in-depth mechanistic study is needed to evaluate whether ICT1 can be used as a novel prognostic marker in gastric cancer patients. In summary, our results demonstrated that knockdown of ICT1 inhibited cell viability and colony formation ability of gastric cells, induced cell-cycle arrest at G2/M phase via a cyclin-dependent pathway, and promoted apoptosis via a caspase-dependent pathway. Our study highlights the crucial role of ICT1 in gastric cancer cell proliferation. However, more investigations are needed to illuminate the exact molecular mechanism of the role of ICT1 in the treatment of gastric cancer. Funding This work was supported by a grant from the Natural Science Foundation of Anhui Province (No. 1508085MH146). References 1. Siegel R. Cancer statistics. CA Cancer J Clin 2014, 64: 9–29. 2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011, 61: 69–90. 3. Wang J, Yu JC, Kang WM, Ma ZQ. Treatment strategy for early gastric cancer. Surg Oncol 2012, 21: 237–246. 4. Cao Y, Depinho RA, Ernst M, Vousden K. Cancer research: past, present and future. 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