ICT1 Knockdown inhibits osteosarcoma cell proliferation by regulating cell apoptosis through targeting BCL-2

Background:Osteosarcoma (OS) which occurs mainly in the young people is a familiar malignant bone tumor. Apoptosis is a kind of programmed and managed cell death and promoting apoptosis of OS cells is an important treatment for OS. However, the pathways of controlling apoptosis in OS remain unclear. Immature colon carcinoma transcript-1 (ICT1) belongs to a family of four putative mitochondrial translation release factors. It controls the termination stage of translation. Methods:In this study, the function of ICT1 was elucidated by a series of in vivo and in vitro assays. The potential downstream targets of ICT1 in the OS was conrmed by Quantitative real ‐ time polymerase chain reaction (qRT-PCR) and western blotting, while immunohistochemical analysis and rescue experiment were performed to conrm this result. Results: ICT1 was signicantly upregulated in osteosarcoma, and higher expression of ICT1 led to the worse survival rate. Furthermore, knockdown of ICT1 could inhibit the cell proliferation, but improved the apoptosis of cells in vitro and in vivo. In addition, our data demonstrated that BCL-2, apoptotic related-genes, is a potential target of ICT1 and the overexpression of BCL-2 partly reduced the negative inuence of ICT1 knockdown on the pro-tumorigenic capabilities of OS cells in vitro. Conclusion: The ICT1 protein was overexpressed in the OS and has identied a novel mechanism by which ICT1 inhibits osteosarcoma cell proliferation by regulating cell apoptosis through targeting BCL-2.


Background
Osteosarcoma is a common bone cancer in children and teenagers and originates in the interstitial cell line [1]. The heterogeneity, genomic instability and high risk of metastasis primarily to the lungs are the basic characteristics of osteosarcoma [2]. Currently, surgery, chemotherapy, immuno-therapy and gene therapy are the main treatments for osteosarcoma [3]. However, the strong proliferative and migratory capacity of osteosarcoma lead to the poor prognosis [4]. Therefore, it is important for us to investigate the novel oncogene which plays a crucial part in the beginning and progress of OS in order to develop better treatments for patients with this devastating disease. ICT1 which is previously named DS-1 controls the termination stage of translation [5]. Besides, more further studies reported that ICT1 played a signi cant part in the development and progression of tumors.
Several evidences have suggested that ICT1 was closely related to the proliferation in some human cancer cells, such as hepatocellular carcinoma, gastric cancer, non-small cell lung cancer and breast cancer [6][7][8][9]. However, the effect of ICT1 in OS is unclear.
Apoptosis belongs to a procedural cell death, which accounts for the vast majority of cell death in biological processes [10]. Proteins of the B-Cell CLL/Lymphoma 2 (BCL-2) family is a crucial part in the management of apoptosis and have pro-apoptotic and anti-apoptotic activities. Some proteins of this family, such as BCL-2, hinder cell death, while other members, including BAX, increase apoptosis. The protein of BCL-2 , playing a crucial part in the control of prolonged cell survival and apoptosis, is a major anti-apoptotic protein [11]. Relevant studies showed that BCL-2 was also a protooncogene. In lung, prostate and laryngeal cancers, BCL-2 expression signi cantly associated with a poor prognosis [12][13][14].
Some recent studies reported the function of BCL-2 in OS. Apatinib promoted apoptosis via VEGFR2/STAT3/BCL-2 signaling in OS [15]. The expression of BCL-2 increased in recurrent pulmonary metastases compared with primary tumor in OS [16]. MiR-326 regulated cell survival and apoptosis by targeting BCL-2 in OS [17]. However, the correction between ICT1 and BCL-2 in OS is unclear.
In this present study, our data demonstrated that the ICT1 protein was observably increased in the OS and has identi ed a novel mechanism by which ICT1 inhibits osteosarcoma cell proliferation by regulating cell apoptosis through targeting BCL-2.

Materials And Methods
Cell culture and reagents Two human osteosarcoma cell lines (MNNG-HOS,143B) and human osteoblast hFOB1.19 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The American Type Culture Collection (ATCC, Manassas, VA, USA) provided the human osteosarcoma U-2OS cell line. All cell lines were cultured under standard conditions. Human osteosarcoma cells were cultured at 37℃ in a 5% CO 2 atmosphere. Meanwhile, the hFOB1.19 cells were cultured in a 5% CO 2 atmosphere at 34.5℃.

Western blotting
Total cellular protein was extracted by making use of a protein extraction buffer (Beyotime, Shanghai, China). Proteins were spilt by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose (NC) membrane (Millipore, USA). After blocking, the membranes were incubated with the primary antibodies at 4 ℃ for one night, Then, the membrane were probed with the secondary antibodies. Finally, the bands were determined using a western electrochemiluminescence substrate (Share-bio, Shanghai, China).

Immunohistochemistry (IHC)
Alena Biotechnology Co., Ltd. (Xi'an, China)provided a microarray containing tissue from 40 OS patients. The IHC assay was carried out as previously described [19]. We employed the corresponding primary antibodies at 1:200 dilutions to detect the Ki67. We took photos of all the sections using a uorescence microscope (Carl Zeiss, Oberkochen, Germany). The nal IHC score was counted by multiplying the intensity score with the quantity score as previously described [20]. These scores were judged independently by two veteran pathologists in a blinded manner.
The infected OS cells were cultured in a 6-well plate at an initial cell density of 1×10 3 cells/well. After two weeks, the ice-cold PBS was used to wash the colonies. Then, the cell pellet was xed with 4% paraformaldehyde and stained with 0.1% crystal violet. Eventually, we took photos and counted cell numbers.

Cell apoptosis
In oreder to cell apoptosis analysis, the adherent cells were separated after culturing for 24 h in serumfree medium. Cells were stained with the Annexin V/FITC Kit (BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Finally, the ow cytometry was use to analyze results.

Xenograft Tumor in Nude Mice
The Balb/c nude mice (no sex limitation; 20-25 g) were fed and all animal experiments were taken place in the East China Normal University and were carried out according to the animal experimental protocols authorized by the Animal Care and Use Committee of the Animal Care and Use Committee of the A liated Hospital of Nanjing Medical University, Changzhou No.2 People's Hospital. The mice were assigned into several groups randomly. In brief, the Balb/c nude mice were anaesthetised with 1.5% pentobarbital sodium. Then, U-2OS cells (1.5×10 6 ) were subcutaneously inoculated into mice. The data of the tumor size was collected every 5 days. All of mice were euthanized by CO 2 After 20 days. All Tumors were removed and the weight and size data of which were recorded.
Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay The percentage of apoptotic cells in the xenograft tumors was quanti ed using the TUNEL kit (Roche, Basel, Switzerland). This assay was carried out as previously reported [20].

Statistical analyses
The Graphpad software was used for statistical analyses. The expression of all data was represented as mean ± SD. The comparison between different groups was performed by Student's t-test. The p< 0.05 was considered to show a statistically remarkable difference.

ICT1 expression increases in OS cell lines and high expression of ICT1 indicates poor survival rate of OS patients
First, the qRT-PCR analysis was used to detected the expression level of ICT1 in hFOB1.19 cells, a common osteoblast cell line, with that in human OS cell lines (MNNG-HOS, 143B and U-2OS). ICT1 was obviously upward in OS cell lines compared to that in hFOB1.19 cell line (Fig. 1A). Then, western blotting showed that ICT1 protein expression was remarkably upregulated in OS cell lines compared with hFOB1.19 cell line (Fig. 1B). Besides, the prognostic value of ICT1 in patients with OS was revealed by using a website (http://hgserver1.amc.nl) which is an online gene expression database. As shown in Fig.  1C, a KaplaneMeier analysis showed that patients with ICT1 overexpression had a signi cantly poor prognosis (p =0.012). In order to investigate the role of ICT1 in OS, The ICT1 stable knockdown cell lines (U-2OS and 143B cells) were built. The e ciency was validated by qRT-PCR and western blotting (Fig. 1D-F).

The knockdown of ICT1 inhibits proliferation and promotes apoptosis of OS cells in vitro
In order to further explore the biological role of the ICT1 in OS occurrence and development, we performed cell proliferation assay and cell apoptosis experiment. Following transfection of U-2OS and 143B cells with sh-NC, sh-1 and sh-2 vectors, we measured cell proliferation in OS cell lines. The consequences of CCK-8 proliferation assay ( Fig. 2A and B) showed that knockdown of ICT1 markedly inhibited the proliferation of OS cells. We found that the proliferation of OS cells obviously fall off when the expression of ICT1 was silenced through using colony formation assay ( Fig. 2C and D). We used ow cytometry to measure the apoptosis and the consequences of cell apoptosis assay also indicated that apoptosis increased on account of the silencing of ICT1 in the OS cells ( Fig. 2E and F).

BCL-2 is a potential target of ICT1
Since the knockdown of ICT1 promoted apoptosis of osteosarcoma cells, we measured the expression of BCL-2 and BAX which were apoptosis-regulating genes after the knockdown of ICT1. As is shown in Fig.   3A and B, in the U-2OS and 143B cell lines, the RNA expression of BCL-2 was markedly reduced while the RNA expression of BAX was remarkably increased after the silencing of ICT1. Then, by use of western blotting, we found that the protein expression of BCL-2 was markedly reduced and the protein expression of BAX was remarkably increased in the U-2OS and 143B cell lines after the silencing of ICT1 (Fig. 3C). Moreover, we found that Cleaved Capase-3 and Cleaved Capase-9 which are the other apoptotic genes were increased in the U-2OS and 143B cell lines (Fig. 3C). To further con rm this result, we used the tissue microarray through immunohistochemical analysis to determine the correlation between the expression of ICT1 and BCL-2. As shown in Figure 3D-3E, ICT1 protein expression levels in OS tissues have a positive correlation with the protein expression levels of BCL-2 (r = 0.523, p < 0.001). However, BAX and ICT1 expression have no obvious correlation. The above results indicated that BCL-2 is a potential target of ICT1.

The knockdown of ICT1 reduces cell growth of osteosarcoma cells in vivo
Moreover, we explored whether knockdown of ICT1 could inhibit proliferation and enhance apoptosis of U-2OS cell line in vivo. Firstly, The nude mice were injected subcutaneously with the stable ICT1 knockdown and control U-2OS cells. The data showed that the speed of the tumor growth was slower in the stable ICT1-knockdown group than in the control group (Fig. 4A-D). As is shown in Fig. 4E, the IHC staining and TUNEL assay indicated descending expression of Ki67 and upward proportion of apoptosis in the subcutaneous tumors of ICT1-knockdown group. Then, we con rmed that ICT1 protein level in xenografted tumor tissues had a positive correlation with the expression level of BCL-2 (Fig. 4F).

ICT1 improves proliferation and inhibits apoptosis by increasing BCL-2 expression in OS cells
The results showed that ICT1 postively regulates OS cell proliferation (Fig. 2) and we gured out whether the effects by ICT1 depend upon its target, BCL-2. First of all, we knockdowned ICT1 in wild-type and stable BCL-2 overexpression cell lines (U-2OS cells and 143B cells). The e ciency of overexpression was detected by western blotting (Fig. 5A). Then, the CCK8 assay and colony formation assay revealed that the overexpression of BCL-2 partly reversed the prohibitive functions of ICT1 knockdown on the protumorigenic features of U-2OS cells and 143B cells in vitro (Fig. 5B-C). In a cell apoptosis assay, the effect of ICT1 knockdown on apoptosis was reversed partly by BCL-2 overexpression (Fig. 5D and E). The above data suggested the signi cant value of BCL-2 as a target gene of ICT1.

Discussion
Some reports have found the biological effects of ICT1 in the regulation of tumor growth and apoptosis. In the hepatocellular carcinoma, ICT1 promoted cell growth via improving cell cycle progression and resisting apoptosis [6]. In the non-small cell lung cancer, depletion of ICT1 inhibited proliferation and promote apoptosis [7]. In addition, ICT1 knockdown restrained cell growth through causing of cell cycle arrest and apoptosis in the breast cancer [9]. A recent study revealed that ICT1 was upregulated in gastric cancer and promoted its invasion and migration [8]. Whereas, the expression and biological effects of ICT1 in OS has not been studied. In our study, the data demonstrated that the levels of ICT1 expression were markedly higher in the OS cell lines by qRT-PCR and western blotting. Additional analysis showed that knockdown of ICT1 inhibited proliferation and promoted apoptosis of osteosarcoma cells in vitro and in vivo.
Apoptosis, a form of programmed and controlled cell death, is a nonin ammatory or silent process [21]. Apoptosis make great contribution to cancer development and therapy and is regulated by many kinds of signaling pathways [22]. Therefore, targeting the apoptosis process has been con rmed to be an available approach for regulating tumor growth and raising anticancer medical treatment. The B-cell lymphoma 2 (BCL-2) family proteins, known as the apoptotic proteins, can be broadly divided into proapoptotic and antiapoptotic. BCL-2 which belongs to antiapoptotic is a typical class of oncogene [23]. Since Fukuhara and Rowley discovered BCL-2 protein, su cient evidence consistently demonstrated that BCL-2 plays a huge part in cancer cell growth, metastasis, angiogenesis and apoptosis [24]. Furthermore, some recent studies con rmed that a few microRNA inhibited OS growth by effecting BCL-2 and some drugs promoted OS apoptosis through BCL-2 [15,[25][26][27]. In our study, ICT1 can improve the cell growth, proliferation and resist the apoptosis of OS cells, which was in consistent with the previous reports. We proved that the knockdown of ICT1 can decrease the expression of BCL-2 in OS cell lines. Moreover, we con rmed that ICT1 protein levels had a positive correlation with the expression levels of BCL-2 in both xenografted tumor tissues and tissues of OS patients. The overexpression of BCL-2 partly reversed the inhibitory effects of ICT1 knockdown on proliferation of OS cell lines. Therefore, BCL-2 is very likely a target of ICT. However, the way of ICT1 regulating BCL-2 is unknown. The previous studies reported that BCL-2 is an anti-apototic molecule, which is phosphorylated at Ser70 by growth factor-activated protein kinases including PKC and the MAPKs (ERK1/2) can actively control the anti-apoptotic function of BCL-2 [28][29][30]. It is unclear that wheather ICT1 leads to phosphoylation of BCL-2 to inhibit apoptosis. This question will be further explored in future studies.

Conclusions
In conclusion, the data of this study con rmed that ICT1 expression was increased in OS cells. ICT1 promoted OS cell growth both in vitro and in vivo by inhibiting apoptosis via increase of BCL-2 expression. This study may provided a biological perception into the evolution of OS and revealed that ICT1 can be thought as a new target for OS treatment in clinic.     The effect of ICT1 knockdown on osteosarcoma cell proliferation was reversed partly by BCL-2 overexpression A. Overexpression e cacy of BCL-2 in sh-ICT1 OS cells (U-2OS and 143B) was detected by western blotting. B. Overexpression of BCL-2 partly reversed the suppressed effects of ICT1knockdown on the CCK8 assay of U-2OS and 143B cells, values are means±SD, ***p <0.01 (Student's ttest). C. Overexpression of BCL-2 partly reversed the suppressed effects of ICT1-knockdown on the colony formation capability of U-2OS and 143B cells, values are means±SD, ***p <0.01 (Student's t-test). D and