Materials Express 2158-5849/2019/9/1106/006 Copyright © 2019 by American Scientific Publishers All rights reserved. doi:10.1166/mex.2019.1601 Printed in the United States of America www.aspbs.com/mex CIAPIN1 is a potential target for apoptosis of multiple myeloma Xiao-Bo Wang1,†,Le-PingYan1,2,†,Li-HuaYuan3,†,BoLu1, Dong-Jun Lin1,∗, and Xiao-Jun Xu1,∗ 1Department of Hematology, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, 518106, PR China 2Scientific Research Center, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, 518106, PR China 3The University of Hong Kong—Shenzhen Hospital, Shenzhen, 518053, PR China ABSTRACT This study firstly aimed to reveal the gene expression differences of CIAPIN1 between myelomas cells from bone marrow cells of multiple myeloma patients and normal human, and subsequently investigate the regu- lation role of this gene on tumorigenicity ability of multiple myeloma (MM) cell line U266 via in vitro colony formation and in vivo xenograftIP: studies. 192.168.39.151 RT-PCR resultsOn: Fri, obtai 01 Octned 2021 from 18:58:12 18 MM patients and 10 health people showed that the expression of CIAPIN1Copyright: gene American was 4 times Scientific higher Publishers in normal human compared to MM patients. Besides, CIAPIN1 siRNA (si-CIAPIN1) transfectedDelivered U266 by cellsIngenta presented higher proliferation ratio and superior colony forming ability than U266 cells and U266 cells transfected with non-coding siRNA (controls) evaluated Article by CCK8 test and soft agar colony formation assay, respectively. In a mice MM xenograft model, the si-CIAPIN1 transfected U266 cells induced the biggest tumor compared to the controls. Furthermore, CIAPIN1 overex- pressed U266 cells were developed and compared with the si-CIAPIN1 transfected U266 cells to study the role of CIAPIN1 in the production of apoptosis related proteins in U266 cells. Results indicated that CIAPIN1 facilitated apoptosis promoting proteins expression in U266 cells, such as upregulation of BAX, BAK, Bcl-xs and BIM, and downregulation of p38, PKC, Bcl-2 and Bcl-xl proteins. Therefore, CIAPIN1 can be a potential suppression target gene in multiple myeloma. Keywords: CIAPIN1, Multiple Myeloma, Apoptosis, Tumorigenicity. 1. INTRODUCTION However, since the pathogenesis of the MM still remains As a type B lymphocyte clone malignant tumor, multi- unclear, it is of essence to explore the underlying ple myeloma (MM) is characteristic of the over-growth mechanism. of plasma cells from bone marrow, insufficient apopto- Cytokine induced apoptosis inhibitor 1 (CIAPIN1 or sis, as well as the abnormal globulin secretion. After anamorsin) was a new cell regulator that found in 2004, more than 20 years of research and practice, the appli- which was independent of Apoptosis-regulatory Genes cation of high-dose chemotherapy and transplantation Bcl2 family and caspase family [4]. In different tumor of autologous hemopoietic stem cell [1], Immunomod- cells, CIAPIN1 played different regulatory roles. Our pre- ulator [2] and proteasome inhibitors [3], have signif- vious study has confirmed that CIAPIN1 could inhibit the icantly extended the survival rates in MM patients. proliferation of multiple myeloma cells [5]. Therefore, we aimed to continually explore the gene expression differ- ences of CIAPIN1 in MM patients and health human, as ∗Authors to whom correspondence should be addressed. Email: [email protected], [email protected] well as its role in regulation of multiple myeloma cells in †These three authors contributed equally to this work. current study. 1106 Mater. Express, Vol. 9, No. 9, 2019 CIAPIN1 is a potential target for apoptosis of multiple myeloma Materials Express Wang et al. 2. MATERIALS AND METHODS corresponding cycle values. The gene copy numbers ratio The current study (including the use of human samples and of CIAPIN1:GAPDH in the samples was employed as the animal study) followed the guideline of the Declaration of relative CIAPIN1 gene expression level. Helsinki and got approval from the Ethics Committee of The Seventh Affiliated Hospital, Sun Yat-sen University 2.5. CCK8 Assay (Shenzhen). All participants signed informed consent and Exponentially growing U266 cells in each group were agreed for the publication of the results. seeded in the 96-well plate (0.8 × 104/mL/well). The cells were Incubated at 37 C for 1 to 4 days. Subsequently, 2.1. Samples of Patients and Normal People the cells were further cultured with 10 L of CCK8 for All the specimens were collected from patients at 4 hours. The optical density was measured under 450 nm The Seventh Affiliated Hospital, Sun Yat-sen Univer- in a microplate reader (iMARK; Bio-Ras, CA, USA). sity (Shenzhen) from 2017 to 2018. The CD138 positive myeloma cells were obtained from the bone marrow in 18 2.6. Soft Agar Colony Forming Assay patients (13 males and 5 females, with age ranged from U266 cells were set up in each group and the cell sus- 45–69 years old) in whom multiple myeloma had been pension of 500 cells/mL was prepared by the RPMI 1640 pathologically confirmed and were purified with CD138 medium (20% FBS). Cell and agarose suspensions were antibody accordingly [6]. The bone marrow mononuclear made by 9 mL of each group cell suspension and 1 mL 3% cells were extracted from 10 normal people (5 males and low melting point agarose solution. The cell and agarose 5 females, with age ranged from 30–57 years old). suspensions were inoculated into the 6-well plate, each well for 3 mL. The 6-well plate were placed in the 4 C 2.2. Cells Culture refrigerator about 10 minutes for solidification, and cul- The U266 cells were obtained from the ATCC (USA) and tured in cell incubator for 16 days. The cells were stained the RPMI 1640 medium (Gibco®, Thermal Fisher Scien- with crystal violet (0.05%) and the numbers of colonies tific, Waltham, MA, USA) containing 15% FBS (Gemini were recorded under microscope observation. Article Bio Products, Broderick, CA, USA) was used for the cell culture. Standard culture conditions were employed 2.7. Nude Mouse Tumorigenicity Assay IP: 192.168.39.151 On: Fri, 01 Oct 2021 18:58:12 (37 C, 5% CO2). Copyright: American Scientific24 female Publishers BALB/c nude mice (age 4–6 weeks, weight Delivered by18–20 Ingenta g,) were randomly divided into 4 groups. 0.2 mL 2.3. Cell Transfection and Screening of 1 × 107/ mL cells were subcutaneously inoculated into The U266 cells were firstly transfected with CIAPIN1- the forelimb of the nude mouse to form the skin rash at small interfering RNA (U266-siRNA), non-coding siRNA ∼3 mm in diameter. The mice were sacrificed after 20 days (U266-NC) for cell proliferation, soft agar colony and of the inoculation. The tumors were measured by a venire nude mice tumorigenicity model studies. In the following caliper and the tumor volume was calculated as: V = A Western blot study, the U266, U266-siRNA and U266- (long diameter)×B2 (short-diameter)/2. The tumor in nude NC cells were further transfected with CIAPIN1 overex- mouse of each group was proved to be a xenograft by pression lentivirus vectors and the names of the groups pathological examination (H&E staining, Fig. 4). were as following: U266-O, U266-NC-O, U266-siRNA- O. The construction of CIAPIN1-small interfering RNA, 2.8. Western Blotting non-coding siRNA and CIAPIN1-overexpressing lentivirus The viable cells were collected and the total protein was vectors, cells transfection procedure were performed as extracted. A BCA Quantitative Kit (Beyotime Biotech- previously described [5]. The nucleotide sequence of nology, CHINA) was employed to test concentrations of CIAPIN1 could be found in the GENBANK database as the proteins. Subsequently, the proteins were run in SDS- (GENBANK: 020313.2 NM). PAGE. After transferring the gel to the PVF membrane (Millipore Corporation, USA), blocking of the membrane 2.4. Real-Time PCR was conducted by using the non-fat dry milk. Dilution Trizol method was used to extract the total RNA of the and incubation of primary antibodies the mouse-anti- cells and the concentrations of total RNA were quan- human P38 (ab170099, Abcam, USA), PKC(ab109539, tified by the spectrophotometer. cDNA was synthesized Abcam, USA), BCL-Xs (PA5-32203, Thermo Fisher Sci- according to the instruction of the Kit (BoShan bio- entific, USA), BAK (ab32371, Abcam, USA), BCL- logical technology co., LTD). Real-time PCR was con- 2(ab185002, Abcam, USA), BCL-xl (ab32370, Abcam, ducted in the ABI 7500 system with the cycle as follow: USA), BAX (ab32503, Abcam, USA), BIM (ab32158, 50 C (2 minutes)-95 C (2 minutes)-95 C (15 seconds)- Abcam, USA) and GAPDH, and monoclonal antibody 60 C (30 seconds) to read board, total 40 cycles. IgG, and secondary antibodies (goat anti-mouse IgG) The GAPDH gene was used as an internal reference. The were adjusted according to the manufacturers’ instruc- amounts of gene copies was calculated according to the tion (PL03b-0297M, PL Laboratories, Port Moody, BC, Mater. Express, Vol. 9, 2019 1107 Materials Express CIAPIN1 is a potential target for apoptosis of multiple myeloma Wang et al. Canada). Blots were obtained using the ECL chemilumi- nescence detection reagent (GE Healthcare, USA). The expression of P38, PKC, BCL-Xs, BAK, BCL-2, BCL-xl, BAX, BIM and GAPPH were quantified by densitometry analysis with ImageJ (National Institutes of Health, USA). 2.9. Statistical Analysis SPSS 20.0 (IBM SPSS, NY, USA) was used for data anal- ysis and the results are displayed as the means ± stan- dard deviation. T -test was used to compare the differences between groups and P<0.05 was considered statistically significant difference. Fig. 1. CIAPIN1 m-RNA gene expressions of bone marrow cells from 3. RESULTS AND DISCUSSION patients with multiple myeloma and adjacent paired normal persons.