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CIAPIN1 Affects Hepatocellular Carcinoma Cell Proliferation Not Been Reported

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CIAPIN1 Affects Hepatocellular Carcinoma Cell Proliferation Not Been Reported European Review for Medical and Pharmacological Sciences 2017; 21: 3054-3060 The study on expression of CIAPIN1 interfering hepatocellular carcinoma cell proliferation and its mechanisms Z. HUANG1, G.-F. SU1, W.-J. HU2, X.-X. BI3, L. ZHANG1, G. WANG1 1Department of Interventional Radiology, Huizhou First Hospital, Huizhou City, Guangdong Province, China 2Department of Interventional Radiology, The Fifth People’s Hospital of Dongguan, Dongguan, Guangdong, China 3Department of Medical Oncology, Huizhou First Hospital, Huizhou City, Guangdong Province, China Abstract. – OBJECTIVE: Liver cancer is one more than 80% of patients have benefited from of the common gastrointestinal cancers. This the first-line chemotherapy, there is still a high study was designed to investigate the effect recurrence rate and low overall survival rate in of the cytokine-induced apoptosis inhibitor 1 patients with advanced liver cancer4. A previous (CIAPIN1) on hepatocellular carcinoma cell pro- 5 liferation and invasion. study found that the development of liver cancer MATERIALS AND METHODS: To establish a is a complicated process involving the interaction low and high expression of CIAPIN1 in hepatoma of various factors, which has a close relationship cell lines, pGPU6/GFP/Neo and CIAPIN1 siRNA with the abnormality in the multi-genes family. vectors were constructed. The growth curve of This suggests that determination of the molecular liver cancer cells with a low and high expression targets should become the goal, which would help of CIAPIN1 was measured by MTT assay and col- ony formation in soft. The effect of overexpres- to understand the development and progression of sion and inhibition of CIAPIN1 on the expressions liver cancer. of cell cycle proteins Cyclin D1, CDK2, CDK4, and Cytokine-induced apoptosis inhibitory factor Cyclin E were detected by western blot. 1 (CIAPIN1), is a new gene identified by Dige- RESULTS: As compared with the low expres- stive Disease Hospital of China’s Fourth Military sion group, the cells in CIAPIN1 high expression Medical University5 through new molecular ge- group showed a significant decrease in prolifer- ation (p < 0.05). In addition, the colony-forming netic screening of patients with gastric cancer ability of cells with high expression of CIAPIN1 in 2004. In 2004, Japanese scholars Shibayama was decreased significantly (p < 0.01). Further- et al6 reported a new mouse-derived anti-apop- more, the expressions of Cyclin D1 CDK2, CDK4, totic gene-Anamorsin, whose structure having and Cyclin E in high expression group were sig- 88% amino acid sequence homology with that of nificantly increased (p < 0.01). CONCLUSIONS: CIAPIN1 played an import- CIAPIN1, both of which are considered to be the ant role in the proliferation of liver cancer cells homologous molecule among different species. through increasing the expressions of cell cycle re- The homolog Dre2 also has been found in yeast7,8. lated proteins Cyclin D1, CDK2, CDK4, and Cyclin E. Since 2004, lots of studies on CIAPIN1 have been Key Words: launched in several laboratories from different Cytokine-induced apoptosis inhibitory factor 1 countries, from which the results suggested that (CIAPIN1), Liver cancer, proliferation, Cell cycle-re- CIAPIN1 played different roles under different lated proteins. physiological and pathological conditions. In the development of several different human tumors, the mechanisms of CIAPIN1 are diffe- Introduction rent. For example, it exerts anti-apoptosis and promoting proliferation effect in ovarian cancer As one of the most common gastrointestinal and large B-cell lymphoma9, but inhibits tumor malignancies1, liver cancer has a very high morta- cell proliferation in renal cell carcinoma and lity rate2. Currently, the main treatments for liver esophageal cancer10. However, the exact role of cancer are surgery and chemotherapy3. Although CIAPIN1 in the development of liver cancer has 3054 Corresponding Author: Ge Wang, MD; e-mail: [email protected] CIAPIN1 affects hepatocellular carcinoma cell proliferation not been reported. In the present study, we aimed PIN1 siRNA plasmid, negative control of pGPU6/ to investigate the distribution of CIAPIN1 in liver GFP/Neo against nuclear and positive control of cancer cells, and further explore the mechanisms pGPU6/GFP/Neo GAPDH were added into 50 μL of CIAPIN1 in liver cancer as well as its relation- Opti-MEM, respectively. Then, 1.5 μl lipofecta- ship with the occurrence of liver cancer. mineTM2000 liposomes were added under gently pipetting, respectively and maintained at room temperature for 5 minutes. DNA and liposomes Materials and Methods are thoroughly mixed at room temperature for 20 Cells minutes; then, the mixture were added into the Human hepatoma cell line QGY-7703 was pur- QGY-7703 cell culture sugar-free DMEM for 5 chased from Sun Yat-sen University. hours. Transfected cells were named as the CIA- PIN1 siRNA, CIAPIN1 siRNA1, CIAPIN1 siR- Reagents NA2 experimental groups, negative control and Methyl thiazolyl tetrazolium (MTT; Sigma-Al- positive control of Con-siRNA GAPDH. drich, St. Louis, MO, USA); DMEM medium (Gi- bco, Grand Island, NY, USA); CIAPINI mouse The effect of CIAPIN1 on QGY-7703 anti-human monoclonal antibody (TNMED1007, Hepatocellular Carcinoma Cell Gene Technology Co., Ltd. Shanghai, China); Proliferation in vitro by MTT Assay Olympus BX51 optical microscope (Olympus, QGY-7703 human hepatoma cells in the logari- Shinjuku, Tokyo, Japan); automatic protein visua- thmic growth phase were digested and resuspen- lizer (Bio-Rad, Hercules, CA, USA). ded to be a single cell suspension. 5 × 103 cells/96 well plate were seeded. Cells were cultured at Construction of pGPU6/GFP/Neo 37°C and 5% carbon dioxide incubator overni- CIAPIN1 siRNA Plasmids ght. After medium replaces, 100 μL medium with 5’ and 3’ terminals of the target sequence of different concentrations of metformin was added the sense strand encoding small interfering RNA into each well. After another 72 hours cultured, 20 have been designed containing BbsI and BamHI μL 5 mg/ml MTT was added to each well. 4 hours restriction sites respectively, while there is an an- later, 150 μL dimethyl sulfoxide (DMSO) solution ti-19nt repetitive DNA sequence encoding siRNA was added into plates, and the plates were shaken antisense strand in the middle of the sequence as for 10 minutes under dark conditions. the hairpin transcript, wherein TTTTTT was ser- ved as transcription termination signals. The loop Clonogenic Assay structure TTCAAGAGA was designed in shR- QGY-7703 human hepatoma cells in the loga- NA template to avoid the formation of a stop si- rithmic growth phase were digested and resu- gnal, while the shRNA transcription termination spended to be a single cell suspension. 1 × 103 sequence was T6 structure. The sticky terminal cells were seeded in 6-well plates and cultured in 5’ terminal of the sense strand of the template at 37°C and 5% carbon dioxide incubator over- could be complementary after BbsI digestion; the night. After medium had replaced in every time, sticky terminal in 5’ GATC of antisense strand 100 μL medium with different concentrations of could be complementary after BamHI digestion. metformin was added into each well. After two Core design includes CIAPIN1 siRNA, CIAPIN1 weeks of culture, medium was removed and, siRNA1, CIAPIN1 siRNA2, Con-siRNA and ne- then, the cells were fixed with 4% DMSO for 15 gative control, with GAPDH as the positive con- minutes, stained with 0.1% crystal purple for 5 trol. minutes. Pictures were taken by a digital camera and archived. Transfection of CIAPIN1 siRNA After trypsin digestion, QGY-7703 cells were Western Blot cultured in high glucose medium containing 10% Human hepatoma cell QGY-7703 was lysed fetal bovine serum (excluding double-antibody) with radioimmunoprecipitation assay (RIPA) in 24-well plates until reaching 80% of the densi- buffer, and the particles were discarded after ty and then washed twice with serum high gluco- centrifugation. The concentration of extracted se Dulbecco’s Modified Eagle Medium (DMEM). protein was measured by the bicinchoninic The liposome/DNA complexes were prepared as acid (BCA) assay. The sample was mixed with follows: antisense plasmid pGPU6/GFP Neo CIA- 5 x loading buffer, and boiled for 5 minutes. 3055 Z. Huang, G.-F. Su, W.-J. Hu, X.-X. Bi, L. Zhang, G. Wang After separated by 10% polyacrylamide gel CIAPIN1 siRNA2, and CIAPIN1 siRNA2) and electrophoresis, the proteins were transferred a negative control -Con-siRNA and a positive to polyvinylidene fluoride (PVDF) membranes; control, namely GAPDH fragment sequences. then, they were blocked with 5% milk at room The pGPU6/GFP/Neo plasmid map was shown temperature for 1 hour. Membranes were incu- in Figure 1. bated with antibody at 4°C overnight. After 3 times rinsing, membranes were incubated with The Efficiency and Effect of CIAPIN1 secondary antibody at room temperature. After siRNA Transfection into QGY-7703 Cells addition of enhanced chemiluminescence (ECL) Image acquisition is completed in an inverted reagent for 5 minutes, the membranes were de- fluorescence microscope 24 hours after green veloped with gel image analyzer. fluorescent labeled with 7703 cells. After the cells had been transfected with a eukaryotic Statistical Analysis expression vector pGPU6/GFP/Neo CIAPIN1 Statistical analysis was conducted by IBM siRNA plasmid, and green fluorescence was ob- SPSS 19.0 software (SPSS Inc., Armonk, NY served under a fluorescence microscope, it indi- USA), and data expresses as mean ± standard de- cated that the expression of CIAPIN1 was found viation (SD). The groups were compared using in cells, and was positively correlated with the the t-test, and count data were compared using expression of GFP. QGY-7703 cells were used the chi-squire test. p < 0.05 was considered stati- as the control group without GFP expression, stically significant. and the number of cell apoptosis is minimal. As shown in Figure 2, the system CIAPIN1 siRNA and Con-siRNA transfection efficiency were re- Results ached to 80%.
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