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@#[ [Abstract] ] Objective: To investigate the biological effects and the related mechanisms of cytokine induced apoptosis inhibitor 1 (CIAPIN1) on the sensitivity of K562 chronic myeloid leukemia cells to imatinib. Methods: Specific short hairpin RNA(shRNA) interference vectors targeting CIAPIN1 (CIAPIN1-shRNA) were constructed. Interference efficiency of interference group (K562 cells transfected with CIAPIN1-shRNA) and control group (K562 cells transfected with scramble-shRNA) was evaluated using Real-time PCR, Western blotting and immunofluorescence staining. The interference group and control group were treated by 2 μmol/L imatinib. Cell viability was detected using MTT assay. Colony formation ability was detected using cell colony forming experiment. Cell cycle and apoptosis was detected using Flow cytometry and Western blotting. Results: CIAPIN1 expression was decreased effectively by specific shRNA targeting CIAPIN1. The CIAPIN1 mRNA content in CIAPIN1-shRNA group accounted (29.74±4.03)% of scramble-shRNA group, while the CIAPIN1 protein content in CIAPIN1-shRNA group accounted (21.57±2.18)% of scramble-shRNA group. CIAPIN1 knock-down significantly enhanced the inhibitory activity of imatinib on proliferation and colony forming ability of K562 cells. The colony number and radius of the CIAPIN1-shRNA+imatinib group was (15.60±1.03) and (2.63±0.55) μm, which were all less than those of the scramble-shRNA+imatinib group. The knock down also increased the activity of imatinib to block the cell cycle at G1 phase and to promot apoptosis of cells. The cell ratio at G1 phase of the CIAPIN1-shRNA+imatinib group was obviously increased while the ratio at S phase was obviously decreased compared with those of scramble-shRNA+imatinib group. Hoechst33258 staining and flow cytometry showed that the proportion of apoptotic K562 cells in the CIAPIN1-shRNA+imatinib group increased. The results of Western blotting showed that CIAPIN1 knock-down not only up-regulated the expressions of apoptosis related proteins (p21, Bid and Bim), but also repressed expressions of cell cycle related proteins (Cyclin D1, Bcl-xl, Bcl-2 and Mcl-1), which had synergistic effects with imatinib. Conclusion: CIAPIN1 knock-down significantly sensitized K562 cells to imatinib treatment, and the mechanism might be related with cell cycle arrest and expression of apoptosis-associated proteins.
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Objective To investigate the expression of cytokine induced apoptosis inhibitor 1 (CIAPIN1) protein in colon cancer tissues and its relationship with the pathological features of tumor.Methods A total of 80 cases of colon cancer tissues and 40 cases of paracancer tissues were collected from our hospital after surgery from January 2014 to January 2017.Immunohistochemical staining and Western blotting were used to detect the expressions of CIAPIN1 protein in the two types of specimens.The relationships between CIAPIN1 protein and the occurrence and development of colon cancer were analyzed.Results The positive rate of CIAPIN1 protein expression in colon cancer tissues was significantly lower than that in paracancer tissues (30.0％ vs.85.0％,x2 =32.303,P ＜0.001).The relative expression level of CIAPIN1 protein in colon cancer tissues was significantly lower than that in paracancer tissues (0.507 ±0.183 vs.1.874 ±0.361,t =22.513,P ＜0.001).The positive expression rate of CIAPIN1 in colon cancer tissues was not correlated with the age (x2 =0.086,P =0.769),sex (x2 =0.779,P =0.377),tumor invasion depth (x2 =0.879,P =0.348),tumor location (x2 =0.725,P =0.395),tumor size (x2 =2.241,P =0.134),tumor differentiation status(x2 =1.372,P =0.241),and the pathological type (P =0.715),but was significantly correlated with lymph node metastasis (x2 =10.813,P =0.001) and clinical stage (x2 =5.610,P =0.018).Conclusion The expression of CIAPIN1 protein in colon cancer tissues reduces significantly,and is correlated with clinical stage and lymph node metastasis.
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PURPOSE: Multidrug resistance (MDR) remains a major obstacle in the treatment of triple-negative breast cancer (TNBC) with conventional chemotherapeutic agents. A previous study demonstrated that hsa-miRNA-143-3p plays a vital role in drug resistance of TNBC. Downregulation of hsa-miRNA-143-3p upregulated the expression of its target protein cytokine-induced apoptosis inhibitor 1 (CIAPIN1) in order to activate MDR, while upregulation of hsa-miRNA-143-3p effectively enhances the sensitivity of drug-resistant TNBC cells to chemotherapeutics. The present study aimed to further verify these findings in vivo. METHODS: We established a hypodermic tumor nude mice model using paclitaxel-resistant TNBC cells. We expressed ectopic hsa-miRNA-143-3p under the control of a breast cancer-specific human mammaglobin promoter that guided the efficient expression of exogenous hsa-miRNA-143-3p only in breast cancer cells. Thereafter, we overexpressed hsa-miRNA-143-3p in xenografts using a recombinant virus system and quantified the expression of hsa-miRNA-143-3p, CIAPIN1 protein, and proteins encoded by related functional genes by western blot. RESULTS: We successfully completed the prospective exploration of the intravenous virus injection pattern from extensive expression to targeted expression. The overexpression of hsa-miRNA-143-3p significantly alleviated chemoresistance of TNBC by inhibiting viability. In addition, we observed that the expression of CIAPIN1 as a hsa-miRNA-143-3p target protein was remarkably decreased. CONCLUSION: We partly illustrated the mechanism underlying the hsa-miRNA-143-3p/CIAPIN1 drug resistance pathway. HsamiRNA-143-3p as a tumor suppressive microRNA may be a novel target to effectively reverse MDR of TNBC in vivo.
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Animals , Humans , Mice , Apoptosis , Blotting, Western , Breast , Breast Neoplasms , Down-Regulation , Drug Resistance , Drug Resistance, Multiple , Heterografts , Mice, Nude , MicroRNAs , Prospective Studies , Triple Negative Breast Neoplasms , Up-RegulationABSTRACT
Aim To Investigate the effect of CIAPIN1 expression on proliferation, cell cycles, apoptosis, in vivo invasion and migration of EC9706, a kind of esophageal cancer cell line, by transfection of recombinant lentiviral vectors for expression and silencing of CIAPIN1 rspectively. Methods EC9706 cells were transfected with CIAPIN1 expression vector and CIAPIN1 siRNA vector respectively, and cell lines with stable over-expression and low expression of CIPAN1 were obtained. RT-PCR and western-blot, MTT, flow cytometry method and transwell were used to study the expression of CIAPIN1, cell proliferation, cell cycles and apoptosis, and cells invasion and migration respectively. Results CIAPIN1 group, expressed inhibited growth of EC9706 cells, decreased cells in S and G2/M phase, increaed cells in G0/G1 phase, elevated apoptosis and reduced cells passing through matrigel compared with control groups. Conclusion Lentiviral vector-mediated over-epression of CIPAIN1 could inhibit the proliferation, promote apoptosis, and reduce invasion and migration of EC9706, a kind of eophageal cancer cells.
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0.05),but had positive relationship with the clinical stages and lymphatic metastasis of the tumor. Conclusion Livin ? is highly expressed in the tissues and peripheral blood cell from the patients of laryngeal squamous carcinoma,which is closely related to the clinical stages and lymphatic metastasis of the tumor.
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Objective To investigate the relationship between the expression of tumor apoptosis inhibitory protein(survivin) and the apoptosis induced by arsenic trioxide(As2O3) in transcatheter arterial chemoembolization therapy.Methods Sixteen Japanese big-ear white rabbits with implanted hepatic VX2 tumor at both right and left hepatic lobes were randomly and equally divided into two groups.Three weeks after the tumor was inoculated,1 ml lipiodol(UFLP) and 2 mg As2O3 were injected via hepatic arterial cannulation into the rabbits of study group,while only 1 ml UFLP was used for the rabbits in control group.Three weeks later,all the rabbits were sacrificed,and the tumor tissue,the tumor-neighboring tissue and the normal liver were separately collected and sent for TUNEL staining and examinations,which included the observation of apoptosis of tumor cells and the assessment of the expression of survivin protein.Results In study group,a large number of yellow apoptosis cells was seen in the tumor tissue but no apoptosis cell was found in the tumor-neighboring tissue or in the normal liver tissue.In the control group,no yellow apoptosis cell was observed in the tumor tissue,tumor-neighboring tissue or normal liver tissue.The survivin protein expression rate of the tumor tissue was 100%(16 / 16) in the control group,including strongly-positive in 12 and weakly-positive in 4 rabbits.In contrast,the survivin protein expression rate of both the tumor-neighboring tissue and the normal tissue was 0%.In study group,the survivin protein expression rate of the tumor tissue was 37.5%(6 / 16),including strongly-positive in 2 and weakly-positive in 4 cases,and the survivin protein expression rate of both the tumor-neighboring tissue and the normal tissue was 0%.Significant difference in survivin protein expression rate of the tumor tissue existed between two groups(P
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Objective To clone human survivin gene and express survivin-an inhibitor of apoptosis proteins in Pichia pastoris eukaryotic expression system. Methods Full length survivin gene was amplified by PCR using survivin specific primers. The verified survivin gene by sequencing was subcloned into pPic9k. The recombinant plasmid was linearized by restriction enzyme cutting. The linear survivin gene was introduced into GS115/ His-cells by electroporation. The transformants were transferred onto YPD plates that contained different concentrations of G418 for screening the positive clones. The integrated survivin gene in positive clones was confirmed by PCR. Selected transformants were cultured in BMMY medium with 1 % methanol for inducing the expression of survivin protein. The expressed survivin protein was analyzed by ELISA. Results The cloned human survivin sequence was the same as that in the GeneBank. The recombinant pPic9k-survivin was constructed in Picha pastoris eukaryotic expression vector. After the linear digestion, the recombinant vector was introduced into GS115/ His-cells, the 6 positive clones against G418(4 mg/mL) were obtained and confirmed by PCR; the highest survivin protein was expressed when expressed for two days in 1 % methanol BMMY medium. Conclusions Improved survivin protein yield may be reached by modifying the experimental conditions. This will help to further study the biological functions of survivin and its roles in tumor developments.
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Objective:Apoptosis inhibitor 6(Api6),also known as AIM(Apoptosis Inhibitor expressed by Macrophage)or SP?(Soluble Protein alpha),is a newly defined member of the group B scavenger receptor cysteine-rich(SRCR)superfamily.Previous studies have suggested its important roles in immune system regulation,but the involvement of Api6 in lipid metabolism is seldomly studied.Methods:The aim of this study was to define the expression pattern of Api6 in lipid loading and inflammatory macrophages.The human monocyte/macrophage lineage THP-1 cells and murine Raw264.7 macrophages were incubated in the presence of 100 ?g/ml oxidized low-density lipoprotein(oxLDL)alone,LDL plus 200 ng/ml lipopolysaccharide(LPS)and LPS alone.Results:Foam cell formation after treatment was evidenced by oil red O staining.Real-time PCR results showed that after LPS or oxLDL treatment,the mRNA levels of Scavenger receptor SRA were increased 6-fold,2.5-fold and 6-fold,44-fold in Raw264.7 cells and THP-1 cellls respectively,but the expression of Api6 in Raw264.7 did not change.The mRNA levels of Api6 increased 2.7-fold after LPS and oxLDL treatment in THP-1 cells.Conclusion:The expression levels of Api6 in these macrophages were highly related to the presence of nuclear receptor liver X receptor(LXR)isoform LXR?.This study revealed that due to the lack of LXR? isoform,the basic expression level of Api6 was low in Raw264.7 macrophages,that makes it an ideal cell line for further study related to pathophysiological function of Api6 in lipid metabolism.