The Functional Characterization Of A Novel Gene CIAPIN1

CIAPIN1,a novel antiapoptotic molecule,which does not show anyhomology to known apoptosis regulatory molecules such as Bcl-2 family,Caspase family,or signal transduction molecules,is proven to be a mediator ofthe RAS signaling pathway and plays a vitally important role in fetal liverhematopoiesis. Microarray analysis showed that Bcl-xL and Jak2 weredownregulated in fetal liver of mCIAPIN1 null mice,suggesting that mCIAPIN1might exert its function by up-regulating the expression of Bcl-xL and Jak2. Inaddition,mCIAPIN1 protects Ba/F3 cells against etoposide,γradiation andstauroporine in vitro. Our previous studies revealed that CIAPIN1 wasupregulated in MDR gastric cancer cell subline SGC7901/VCR at mRNA level.Furthermore,CIAPIN1 was found to act as an antiapoptotic molecule in vivobecause mCMPIN1-/-mice died in late gestation from defective definitivehematopoiesis in the fetal liver. However,although mCIAPIN1 as an anti-apoptosis molecule has been observed in the mouse cell lines,the basicbiological function of CIAPIN1 is still unclear. Here,we want to explore the basic biological function of CIAPIN1 and to examine its effect on theembryogenesis,multidrug resistance,cancer development and so on. We hopethat those studies could reveal the basic biologivcal function of this novel gene-CIAPIN1.【Objectives】In order to explore the basic biological function of CIAPIN1,in thefollowing studies,we first examined the expression trend and location ofCIAPIN1 in mammal development and its subcellular location in differential celllines. Then,we wanted to focuse on the effect and mechanism of CIAPIN1 ongastric cancer MDR,cancer development and process. Finally,we hoped thatwith the 2-DE,gene chip,TAP and MALDI-TOF-MS technologies,we couldfind the possibal signal pathways and interaction molecules of this novel gene-CIAPIN1.【Methods】(1) Immunohistochemistry was used to analyze the expression trend ofCIAPIN1 in heart,liver,brain,kidney and skeletal muscle during mousepostnatal development from P0 to P28 and the various tissues of adult mouse.Immunochistochemistry was also used to analyze the expression distribution ofCIAPIN1 in the various tissues from five month human embryos and adulthuman. (2) Bioinformatic prediction disclosed a putative nuclear localizationsignal and a putative nuclear export signal within both human and mouseCIAPIN1. Here,we want to examine the distribution of CIAPIN1 in normal fetaland adult human tissues by immunohistochemical (IHC) staining with an in-house-produced we further performed immunofluorescence,6×His-taggedfusion protein expression,and analytical cell fractionation in our present study. (3) CIAPIN1 was found to be overexpressed at the mRNA level in thevincristine induced MDR gastric cancer cell SGC7901/VCR compared to itsparental cell line. In this study,with the gene transfected,in vitro drug sensitivityassay,flow cytometry detect apoptosis,RT-PCR,Western Blot and Luciferasereporter assay methods,we investigated the role of CIAPIN1 in multidrugresistance (MDR) of gastric cancer cells and the possible underlyingmechanisms. (4) CIAPIN1,CIAPIN1-siRNA and their control vectors andadenovirus were constructed and packaged. In order to explore the relationshipof CIAPIN1 and gastric carcinoma,CIAPIN1 related adenoviruses were used toinfect gastric cancer cells. (5) The downstream effectors were screened by two-dimensional electrophoresis and MALDI-TOF-MS. (6) The downstreameffectors were screened by microarray. (7) The interaction molecules werescreened by TAP.【Results】1. The distribution of CIAPIN1 in tissues of mouse postnatal of differentialtime pointDuring mouse postnatal development,CIAPIN1 immunoreactions in theheart,brain,liver,kidney and skeletal muscle have some upregulated between P0and P7. CIAPIN1 immunoreaction in the liver,kidney and skeletal musclebecomes slightly low,but in the heart and brain higher between P7 and P14.Between P14 and P21,CIAPIN1 immunoreaction in the brain,heart and liverbecame much lower. However,between P21 and P28,CIAPIN1 immunoreactionin the heart,brain,liver and skeletal muscle became much lower,while with thekidney development,CIAPIN1 immunoreaction in the kidney became higher. Invarious tissues from adult mouse,CIAPIN1 immunoreaction could be seen incardiac muscle cell,brain,hepatocyte,epithelium of renal tubule,skeletal muscle,lung tissue,gastric mucosa and gland,acinus lienalis.2. Distribution of CIAPIN1 in normal fetal and adult human tissuesTo reveal the possible physiological role of CIAPIN1,we examined theexpression and distribution of CIAPIN1 in fetal and adult human tissues usingimmunohistochemistry. We found that CIAPIN1 was ubiquitously distributed infetal and adult tissues,and was localized in both the cytoplasm and the nucleus.The expression patterns of CIAPIN1 were similar in fetal and adult tissues andcorrelated with the previously described expression pattern of Ras. Theseobservations suggested that CIAPIN1 expression appears to be involved in celldifferentiation,and it might exert universal and possibly important physiologicalfunctions in the regulation of Ras in humans.3. Subcellular localization of CIAPIN1Our previous studies have demonstrated that CIAPIN1 is ubiquitouslyexpressed in normal fetal and adult human tissues. However,fundamentalbiological functions of CIAPIN1 have not been elucidated. In this study,we firstpredicted the subcellular localization of CIAPIN1 with bioinformatic approachesand then characterized the intracellular localization of CIAPIN1 in both humanand mouse cells by a combination of techniques including (a)immunohistochemistry and immunofluorescence,(b) His-tagged CIAPIN1expression,and (c) subcellular fractionation and analysis of CIAPIN1 in thefractions by Western Blot. All methods produced consistent results;CIAPIN1was localized in both the cytoplasm and the nucleus and accumulated in thenucleolus. Bioinformatic prediction disclosed a putative nuclear localizationsignal and a putative nuclear export signal within both human and mouseCIAPIN1. These findings suggested that CIAPIN1 may undergo acytoplasm-nucleus-nucleolus translocation. 4. CIAPIN1 confers MDR of gastric cancer cellsThe overexpression of CIAPIN1 gene has been previously reported inSGC7901/VCR cell line compared with its parental cell line SGC7901. In thepresent study,we constructed the siRNA eukaryotic expression vectors ofCIAPIN1 and transfected them into SGC7901/VCR cells to examine whether thedownregulation of CIAPIN1 increased cell sensitivity towards chemotherapeuticdrugs. After transfection,the expression of CIAPIN1 was dramatically decreasedin CIAPIN1-siRNA transfectants compared with that in parental cells and emptyvector control cells. The downregulation of CIAPIN1 significantly enhanced thesensitivity of SGC7901/VCR cells to vincristine (VCR),adriamycin (ADR) andetoposide (VP-16),but not to 5-fluorouracil (5-Fu) and cisplatin (CDDP). Cellcapacity to efflux adriamycin decreased markedly in CIAPIN1-siRNAtransfectants,and the correlation between CIAPIN1 downregulation anddecreased MDR-1 transcriptional activity was observed. Inhibited the expressionof CIAPIN1 could significantly downregulate the expression of Bcl-2,andupregulate the expression of Bax,but does not alter the expression of PTEN ingastric cancer cells. These observations suggested that the CIAPIN1-siRNAcould effectively downregulate the expression of CIAPIN1 and reverse theresistant phenotype of gastric cancer cells. Further study of the biologicalfunctions of CIAPIN1 may be helpful for understanding the mechanisms ofmultidrug resistance of gastric cancer and in developing possible strategies totreat gastric cancer.5. CIAPIN1 inhibits gastric cancer cell proliferation and cell cycleprogressionCIAPIN1 was found to be downregulated in human gastric cancer tissues ascompared to the matched adjacent nonneoplastic tissues. In this study,we investigated the effect of CIAPIN1 on in vitro and in vivo proliferation of gastriccancer cells. Ectopic expression and knockdown of CIAPIN1 in gastric cancercells SGC7901 and MKN45 were achieved by transduction with recombinantadenoviruses expressing human CIAPIN1 (Ad-CIAPIN1) and humanCIAPIN1-specific small interference RNA adenovirus (Ad-CIAPIN1siRNA).Gastric cancer cells with upregulated expression of CIAPIN1 exhibitedsignificantly decreased proliferation,while cells with downregulated CIAPIN1expression showed significantly quicker growth rate as compared with theirrespective controls in both in vitro and in vivo tests. Also,CIAPIN1 couldsignificantly suppressed colony formation of SGC7901 and MKN45 cells in softagar cloning test. Furthermore,cell cycle distribution analysis revealed thatCIAPIN1 induced cell cycle arrest at G1/S phase. Downregulation of CyclyinD1and upregulation of P27 might contribute,at least in part,to this altered cellcycle distribution. This study demonstrated that CIAPIN1 is a suppressor ofgastric cancer cell proliferation and suggested that CIAPIN1 might play animportant role in the development of human gastric cancer.6. Screening of the downstream effectors of CIAPIN1The proteomic maps of SGC7901-pSiCIAPIN1 and SGC7901-pSi weresuccessfully acquired through two-dimensional electrophoresis. Most of theprotein spots were distributed in the center of the map. Melanie3 2D softwarewas used to analyze the two proteomic maps. Compared to the map ofSGC7901-p Si,there are 13 protein spots in SGC7901-p Si CIAPIN1 which werestrongly stained and 7 spots weakly stained. All the spots were treated bydestaining of silver,in-gel digestion and then analyzed by MALDI-TOF-MS.After MASCOT database searching,eight proteins were identified. They wereP66,If-A20,Twist,IGHD,CACNA1B,GSDM1,IFT20 and RGS,among which P66,RGS,Twist,CACNA1B,GSDM1 and IGHD were downregulated,IFT20and If-A20 were upregulated by CIAPIN1. The total RNA of SGC7901-pCIAPIN1 and SGC7901-pc were extracted. The quality of extracted RNA wasevaluated by agarose electrophoresis and analysis of Lab-on-chip. Aftermicroarray hybridization and data normalization,84.6 % spots out of 14,000genes were positively detected,among which in gastric cancer cell lines 411genes were up-regulated while 288 genes were down-regulated by CIAPIN1.7. Screening of the interaction molecules of CIAPIN1Identification of protein-protein interactions is at the core of understandingbiological processes occurring in living cells. Traditionally,potential interactingproteins have been identified by genetic methods (two-hybrid screens) withsubsequent verification of the interaction by co-immunoprecipitation. While thismethod has been successful for detection of two interacting proteins,it is oflimited utility when more complex protein aggregates such as ribosomes,spliceosome complexes,or transcription complexes are investigated. Toovercome this limitation,an alternative method was developed for purification ofyeast protein complexes. This tandem affinity purification (TAP) methodcombines purification of a protein complex of interest using affinity purificationtags with subsequent mass spectrometry identification of unknown proteincomplex components. The key feature of this technology is the use of twodifferent affinity purification tags that are fused to at least one known componentof the protein complex of interest by genetic methods. Performing twoconsecutive purification steps using affinity purification tags that have gentlewashing and elution conditions allows for isolation without disrupting thetargeted complex. In this study,we constructed the pCTAP expression vector ofCIAPIN1 and trasfected the vectors into SGC7901 cells. With the TAP and SDS- PAGE,the spots were treated by destaining of Coomassie brilliant blue,in-geldigestion and then analyzed by MALDI-TOF-MS. After MASCOT databasesearching,one protein molecule-TXNL2 interacted with CIAPIN1 wasidentified.【Conclusion】In conclusion,we provide evidence of several new biological function ofnovel gene CIAPIN1. Although CIAPIN1 plays significantly important effects onapoptosis,MDR and mmoregeneis,the basic biological function of this newgene is still unclear,the further research could be added the new clue to CIAPIN1.