The Roles Of ZNRD1 In Carcinogenesis And Multidrug Resistance Of Gastric Cancer

【Background】The carcinogenesis and multidrug resistance (MDR) of gastric cancer is controlled by a complex signal network, which remains largely unknown. Although the signal network controlling the carcinogenesis of gastric cancer may be quite different from that of MDR of gastric cancer, there may exist some molecules who are involved in both networks. Further research is needed to investigate the molecules related to gastric cancer.ZNRD1 (zinc ribbon domain-containing 1) gene is cloned from the human HLA, with potential roles in MHC gene therapy. The key function of ZNRD1 is to promote the nascent transcript and read through the blockage to elongation, indicating that ZNRD1 may play important roles in many physiological and pathological processes. Our laboratory has previously found that ZNRD1 was associated with MDR of gastric cancer. Here we further investigate the functions and mechanisms of ZNRD1 in carcinogenesis and MDR of gastric cancer. This study will push further insight into the biological functions of ZNRD1, and help find potential targets for gene therapy. 【Aims】To investigate the functions and mechanisms of ZNRD1 in carcinogenesisand MDR of gastric cancer.【Methods】1) The monoclonal antibody specific to the native form of ZNRD1 protein isprepared; 2) RT-PCR, Western blot and immunohistochemical analysis are used toinvestigate the expression of ZNRD1 in gastric cancer cells, normal gastricepithelium cells, gastric cancer tissues, adjacent non-neoplastic gastric tissues,gastritic tissues, normal gastric tissues; 3) The small interference RNA vector ofZNRD1 is constructed and further identified by sequencingT; T4) Liposome is usedto transfect the eukaryotic expression vector of ZNRD1 and the correspondingempty vector into the gastric cancer cells (AGS, SGC7901 and MKN28) andNIH3T3 cells. The stable clones are further identified by RT-PCR and WesternblotT; T5) Liposome is used to transfect the RNAi vector of ZNRD1 and thecorresponding empty vector into the SGC7901 cell line, normal gastric epithelialcell line GES-1, vincristine resistant gastric cancer cell line SGC7901/VCR,leukemia cell line HL-60, and vincristine resistant leukemia cell line HL-60/VCR.The stable clones are further identified by RT-PCR and Western blot; 6) MTTassay is used to investigate the effect of ZNRD1 on the cell growth of cells(AGS,SGC7901, MKN28, NIH3T3, GES-1)T ; T7) Soft agar assay is used to investigatethe effect of ZNRD1 on the clonality of cells (AGS, MKN28)T ; T8) In vivo usingnude mice assay is used to investigate the effect of ZNRD1 on the cell growth ofgastric cancer cells (AGS, MKN28)T ; T9) Flow cytometry assay is used toinvestigate the effect of ZNRD1 on the cell cycle distribution of cells (AGS, MKN28, NIH3T3, GES-1)T ; T10) Flow cytometry assay is used to investigate theeffect of ZNRD1 on the cell apoptosis of cells (AGS, MKN28, NIH3T3)T ; T11)MTT assay is used to investigate the effect of ZNRD1 on the drug sensitivity ofcancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR) in vitroT; T12) SRCAmice assay is used to investigate the effect of ZNRD1 on the drug sensitivity ofgastric cancer cells(SGC7901, SGC7901/VCR) in vivoT; T13) Flow cytometry assayis used to investigate the effect of ZNRD1 on adriamycin accumulation of cancercells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR)T; T14) Transmissionelectron microscope is used to investigate the effect of ZNRD1 on the sensitivityof SGC7901 cells towards drug-induced apoptosisT; T15) Flow cytometry assay andDNA ladder assay are used to investigate the effect of ZNRD1 on the sensitivityof cells (SGC7901, SGC7901/VCR, HL-60/VCR) towards drug-inducedapoptosisT; T16) Microarray is used to investigate the Tprofiling ofZNRD1-responsive genesT in gastric cancer cells (AGS, MKN28, SGC7901,SGC7901/VCR)T ; T17) RT-PCR and Western blot are used to identify the results ofmicroarray; 18) Reporter gene assay is used to investigate the Teffect of ZNRD1 onthe transcriptional activity of Cyclin D1; T19) Reporter gene assay is used toinvestigate the Teffect of ZNRD1 on the transcriptional activity of MDR1; T20)Kinase assay is used to investigate the Teffect of ZNRD1 on the activity of cyclinE-CDK2 kinase; T21) The antisensenucleic acids of p21 is used to inhibit theexpression of p21, and flow cytometry assay is used to investigate the Teffect ofp21 on ZNRD1-induced cell cycle arrest in gastric cancer cells; T22) Theantisensenucleic acids of p27 is used to inhibit the expression of p27, and flowcytometry assay is used to investigate the Teffect of p27 on ZNRD1-induced cell cycle arrest in gastric cancer cells; T23) Liposome is used to up-regulate theexpression of Skp2, and flow cytometry assay is used to investigate the Teffect ofTSkp2T on ZNRD1-induced cell cycle arrest in gastric cancer cells; T24) Western blotis used to investigate the Teffect of ZNRD1 on the stability of TSkp2T and p27 ingastric cancer cells; T25) MVD assay is used to investigate the Teffect of ZNRD1 onthe Tangiopoietic activity Tof gastric cancer cells; T26) ELISA assay is used toinvestigate the Teffect of ZNRD1 on the Texpression Tof TVEGFB165 TBin gastric cancercells; T27) The roles of DARPP-32 in MDR of gastric cancer cells are investigatedusing gene transfection, MTT assay, SRCA mice assay, Flow cytometry assay andDNA ladder assay. The roles of TZNRD1T in DARPP-32-mediated MDR of gastriccancer is investigated using gene transfection and MTT assay.【Results】1) The monoclonal antibody (H6) specific to the native form of ZNRD1protein is successfully established; 2) ZNRD1 is found downregulated in gastriccancer cells, compared with normal gastric epithelium cells. ZNRD1 is founddownregulated in gastric cancer tissues, compared with normal gastric tissues.ZNRD1 is found downregulated in gastric cancer tissues (9%), compared withnormal gastric tissues (63%)T; TThe expression of ZNRD1 is found significantlyhigher in gastritic tissues (81%), compared with that in normal gastric tissues; 3)The RNAi vector of ZNRD1 is successfully established; 4) As identified byRT-PCR and Western blot, the gastric cancer cell models with increased ZNRD1expression is successfully established; 5) As identified by RT-PCR and Westernblot, the gastric cancer cell models with decreased ZNRD1 expression issuccessfully established; 6) As the results of MTT assay show, up-regulation of ZNRD1 may inhibit the growth of gastric cancer cells and NIH3T3 cells, whiledown-regulation of ZNRD1 may promote the growth of normal gastric epitheliumcells; 7) As the results of soft agar assay show, up-regulation of ZNRD1 mayinhibit the clonality of of gastric cancer cells; 8) As the results of nude mice assayshow, up-regulation of ZNRD1 may inhibit the growth of gastric cancer cells invivo; 9) As the results of FCM assay show, up-regulation of ZNRD1 lead to G1cell cycle arrest in gastric cancer cells, while down-regulation of ZNRD1 promotethe normal gastric epithelium cells from G1 to S phase; 10) As the results of FCMassay show, up-regulation of ZNRD1 may not induce the apoptosis of gastriccancer cells; 11) As the results of MTT assay show, up-regulation of ZNRD1 mayinhibit the drug sensitivity of cancer cells in vitro, while down-regulation ofZNRD1 may increase the drug sensitivity of cancer cells in vitro; 12) As theresults of SRCA mice assay show, up-regulation of ZNRD1 may inhibit the drugsensitivity of gastric cancer cells in vivo, while down-regulation of ZNRD1 mayincrease the drug sensitivity of gastric cancer cells in vivo; 13) As the results ofFCM assay show, up-regulation of ZNRD1 may inhibit the drug accumulation andincrease the drug release index of cancer cells, while down-regulation of ZNRD1may increase the drug accumulation and decrease the drug release index of cancercells of cancer cells; 14) As the results of transmission electron microscope show,up-regulation of ZNRD1 may increase the apoptotic sensitivity of gastric cancercells; 15) As the results of flow cytometry assay and DNA ladder assay show,up-regulation of ZNRD1 may increase the apoptotic sensitivity of cancer cells,while down-regulation of ZNRD1 may decrease the apoptotic sensitivity of cancercells; 16) As the results of microarray show, ZNRD1 may mediate 263 kinds of genes, including 38 kinds of gene significantly upregulated and 24 kinds of genesignificantly downregulate; 17) As identified by RT-PCR and Western blot,ZNRD1 may upregulate the expression of P21, P27, pRB, IMPDH2, P-gp andBcl-2, and downregulate the expression of cyclin D1, CDK4, DARPP-32 andVEGFB165B; 18) Upregulation of TZNRD1 Tmay increaseT the transcriptional activity ofCyclin D1; T19) Upregulation of TZNRD1 Tmay increaseT the transcriptional activityof MDR1.T TDownTregulation of TZNRD1 Tmay decreaseT the transcriptional activity ofMDR1; T20) TZNRD1 Tmay decreaseT on the activity of cyclin E-CDK2 kinase; T21)The antisensenucleic acids of p21 may significantly inhibit the expression of p21,and dTownTregulation of Tp21 Tshow little Ton ZNRD1-induced cell cycle arrest ingastric cancer cells; T22) The antisensenucleic acids of p27 may significantlyinhibit the expression of p27, and dTownTregulation of Tp27 Tmay significantlyreverseT ZNRD1-induced cell cycle arrest in gastric cancer cells; T23) The cellmodels with increased Skp2 expression are successfully established, andupregulation of Skp2T Tmay significantly reverseT ZNRD1-induced cell cycle arrestin gastric cancer cells; T24) TZNRD1 Tmay increaseT the stability of p27T, Tand inhibitthe stability of TSkp2T in gastric cancer cells; T25) UpregulationT of ZNRD1 TmayinhibitT the Tangiopoietic activity Tof gastric cancer cells; T26) UpregulationT ofZNRD1 Tmay inhibitT the Texpression Tof TVEGFB165 TBin gastric cancer cells; T27)Upregulation of DARPP-32 may increase the drug accumulation of cancer cells,and decrease the apoptotic sensitivity and drug release index of gastric cancercells. Upregulation of TZNRD1 Tmay significantly promote DARPP-32-mediatedMDR of gastric cancer, while downregulation of TZNRD1 Tmay significantlyreverse DARPP-32-mediated MDR of gastric cancer. 【Conclusions】1) ZNRD1 is found downregulated in gastric cancer cells and gastric cancertissues, compared with normal gastric epithelium cells and normal gastric tussues.Up-regulation of ZNRD1 may reverse the malignant phenotyoe of gastric cancercells, resulting in cell growth arrest in G1 phase. Down-regulation of ZNRD1 maypromote the normal gastric epithelium cells from G1 to S phase. All these datareveal that ZNRD1 may be a novel candidate for tumor suppressor gene, and itmay be considered as a valuable target for gene therapy in the future.2) ZNRD1 may play important roles in gastric carcinogenesis throughregulation of cell cycle and angiogenesis.The mechanisms underlying the roles of ZNRD1 in cell cycle regulation maybe as following: ZNRD1 may reduce the promotor activity PPof cyclin D1, whichlead to the decrease of the association of cyclin D/CDK4 complexes with Tp21Tand/or Tp27T. With limited sequestration of the CIP/KIP proteins, increasedPPlevels ofTp27T and/or Tp21T would be free to decrease the ability of cyclin E/CDK2 complexes.Downregulation of Cyclin E/CDK2 activity result in the increasedhypo-phosphorylated form of pRb, which may inhibit the release of criticaltranscription factors, such as E2F. ZNRD1 may inhibit the expression of TSkp2through increase the stability of Skp2T, Tand Tmay TupregulateT the expression of p27Tthrough TincreaseT the stability of p27. TThe mechanisms underlying the roles of ZNRD1 in the regulation ofangiogenesis may be as following: up-regulation of ZNRD1 may inhibit thePPVEGF production by tumor cells, thus reduce the vascular densityPPwithin thetumors formed by gastric cancer cells. Such an enhanced anti-angiogenicPPeffect may contribute to the inhibition ofPPthe tumorigenesis in nude mice.3) Down-regulation of ZNRD1 may reverse the MDR phenotyoe of gastriccancer cells, and up-regulation of ZNRD1 may promote the MDR phenotyoe ofgastric cancer cells.4) The mechanisms underlying the roles of ZNRD1 in MDR of gastric cancermay be as following: ZNRD1 may mediate the drug accumulation and the drugrelease index of cancer cells through regulation of P-gp; it may mediate the cellapoptosis of cancer cells through regulation of Bcl-2; it may mediate the DNAsynthesis of cancer cells through regulation of IMPDH2; it may mediate thephosphorylation of protein through regulation of DARPP32; it may mediate thegrowth of cancer cells through regulation of cell cycle.5) DARPP-32 is proved to be a novel MDR related molecule of gastric cancer.ZNRD1 play important roles in DARPP-32-mediated MDR of gastric cancer.ZNRD1 and DARPP-32 may be used jointly in the gene therapy of MDR.