Molecular Mechanism Of Bifidobacterium-mediated CTP-NPRL2 For Treating Renal Cancer Of Nude Mice

Renal cancer derives from uriniferous tubule epithelium of renal parenchyma.It is a common malignant tumor of adults.Surgery is the main treatment for patients in the early stage,but a part of these patients have postoperative metastasis.Patients in advanced stage lose the chance of surgery and are lack of effective therapies.Although cytokines and molecular targeted drug have a certain effect for treating renal cancer,the effect is unsatisfactory.However,the occurrence and development of malignant tumor is closely related to functional change of gene,and it is meaningful to explore gene therapy which is also the long-term research hotspot of renal cancer.NPRL2 is one of newly found and hotly researched cancer suppressor gene.It is closely related to the genesis,development and prognosis of many kinds of tumors.Our early study demonstrated the expression of NPRL2 was significantly lower in renal cancer tissue than adjacent normal tissue,and inhibiting effect occurred when the plasmid containing NPRL2 gene was transfected into human renal cancer cells.The result implies that NPRL2 has an important role to renal carcinoma in the process of its genesis and development.However,there is a series of questions to use traditional transgenic technology for transfecting exogenous gene into cancer cell,such as low transfection efficiency,gene mutation and lost.But the CTP may solve these questions because it can efficiently mediate proteins to penetrate cell membrane and specifically locate in cytoplasm.In the research of gene therapy,the vector systems mainly contain two types: viral vector and non-viral vector,for example,liposomes.The former has a series of disadvantages,such as low virus titer,lack of security and gene mutation and so on.The latter also has some shortcomings,such as lack of tumor targeting and low transfection efficiency and so on.However,as an important probiotics of intestinal tract and one of obligate anaerobic bacteria,Bifidobacterium has a specific targeting to the anaerobic necrotic area of tumor tissue and is a good targeting vector for gene therapy of tumors.On the basis of our early study,this research aims to have a further exploration on cancer suppressor gene NPRL2.Using the remarkable location in cytoplasm and highly efficient transduction potential of CTP,it is the first to put forward strategy that exogenous NPRL2 protein can be endogenous through fusion expression of NPRL2 and CTP.After the prokaryotic expression of fusion protein CTP-NPRL2,the change of biological behavior of renal cancer cells was observed after NPRL2 protein entered into cells using the transduction potential of CTP.At the same time,animal experiment on nude mice with renal cell carcinoma was done for the assessment of curative effect which using Bifidobacterium as vector.Finally,some techniques like gene microarray and protein spectrum were used for initially uncovering the molecular mechanism of Bifidobacterium-mediated CTP-NPRL2 for treating renal cancer on nude mice to provide a new method for treating renal cancer,especially advanced renal cancer.This paper contains two parts in all.Part One BIOLOGICAL CHARACTERISTICS OF HUMAN RENAL CANCER CELL 786-O AFTER CTP-MEDIATED NPRL2 PROTEIN TREATMENTObjectives: To certify NPRL2 protein can be transferred into human renal cancer cell 786-O by CTP and has an effect on the biological characteristics of cells.Methods:1.The pET15b-CTP-NPRL2,pET15b-NPRL2 were constructed and respectively transformed into competence Escherichia coli BL21(DE3),and then the expression of proteins was induced by IPTG and identified by Western blot.2.The proteins CTP-NPRL2 and NPRL2 were marked with the use of FITC,and then their localization in 786-O was observed by Laser Scanning Confocal Microscope,also transduction efficiency was detected by FACS.3.CTP-NPRL2 group,NPRL2 group and blank control group were established according to different protein acted on 786-O.Cell proliferation was detected by CCK-8 method.Cell cycle and apoptosis were detected by FACS.Cell invasion and migration ability were detected by Transwell method.Results:1.Recombinant plasmids pET15b-CTP-NPRL2 and pET15b-NPRL2 expressed objective protein after being transformed into Escherichia coli BL21(DE3)which was identified by Western blot.2.Green fluorescence appeared in cells of CTP-NPRL2 group but not NPRL2 group and blank control group,also significantly located in cytoplasm.The result of FACS showed that fusion protein CTP-NPRL2 transferred into renal cancer cell 786-O at a high efficiency around 75%,but NPRL2 protein hardly entered into cells.3.Cell proliferation was weaker(P<0.05),cell invasion and migration ability were significantly suppressed(all P<0.01),cell apoptosis rate was higher(P<0.01)and the percentage of G0/G1 phase cell was larger(P<0.05)in CTP-NPRL2 group compared with NPRL2 group or blank control group.And in CTP-NPRL2 group,the function of protein CTP-NPRL2 enhanced along with higher concentration.Between NPRL2 group and blank control group,there was no significant difference in all kinds of results above(all P>0.05).Conclusion: Protein NPRL2 mediated by CTP efficiently enters into renal cancer cell line 786-O and has a significant tumor suppression function on cells,also the function enhances along with higher concentration of CTP-NPRL2 fusion protein.Part Two THE CURATIVE EFFECT AND MOLECULAR MECHANISM OF BIFIDOBACTERIUM-MEDIATED CTP-NPRL2 FOR TREATING RENAL CANCER OF NUDE MICEObjectives: To discuss the curative effect of Bifidobacteriummediated CTP-NPRL2 for treating renal cancer of nude mice,and then explore its molecular mechanism using the strategy of comprehensive analysis of gene chip and protein spectrum,finally uncover the action principle of this therapeutic method from the overall level and screen out key target gene.Methods:1.Mouse models of renal cancer were constructed using subcutaneous injection of 786-O cell suspension and identified by pathological section.2.Bifidobacterium Infantis was cultivated by anaerobic method,and then recombinant plasmid pET15b-CTP-NPRL2,pET15b-NPRL2 and blank plasmid were transformed into competent Bifidobacterium by electroporation,finally the expression of target proteins were identified by Western blot.3.Forty mouse models of renal cancer were randomly and equally divided into five groups according to different treatment factors: Bifidobacterium+pET15b-CTP-NPRL2 group,Bifidobacterium+pET15bNPRL2 group,Bifidobacterium+pET15b group,Bifidobacterium group and normal saline group.All mouse models of renal cancer were injected bacteria liquids or normal saline via caudal vein once a week for four weeks.4.Nude mice weight,tumors weight and double kidneys weight were detected for the assessment of curative effect.The apoptosis of tumors were detected by TUNEL method.5.Differentially expressed genes and proteins of renal cancer tissue on nude mice after the treatment of Bifidobacterium-mediated CTP-NPRL2 were sought by gene chip and protein spectrum detection,and then the results were used for bioinformatics analysis,such as GO,Pathway,Pathway-network.The molecular mechanism of Bifidobacterium-mediated CTP-NPRL2 for treating renal cancer was deduced after reading many papers on the basis of the results of gene ships and protein spectrum.6.The targeted genes which were closely related to Bifidobacteriummediated CTP-NPRL2 for treating renal cancer were screened out,and then proved by Real time-PCR and Western blot.7.Representative targeted gene was selected for constructing three siRNAs and a siRNA with no meaning which was used as negative control,and then the best one was selected by Real time-PCR.The recombinant plasmid was constructed for the gene.8.Five groups were divided for exploring the effect of the representtatively targeted gene on biological characteristics of human renal cancer cells 786-O: siRNA group,siRNA with on meaning group,blank control group,recombinant plasmid group and blank plasmid group.The expression of gene was detected by Real time-PCR and Western Blot.Cell proliferation was detected by CCK-8 method.Cell cycle and apoptosis were detected by FACS.Results:1.The tumors,which was identified as renal cell carcinoma by pathological section,grown out under skin of nude mice.2.The Bifidobacterium grown well after the electroporation of pET15b-CTP-NPRL2,pET15b-NPRL2 and blank plasmid,and then the target protein expressed and identified by Western blot.3.In Bifidobacterium+pET15b-CTP-NPRL2 group,weight of nude mice was improved,also the weight of tumors was lighten,and there were significant difference compared with each one of other groups(all P<0.01),but not among other groups(all P>0.05).There was no significant difference in the weight of double kidneys between any two of the groups(all P>0.05).The apoptosis rate renal cancer tissue in Bifidobacterium+pET15b-CTP-NPRL2 group(26.8±4.15)% was higher than in normal saline group((11.6±3.58)%,P<0.01).4.Gene ships screened out 565 genes which had a differential expression no less than double between Bifidobacterium+pET15b-CTPNPRL2 group and normal saline group.Among these genes,313 with an up-regulation expression and 252 with a down-regulation expression appeared in Bifidobacterium+pET15b-CTP-NPRL2 group.The results of GO analysis showed terms which had a significant enrichment of genes and were related to tumors,such as cell proliferation,cell migration,cell movement,regulation of cell proliferation,regulation of cell migration and so on.The terms showed by Pathway analysis and related to tumors were organization of extracellular matrix,early stage of G0/G1,adherence among cell surface mediated by integrin,cell cycle and so on.The results of Pathway-network analysis showed terms,which were in central position,were MAPK signal pathway,PI3K-Akt signal pathway and cell cycle.5.Protein spectrum screened out 560 proteins which had a differential expression no less than 1.5 times between Bifidobacterium+pET15b-CTPNPRL2 group and normal saline group.Among these proteins,79 with an up-regulation expression and 481 with a down-regulation expression appeared in Bifidobacterium+pET15b-CTP-NPRL2 group.The results of GO analysis showed terms which had a significant enrichment of proteins and were related to tumors,such as extracellular vesicle,Rac protein signal transduction,translational initiation,extracellular matrix component and so on.The terms showed by Pathway analysis and related to tumors were Beta1 integrin cell surface interactions,Ras pathway,C-MYB transcription factor network,collagen degradation and so on.The results of Pathway-network analysis showed terms,which were in central position,were MAPK signal pathway and PI3K-Akt signal pathway.6.ANXA1,PRMT1,TIAL1 and ANGPTL2 were four genes which were closely related to the genesis and development of tumors and screened out.The result of Realtime-PCR showed the expression of ANXA1 increased 1.86 times in the level of mRNA in Bifidobacterium+pET15bCTP-NPRL2 group compared with normal saline group,also the expression of TIAL1,PRMT1 and ANGPTL2 decreased 1.77,2.24 and 19.56 times,respectively.The result of Western blot was consistent with the result of Real time-PCR.7.ANGPTL2 was the representative gene which was closely related to tumors and had a differential expression around 20 times between Bifidobacterium+pET15b-CTP-NPRL2 group and normal saline group.The result of CCK-8 showed up-regulating the expression of ANGPTL2 can increase the cell proliferation of 786-O,but down-regulating its expression decreased the cell proliferation.8.The results of FACS showed cell apoptosis rate of groups were(10.06±0.27)%,(2.93±0.30)%,(2.99±0.11)%,(1.98±0.11)% and(3.13± 0.08)%,respectively.There was significant difference in cell apoptosis rate in recombinant plasmid group compared with each one of other groups(all P<0.01),and the same to siRNA group.However,there was no difference between any two group of siRNA with on meaning group,blank control group and blank plasmid group(all P>0.05).The percentage of G0/G1 cells in groups were(69.32±2.45)%,(54.89±0.48)%,(54.22±2.7)%,(46.57± 0.13)% and(56.05±4.29)%,respectively.There was significant difference in percentage of G0/G1 cells in recombinant plasmid group compared with each one of other groups(all P<0.01),and the same to siRNA group,but no difference between any two group of other three groups(all P>0.05).Conclusion:1.Bifidobacterium-CTP-NPRL2 can inhibit the growth of renal cancer of nude mice,increase the apoptosis of renal cancer tissue and improve the weight of nude mice.2.Bifidobacterium-CTP-NPRL2 regulates some genes containing ANXA1,PRMT1,TIAL1 and ANGPTL2 which are closely related to the genesis and development of tumors.These genes involve oncogene,cancer suppressor gene,proliferation and apoptosis related gene,cell cycle related gene and angiogenesis gene and so on.3.ANGPTL2 has the function of oncogene which can accelerate the proliferation of renal cancer cell 786-O and decrease apoptosis after up-regulating its expression,and slow the proliferation of 786-O and increase apoptosis after down-regulating its expression.At the same time,down-regulating the expression of ANGPTL2 can inhibit the cell cycle of 786-O at G0/G1 phase,and on the contrary,the opposite result appears.4.In the genes which are closely related to the genesis,development of tumors and regulated by Bifidobacterium-CTP-NPRL2,the downregulation of the expression of ANGPTL2 may play an important role.5.Bifidobacterium-CTP-NPRL2 may play inhibition on renal cancer of nude mice mainly through MAPK and PI3K-Akt signal pathway.