Study On Telomerase And Related Signaling Pathway After MicroRNA-mediated NBS1 Gene Silencing

ObjiectiveNBS1 is an important gene of MRN complex(MRE11/RAD50/NBS1 complex), the most important participants of DNA double-strand break repair.In recent years some studies show that the MRN complex to maintain the survival of telomerasenegative tumor cells.NBS1 dysfunction will dissociate MRN complex leading to its functions loss,damage of homologous reorganization-mediated alternative lengthening mechanism of telomeres(ALT) in telomerase-negative cells,and suppression of its proliferation.Its overexpression can be induced transformation and tumorigenesis,and some closely related to tumor prognosis.The study of the relationship between NBS1 and mechanisms of telomerase has few.Recent studies have shown that the reduction of MRN resulted in a transient shortening of G-overhang length in telomerase-positive cells.They found that the reduction in overhang length was not seen in telomerase-negative cells,but was observed after the expression of exogenous telomerase,which suggested that the MRN complex might be involved in the recruitment or action of telomerase.On the study of the relationship between NBS1 and signaling pathway has present limited.The study on head and neck cancer showed that the expression level of NBS1 correlated with the phosphorylation levels of Akt,and repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Ratla cells overexpressing NBS1.Another think that NBS1 can function as an adaptor/activator of PI3-kinase through a novel activation motif.The studies of the relationship between NBS1 and ERK1/2 signaling pathway is not carried out. In this study using a novel tool and experimental tools of RNA interference research-microrna interference of gene expression,the first study of the important ingredients in MRN complex-NBS1 gene silencing,and the relations between it and telomerase activity of telomerase-positive tumor cells,PI3K/Akt and ERK1/2 signaling pathways.Methods1.Design,Synthesis and Construction of targeting NBS1 microrna gene sequenceTo select the 21 nt fragment of NBS1 gene mRNA sequence for suitable microRNA target sequence,the 64nt pre-microRNA oligonucleotide sequence targeting four different regions of NBS1 were designed,synthesized and builded into pcdna6.2-GW/emGFP-miR eukaryotic expression vector,were transformed into E. Coli DH5α.All recombinants were named NBS1mi-1,NBS1mi-2,NBS1 mi-3, NBS1mi-4.2.Identification of recombinantsTo culture recombinants on LB plate at first.The positive clone colony is picked from LB plate,performed on a colony PCR and agarose gel electrophoresis for a preliminary identification of recombinants.Positive clones strain in LB medium culured.The plasmid were extracted,purified,and carried out sequencing by company.3.Cell culture and transfectionTo choice Hela cells of confluent of above 70%,inoculate in the culture plate. The cell above 80%of growth density were transfected.Experimental group is null-vector control,negative control(normal cells),NBS1mi-1,NBS1mi-2,NBS1mi-3, NBS1mi-4.Each have three parallel hole.4.Detection of transfection efficiencyTo collect the cells 12 h after transfection,determine transfection efficiency with Real-Time PCR of GFP reporter gene.5.Detection of NBS1 mRNA expression levelTo collect the cells 12h after transfection,determine NBS1 mRNA level with Real-Time PCR.6.Western blot analysis of NBS1 gene protein expression levelThe cells 24 h after transfection were collected,detected NBS1 gene protein expression level of NBS1mi-1,NBS1mi-2,NBS1mi-3,NBS1mi-4 recombinant group by Western blot.According to their NBS1 mRNA and protein expression results,to select the best one of NBS1 interference effects in four microRNA eukaryotic expression recombinants vector for the next experiment.7.Detection of telomerase activity with TRAP-EB-PCRTo explore the relationship of microRNA-mediated NBS1gene silencing and telomerase activity in Hela cell.8,MTT assayTo explore the relationship of microRNA-mediated NBS1gene silencing and proliferation in Hela cell.9,Detection of Akt,ERK1/2 phosphorylation and protein expression levels with Western blotTo explore the relationship of microRNA-mediated NBS1gene silencing and PI3K/Akt,ERK1/2 signaling pathway.10.Statistics AnalysisThe experimental result is express to(?)±s,statistics software SPSS 13.0 was used to T-test,P＜0.05 is express to difference have statistics significance.Results1.The design of four microRNA sequences targeting NBS1mRNA Their non-specific Homology were ruled out through NCBI Blast,and can have complementary with cohesive end of pcDNA6.2-GW/EmGFPmiR eukaryotic expression vector.2.Identification results of recombinantsThe amplified fragments with colony PCR and agarose gel electrophoresis were above 250 bp,were consistent with the expected results.Sequencing analysis shows that the presence of insert fragment in recombinants,and its integrity,as well as loss mutations of base.3.Transfection efficiencyReal-Time PCR analysis showed that transfection efficiency of negative control group,null vector control group,NBS1mi-1,NBS1mi-2,NBS1mi-3,NBS1mi-4 respectively were(57.26±1.04)%.(55.41±3.68)%.(45.51±2.62)%.(47.55±1.35)%. (50.23±3.27)%.(43.56±2.56)%12h after transfection.The difference of transfection efficiency was not significant(P＞0.05) by way of null vector control group,NBS1mi-1, NBS1mi-2,NBS1mi-3,NBS1mi-4 compared with negative control group by independent samples T-test analysis,also no significant difference(P＞0.05) through the comparison between them.This suggested that amounts of GFP mRNA amplification cycles and initial template between every group was not notable different.Therefore there is not significant distinction about transfection efficiency.4.The changes of NBS1 mRNA expression levelThe mRNA expression levels of NBS1gene respectively were 0.86±0.16,0.78±0.16,0.24±0.17,0.12±0.12,0.41±0.97,0.48±0.93 in negative control group, null vector control group,NBS1mi-1,NBS1mi-2,NBS1mi-3,NBS1mi-4 12 h after transfection.The NBS1 mRNA expression levels of NBS1mi-1,NBS1mi-2,NBS1mi-3 and NBS1mi-4 compared with the negative control group and null vector control group were significant differences(P＜0.05).The NBS1 mRNA expression level of null vector control group compared with negative control group was no significant difference(P＞0.05).The NBS1 mRNA expression level of NBS1mi-2 recombinant group among NBS1mi-1,NBS1mi-2,NBS1mi-3,four NBS1mi-4 recombinant groupwas the lowest.5.The changes of NBS1 protein expression levelCompared with the negative control group NBS1 gene expression levels in NBS1mi-1,NBS1mi-2,NBS1mi-3,NBS1mi-4 were decreased,which of NBS1mi-2 was the lowest.,and the difference between null vector control group and negative control group was not significantly,combined together NBS1 mRNA and protein expression results we determined that NBS1mi-2 of the four recombinants has the best gene silencing effect targeting NBS1.6.The changes of telomerase activity in Hela cellsAccording NBS1 gene mRNA and protein expression results,NBS1mi-2 possesing the best interferencere effect was choiced and detected telomerase activity. The telomerase activity of negative control group compared with NBS1mi-2 decreased significantly(P＜0.05) 24 h,48h,72h after transfection..The telomerase activity of Hela cells had been gradually declining following transfected time of NBS1mi-2,and which was significant(P＜0.01) 72h after transfection.The difference between null vector control group and negative control group was not significant.7.Cell growth curveHela cell growth curve after transfection of NBS1mi-2 1 group compared with the negative group and the null vector was slowed down.The proliferation of Hela cell was inhibited.8.Changes of phosphorylation Akt levelThe phosphorylation Akt protein expression levels of NBS1mi-2 compared with the negative control group were significantly lower(P＜0.01),and which of the null vector control group was not significant changes(P＞0.05),and the difference between them was significant. 9.Changes of phosphorylation ERK1/2 levelThe phosphorylation ERK1/2 protein expression levels of NBS1mi-2 compared with the negative control group were significantly lower(P＜0.01),and which of the null vector control group was not significant changes(P＞0.05),and the difference between them was significant.Conclusion1.The designed pre-microRNA fragments of target mRNA were inserted correctly into the pcDNA6.2-GW/EmGFPmiR eukaryotic expression vector.MicroRNA eukaryotic expression vector targeting NBS1 gene was constructed successfully.2.The transfection efficiency of every experimental group is not the same,but there is no significant difference between each other.This eliminated the error of indicators with the transfection efficiency of every groups.Thus,microRNA eukaryotic expression vector targeting NBS1 gene was transfected successfully.This laid the foundation for the next experiment.3.NBS1 gene silencing can lead to the inhibition of telomerase activity in telomerase positive-Hela cells.4.NBS1 gene silencing can lead to that the proliferation of Hela cell was inhibited.5.The expression of phosphorylation Akt protein and ERK1/2 will be depressed after NBS1 gene silencing by microRNA.6.On the basis of above conclusion,we propose that:NBS1 gene silencing by microRNA interference can lead to the decrease of telomerase activity and inhibition of cell proliferation through PI3K/Akt pathway and ERK1/2 pathway.NBS1 gene may become a novel gene therapy target of telomerase-positive tumors.