1.A Preliminary Study On The Molecular Mechanism Of Tetraspanin31 Regulating CDK4 2.Construction And Application Of Plasmid Type Conditional Autolytic Bacteria Based On MS2 Phage Gene Lys

Objective:Naturally antisense transcripts(NATs)are regulatory RNAs that complement their sense transcripts to form sense-antisense double stranded RNA(ds RNA).They regulate physiological and pathological processes through genomic imprinting,selective splicing and termination,and affecting m RNA stability.However,the biological functions and related mechanisms of NATs in tumor cells are still unclear.According to NATsDB databases,Tetraspanin31 and CDK4 are a pair of cis-NATs.Therefore,this study focuses on the effects of Tetraspanin31 on CDK4 and its related Cyclin D1 and downstream molecule E2F1 in liver cancer cells,and preliminarily studied the molecular mechanism of the regulation of CDK4 by Tetraspanin31.Method:The Tetraspanin31 gene and CDK4 gene were consulted through NCBI database and NATsDB database.We designed and synthesized si RNA for the non complementary regions of Tetraspanin31 and CDK4 and used the si RNA to interfere Tetraspanin31 specifically in hepatoma cells.Then we take advantage of q RT-PCR and Western blot technology to detect the expression levels of CDK4 at transcription and translation levels.Afterwards we take advantage of Western blot technology to detect the expression levels of Cyclin D1 and E2F1 at the translation level.In addition,we constructed over expression plasmids of the 3’untranslated region(3’UTR),the coding region(CDS)and the full length(Full Length,FL)of Tetraspanin31,and transfected into HEK293 T cells and Hela cells,so that preliminarily determined the regulatory region of Tetraspanin31 on its target genes CDK4.Result:According to the NCBI database,Tetraspanin31 and CDK4 are both located on human chromosome 12,and their m RNA have 517 bases in the 3’UTR region that can complement and pair.According to the NATsDB database,Tetraspanin31 and CDK4 are a pair of cis-NATs.In hepatoma cells,after si RNA interfere Tetraspanin31,the relative expression level of Tetraspanin31 protein was significantly reduced(P<0.05),the relative expression levels of CDK4 m RNA and protein were significantly increased(P<0.05),and the relative expression levels of Cyclin D1 and E2F1 protein were increased(P<0.05).In HEK293 T cells and Hela cells,overexpression of Tetraspanin31-3’UTR and overexpression of Tetraspanin31-FL significantly reduced the relative expression of its target genes CDK4 m RNA and protein(all P<0.05).On the contrary,there was no significant difference in the relative expression of its target genes CDK4 m RNA and protein after overexpressing Tetraspanin31-CDS(all P>0.05).Conclusion:Tetraspanin31 can regulate the expression of Cyclin D1,CDK4 and E2F1 proteins in hepatoma cells.The regulatory effect of Tetraspanin31 on its target genes CDK4 may be mainly through the 3’UTR of transcription products.Objective: The prokaryotic expression and purification of traditional exogenous protein need to go through the process of engineering strain culture,induced expression of exogenous protein,bacterial recovery,bacterial fragmentation and purification,etc.Among them,bacterial fragmentation is an indispensable step to obtain intracellular target protein,which needs ultrasonic breakage or enzymolysis.The process is complicated.Lys gene encoded protein in MS2 phage has the function of cracking host bacteria.This project aims to construct L-arabinose-induced plasmid type conditional autolytic bacteria based on Lys gene of MS2 phage,simplify the purification process of exogenous protein,and explore the effect of conditional autolytic bacteria on the expression of exogenous protein.Method: According to the codon preference of E.coli,we optimized Lys gene of MS2 phage,designed and synthesized Optilys gene.And we further constructed two expression vector of p-BAD-Optilys and p CDF-BAD-Optilys.In BL21(DE3)strain,we respectively transformed the plasmids of p-BAD-Lys,p-BAD-Optilys or p CDF-BAD-Optilys,and constructed three L-arabinose-induced plasmid type conditional autolytic bacteria.We tested its growth curve and colony forming units(CFU)to study its lysis rate.We transferred p ET28a-EGFP plasmid into BL21(DE3)which contains p CDF-BAD-Optilys expression vector,and make it express enhanced green fluorescent protein(EGFP).After the host was lysed by L-arabinose,the release of host intracellular EGFP into the medium was detected,and the EGFP in the medium was purified and identified.In addition,We transferred chitinase plasmid(R-46,independently constructed in our laboratory)or mitochondrial fusion protein 2(mitofusin 2,MFN2)plasmid into BL21(DE3)which contains p CDF-BAD-Optilys expression vector,and tested its effect on the soluble expression of the chitinase(R-46)or the mitochondrial fusion protein 2(MFN2).Result: Through the design and synthesis of Optilys gene by optimizing Lys gene codon,the p-BAD-Optilys and p CDF-BAD-Optilys expression vectors were constructed,and the autolytic bacteria BL21(DE3)containing p-BAD-Lys(Laboratory storage),p-BAD-Optilys or p CDF-BAD-Optilys expression vectors was further constructed.Compared with the control group,the growth curves and the plate colony forming unit(CFU)of p-BAD-Lys BL21(DE3),p-BAD-Optilys BL21(DE3)and p CDF-BAD-Optilys BL21(DE3)showed a significant downward trend after L-arabinose induction during the experimental period(P<0.05).The lysis rate of host bacteria can over 99.9%.Compared with Lys gene,the optimized Optilys gene lyses the host more significantly.However,there was no significant difference in host efficiency induced by different L-arabinose concentrations(0.2%,0.02% and 0.002%).Studies on the expression of EGFP in BL21(DE3)which contains p CDF-BAD-Optilys showed that the plasmid p ET28a-EGFP transferred into this host did not affect L-arabinose induced host lysis.At 1h,2h and 3h after L-arabinose induction,EGFP was released from the host cell into the medium,and the fluorescence intensity results showed that the release rates were 44.75%,59.42% and 63.47%,respectively.After purificating the EGFP of the medium by Ni-NTA,SDS-PAGE showed that a single protein band of about 30 k Da.It could be identified as EGFP of recombinant expression.In the study of protein soluble expression of chitinase(p ET28a-R46)or mitochondrial fusion protein 2(p ET28a-MFN2),SDS-PAGE and Western Blot results showed that L-arabinose induced lysis in BL21(DE3)which contains p CDF-BAD-Optilys could not improve the soluble expression of chitinase(R-46)or mitochondrial fusion protein 2(MFN2).Conclusion: BL21(DE3)containing p-BAD-Lys,p-BAD-Optilys or p CDF-BAD-Optilys expression vectors could effectively lyse the host bacteria under the induction of L-arabinose(final concentration of 0.2%),and the optimized Optilys gene had better effect.In addition,L-arabinose induced lysis of BL21(DE3)which contains p CDF-BAD-Optilys could release more than 63% of intracellular EGFP into the medium,and Metal ion chelation chromatography can purify EGFP directly from the medium and simplify the process of traditional target protein.However,the method is difficult to the purification of chitinase(R-46)and mitochondrial fusion protein 2(MFN2)which soluble protein has low expression.