Rna Interference Technology For Reporter Gene In Mammalian Cells, Inhibition Of Telomerase-associated Gene Expression Study

In post-genome era, the primary task we now confront is systematically to analysis the functions of those newly separated or validated genes so as to make clear the spatio-temporal expression of genes, regulation mechnisam and biological roles that coding proteins exert in special physical or pathological condition. Therefore, it is extraordinarily necessary to establish or empolder newly technique platform for the studies of gene functions.RNA interference is a study tool that can specifcially inhibit the expression of genes. RNAi is the post-transcriptional gene silencing (PTGS) mechanism whereby the introduced dsRNA directs the specific degradation of cognate mRNA. For the mechanism, It is now generally accepted that the long double-stranded RNA(dsRNA) is primarily processed to active small interfering RNAs (siRNAs) in vivo through the action of RNase Ill-like protein Dicer, the resulting 21- to 23-nt siRNA are subsequently utilised by the RNA-induced silencing complex (RISC), the active RISC couples with cognate mRNA transcripts through accurate base pairing, and finally capable of inducing the degradation of homologous mRNA.RNAi technology has acquired obvious developments these years. It can be notable that the ever-increasing publications that display RNAi data have no sufficient controls despite the apparent ease of RNAi, such as a mismatchor scrambled siRNAs control, a basic control to show reduction of expression at the mRNA and protein level, a quantitative control to denote the inhibitory effect at mRNA and protein level, as well as a multiplicity control to demonstrate a similar effect with two or more siRNAs targeted sites.In addition, for the application studies of RNAi, it is rather important to ascertain the most optimal time, the most appropriate concentration of siRNA or shRNA functioned, as well as the suited dosage to largely decrease the side effect resulted from RNAi. However, in cultured mammalian lines, studies on the time- and dose- effect have not be addressed as yet.We selected a destabilized vector pdlEGFP with a half-life of approximately one hour, a pGL3-control vector that expresses luciferase and pDsRed2, a prokaryotic expression vector that encodes red fluorescent protein in transfected cell lines as target vectors, and constructed a series of short hairpin RNA(shRNA) expression vectors by using pAVU6+27 as expressing cassette. The expression vectors carrying reporter component were cotransfected with the plasmids coding short hairpin RNAs(shRNAs) into mammalian cell by setting a series of experimental controls, the cells were harvested at scheduled time after transfection, and the consequent inhibitive effect mediated by RNA interference was estimated using RT-PCR and western blot analysis.These results showed as following:1) We successfully constructed a series of pAV-shRNA expression vectors, including one construct targeting c-myc, RFP, Dicer and luciferase gene respectively, two 1-2 mismatched constructs targeting GFP, two single-stranded construct targeting GFP gene( sense only, antisense only), three sequence-differed constructs targeting GFP.2) Optimization of transfection condition, detction of transfection efficiency : the appropriate proportion of DNA/LF2000 value was obtainedat 1:2.5 in transiently transfected HEK293H cell, while in HeLa cells this proportion was altered to 1:3. Under optimized transfection described as above, the average transfection efficiency was arrived at 91.6% in HEK293H cell, and 82.6% in HeLa cell by in situ β -galactosidase staining.3) The effect of RNAi mediated by transient expression of shRNA vectors: the expression of EGFP and RFP was obviously inhibited by transient cotransfections of psh-d1EGFP and pd1-EGFP, psh-PFP and pDs-Red2 respectively; the expression of RFP was not affected by cotransfections of psh-d1EGFP and pDsRed2, psh-luciferase and pDsRed2 respectively; the expression of EGFP was not affected by cotransfections of psh-RFP and pd1EGFP, psh-luciferase and pd1EGFP respectively.4) The effect of RNAi mediated by three sequence-differed constructs targeting EGFP: the expression of EGFP, both at mRNA or protein level, was obviously decreased by cotransfecting either of three sequence-differed constructs targeting EGFP with corresponding pd1EGFP target; the inhibitory effect caused by cotransfections of two sequence-differed constructs was lower than either of constructs functioned alone.5) The effect of RNAi mediated by single-stranded only, base-mismatched shRNA expression vectors: the expression of EGFP, neither at mRNA or protein level, was decreased by cotransfecting of single-stranded only or 1-2 base-mismatched constructs targeting EGFP in HEK293H cell, while the expression of EGFP could be obviously inhibited by cotransfection of positive construct psh-EGFP3 and pd1EGFP target.6) The time-dependent effect midated by psh-d1EGFP: the mRNA expression of EGFP was decreased slightly after 12 hours from initial contransfection of pd1EGFP and psh-d1EGFP, but the inhibiton of the expression of EGFP, either at mRNA or protein level, became obvious at 24 hours following initial transfection. The numbers of cells that expressedEGFP as well as the corresponding fluorescence intensities decreased obviously during from 48 to 72 hours. After 96 hours' transfection, the expression of EGFP basically got back compared with that at previous time described as above, and the fluorescence intensities also enhanced significantly than that of 72 hours from initiative transfection.7) The dose-dependent effect mediated by psh-d1EGFP: the expression of EGFP was slightly inhibited by cotransfection of a lower dosage of psh-d1EGFP denoting at 0.2μg and pd1-EGFP. The inhibitory effect mediated by psh-EGFP increased gradually accompanied with an increase of the dosage of psh-d1EGFP, the inhibitory efficiency reached 82.5% at mRNA level and 73.8% at protein level respectively with a dosage of psh-d1EGFP denoting at 1.6/zg, but the inhibitory effect of RNAi took on similar"platform effect" subsequently.8) Time- and dose- dependent RNAi effect mediated by psh-pGL3: HEK293H cell was cotransfected with psh-pGL3 and pGL3-control and lysed for luciferase activity analysis subsequently, the inhibitory efficiency of RNAi reached 41.0%, 79.2%, 82.0%, 60.0%, 40% at 24, 48, 60, 72, 96 hours following initial transfection respectively. However, for HeLa cell the inhibitory efficiency reached 36.2%, 70.0%, 72.3%, 56%, 42.7% respectively. In addition, a series of proportional dosage of psh-pGL3 and pGL3-control were cotransfected into HEK293H or HeLa cells for 48 hours, the results showed that a dosage of 0.2μg psh-pGL3 could inhibit the expression of luciferase slightly. However, with a increase of the dosage of psh-pGL3, the inhitory efficiency elevated gradually. The inhibitory efficiency reached 68.4% (for HeLa) and 78.2%(for HEK293H) respectively with a dosage of psh-pGL3 denoting at 1.6μg, but the inhibitory effect of RNAi took on similar "platform effect" subsequently.9) The effect of RNAi in HEK293H cell stably expressing EGFP: thefluorescence intensities in HEK293H stably expressing EGFP cells weakened at a certain extent after 24 hours' transfection of psh-dlEGFP3, subsequently the inhibition of EGFP became more and more obvious during from 48 hours to 72 hours, and the numbers of cells that expressed fluorescence decreased distinctly. The fluorescence of EGFP began to gradually get back at 96 hours from initiative transfection, and basically got back to the initial level at 144 hours. Similarly, a dose-dependent effect was also observed by transfection of a series of dose-proportional psh-d1EGFP into HEK293H cells stably expressing EGFP.10) The time- and dose- dependent effect of RNAi by inhibition of endogenous c-myc gene: the expression of c-myc in HeLa cell, either at mRNA or protein level, reduced slightly after 24 hours from initial transfection, the expression of c-myc was further decreased from 24 hours to 48 hours. The inhibition of c-myc mediated by psh-c-myc reached the maximal degree, while restored gradually from 72 hours. We transfected a series dose-proportional psh-c-myc into HeLa cells, the results indicated that the inhibitory efficiency was related to the dosage of psh-c-myc within a limited range. Similarly, the effect of RNAi took on "platform effect" subsequently.11) The effect of RNAi targeting Dicer gene: the green or red fluorescent intensities decreased obviously when we cotransfected with pd1EGFP and psh-d1EGFP, pDsRed2 and psh-RFP respectively after 48 hours. On the contrary, the green or red fluorescent intensities remained unchanged when cotransfections of pd1EGFP, psh-d1EGFP and psh-Dicer, or pDsRed2, psh-RFP and psh-Dicer respectively.These results conformed to the conclusions verifed from RT-PCR and western blot analysis.Taken together, it concluded that pAVU6+27 was a highly effective short hairpin RNA expression vectors, the short hairpin RNAs transcribedunder the control of U6 promoter could obviously inhibit the expression of corresponding genes in mammalian cells, characterized by highly efficiency and specificity; Taget-sited effect was observed in the process of RNAi, i.e. various sequences of siRNA on coding region of specific genes could mediate different inhibitory effect of RNAi, this result indicated that the inhibitory efficiency of RNAi was not only pertinent to the combinative abilities between RNA and target mRNA, but also the sequence of combinative target itself; Either enhanced or lessened effect of RNAi was not observed when cotransfecting with two different expressing vectors, which indicated that vector-based RNAi took on a "threshold effect"; In transfected mammalian cells, shRNA expression vectors could obviously inhibit the the expression of exogenous reporters and endogenous genes, the effect of RNA interference underwent a tendency of from weak to strong, then from strong to weak, and ultimately disappeared gradually, exhibiting an obvious time-dependent effect; The efficiency of inhibition caused by RNAi was related to the dosage of RNA interfering vector within a confined limit, whereas it nearly kept constant when the dose of interfering plasmid was sufficient to depress the expression of extraneous genes, exhibiting an obvious dose-dependent effect; The gene of Dicer enzyme is a critical component in the pathway of RNAi mediated by shRNA, the normal process of RNAi will be blocked owning of the inhibition of Dicer gene itself; Effective experimental controls are necessary for the studies of RNAi for they could quantitatively estimate the efficiency of RNAi. Besides, it also played important role in specificity validation of RNAi, distinguishing RNAi from microRNA pathway.