An Experimental Study On The Inhibitory Effect Of SiRNA On The Expression Of PPARγ In Marrow Stromal Cells Of Rabbit

ObjectiveThe mobility of alcohol-induced osteonecrosis of the femoral head (ONFH) isnow increasingly high. The patients suffering from alcohol-induced ONFH usuallyreceive conservative treatment in the early stage and total hip arthroplasty in theadvanced stage. However, the former has less effect and the latter has somecomplications.The purpose of this study was to observe the effect of the technology of RNAinterference (RNAi) on the treatment of alcohol-induced ONFH and to explore thepossibility of employing RNAi technology upon the treatment of alcohol-inducedONFH.Recent studies indicate that alcohol in vitro may enhance the expression ofperoxisome proliferator-activated receptor-γ(PPARγ) in MSCs,induce thedifferentiation of MSCs into adipocytes and ultimately reduce osteogenicdifferentiation.This process just like the pathological changes such as adipocytesincreasing,osteoporosis in the femoral head in the case of alcoholism in vivo.The PPARγis an adipogenic transcription factor,which regulates thedifferentiation of pre-adipocytes into adipocytes.While the PPARγdoesn't express inthe pre-adipocytes.It expresses dramatically in the differentiation of adipocytes, priorto the expression of the other adipogenesis mRNAs.The differentiation of pre-adipocytes into adipocytes can not accomplish without PPARγ.Therefore,PPARγplays a key role in the differentiation of pre-adipocytes and adipogenesis.Theexpression of PPARγin MSCs exists in the differentiation of pre-adipocytes intoadipocytes.Therefore we can assume that inhibition of PPARRγexpression in MSCscould help preventing MSCs from differentiating into adipocytes,making it possiblethat MSCs convert into osteoblast which can produce new bone.So the geneticpathogenesis of alcohol-induced ONFH could be answered.Today,as the technology of RNA interference (RNAi) maturing,it is possible toemploy it to suppress the alcohol-induced expression of PPARRγin MSCs andprevent alcohol-induced MSCs from differentiating into adipocytes,consequently tofacilitate osteogenic differentiation and promote the repair and reconstruction ofbone.This provides us with the etiotropic method of treating and curingalcohol-induced ONFH.This experiment observed the effect of the maturedtechnology of RNA interference (RNAi) on the inhibition of alcohol-inducedexpression of PPARRγin MSCs and the prevention of alcohol-induced MSCs fromdifferentiating into adipocytes,explored the possibility of prevention and treatment ofalcohol-induced ONFH with RNAi. The study included two parts.PART 1. Construction of the target siRNA vector for rabbit PPARγMethods:Firstly,the target sequence analysis design system of Takara,Ambion andpromega siRNA was employed to scan three specific target sequences from rabbitPPARγmRNA sequence(AF013266).Secondly,the autoploidy homology betweenAF013266 and other gene family was analyzed by BLAST, according to the principleof siRNA design.Three exons were taken as the siRNA target sequence of 19bases,and a control siRNA sequence was assorted randomly.Then,DNA single strandof encoded short hairpin RNA (shRNA) sequence was synthesized with the enzymecutting site of BamHI and XhoI being added to the two ends.Thirdly,the four pairs ofsynthesized hairpin DNA were renaturated separately and cloned into pGEM-Tvector,thus the pGEM-T-siPPARγ-1,pGEM-T-siPPARγ-2,pGEM-T-siPPARγ-3 and pGEM-T-C were obtained through PCR amplification and filtering,with T7/SP6 beingthe primer.The pGEM-T-siPPARγ-1,pGEM-T-siPPARγ-2,pGEM-T-siPPARγ-3,pGEM-T-C and the siRNA vector pRNAT-U6.2/Lenti were cut by BamHI and XhoI.After that,double sticking hairpin DNA fragments and filiated double sticking siRNAvector were recovered respectively.Then,the four double sticking hairpin DNAfragments were subcloned into siRNA vector pRNAT-U6.2.Then, by means ofBamHI/XhoI cutting,PCR filtering,and DNA sequence analysis, the pRNAT-U6.2/Lenti-siPPARγ-1, pRNAT-U6.2/Lenti-siPPARγ-2, pRNAT-U6.2/Lenti-siPPARγ-3 and pRNAT-U6.2/Lenti-siPPARγ-C of Plasmid type siRNA expressionvetor was obtained.The constructed siRNA expression vector and assistant pack vectorwere packed by bangosome Lipofectamine^TM 2000.Then the 293FT cells werecotransfected by the packed vector to produce the Lentivirus virus particles containingsiRNA expressions.Finally,these Lentivirus virus particles were purified and then thevirus titre was tested.Results1. The filtering of the target site of PPARγsiRNA:Three siRNA target sequencesfrom rabbit PPARγmRNA were obtained:GGCCTCCTTGATGAATAAA(677-695),GCAGGAGCAGAGCAAAGAA(497-515),andGGAAAGACG ACAGACAAAT (402-420).2. The cloning of hairpin DNA fragment:After PPARγand the renaturaed controlhairpin siRNA single DNA being connected with pGEM-T Easy,they weretransformed into JM109 and filtered by Blue and White filter method.Then 10colonies were chosen randomly and amplified by PCR with T7/SP6 being theprimer.Among all these colonies,positive clones containing 252bp amplificationfragments were obtained.3. The Subclone into siRNA vector:The products of ligase (pRNAT-U6.2/Lenti-siPPARγ-1, 2, 3 and pRNAT-U6.2/Lenti -siPPARγ-C) were transformed intoJM109 on the Amp⁺ LB plate.Each of them produced more than 10colonies.Then,we screened them by PCR and obtained 4 siRNA expressvectors.Ecombinant plasmid were extracted respectively and identified through BamHI/XhoI digesting. The result confirmed us that we obtainedpRNAT-U6.2/Lenti-siPPAR-1, 2, 3 and pRNAT-U6.2/Lenti-siPPARγ-Csuccessly and that the designed sequence was consistent with the insertedsequence of DNA.4. The packaging of Lentivirus virus expressing siRNA: The siRNA expressvectors and packaging plasmid cotransfected 293FT cell.The virus particleswhich expressed siRNA and whose tite were 2.1×10⁷ IU/ml -4.3×10⁷ IU/mlwere obtained.PART 2. Observe the inhibitory effect of siRNA on the expression of PPARγinMSCs induced by alcohol of RabbitMethods:MSCs were firstly obtained and nurtured from New Zealand Rabbit. MSCs ofsecond filial generation were randomly divided into 7 groups which had 6 specimen.In group N (Normal, N), the cells were treated neither with alcohol nor with thevector of pRNAT-U6.2/Lenti virus. In group M (Model, M), the cells were treatedwith 0.09 mol/L alcohol only. In group C0(Control 0, C0), the cells were treated with0.09 mol/L alcohol and packed Lentivirus virus of no-load vetor pRNAT-U6.2/Lenti.In group C1(Control 1, C1), the cells were treated with 0.09 mol/L alcohol andLentivirus virus packed with pRNAT- U6.2/Lenti-siPPARγ-C. In group S1 (siRNA1,S1), the cells were treated with 0.09 mol/L alcohol and packed with pRNAT-U6.2/Lenti-siPPARγ-1. In group S2 (siRNA2, S2), the cells were treated with 0.09mol/L alcohol and packed with pRNAT- U6.2/Lenti-siPPARγ-2.In group S3(siRNA3,S3),the cells were treated with 0.09 mol/L alcohol and packed pRNAT-U6.2/Lenti-siPPARγ-3. MSCs of second filial generation were planted and nurtured in6 wells of the plate or in the bottle of 50ml in demand. In each well or bottle wherethe packed virus were placed, it was as 10 times as the nurtured MSCs. MSCs infectedwith virus for 12 hours. Then complete high glucose DMEM medium was added intothe well or bottle. At the same time the final concentration of 0.09mol/L of alcoholwas added into the medium of the groups above which needed alcohol inducing. Thevery concentration of alcohol was added into the new medium everytime when it was replaced. The state and growing condition of the MSCs were observed through theinverted microscope. On the 3rd, 5th, 7th and 14th day of the first alcohol inducement,the expression of the PPARγmRNA and the amount of protein were determined usingthe method of fluorescent quantification Reverse Transcription PCR (RT-PCR) andthe method of Western-blot respectively. On the 14th day, the adipocytes induced bythe alcohol in the nurtured MSCs were stained with the SuDanâ…¢and counted undera microscope; the contents of triglyceride (TG) and alkaline phosphatase (ALP)activity in MSCs were determined by biochemical assay; the content of osteocalcin(OC) in the media were determined by radioimmunoassay. All data was representedwith mean±standard deviation (SD) and analyzed statistically with one-wayANOVA using the software of SPSS (Ver.12.0) (a=0.05).Results:1. Observation of MSCs infected by the packed virus. All the MSCs infected by thevirus in the group C1, S1, S2 and S3 gave off green fluorescence when they wereobserved under the fluorescent microscope. It showed a significant infectiouseffect of the packed virus on MSCs. However, no fluorescence was observed inthe group N and M.2. Fluorescent quantification RT-PCR: In group M, CO and C1, the expression levelof PPARγmRNA increased on the 3rd day and maintained a high level with aconstant alcohol inducement. The expression level of PPARγmRNA in the MSCsin group N remained constantly low during the 14 days of cell nurture.In group S1,S2 and S3, the expression of PPARγmRNA in MSCs was all suppressed to somedegree. The suppressive effect on group S1 was the strongest, on group S2 was theweakest. In addition, the suppressive effect for the above 3 groups decreased dayby day until the 14th day when the expression level of PPARγmRNA in MSCs ofgroup S1, S2 and S3 was lower than that in MSCs of group M, CO and C1. Thedifference had statistical significance (P＜0.05).3. Western-blot: On the 3rd, 5th, 7th and 14th day of alcohol inducement, theimmunoblotting showed that the blot of PPARγprotein in group N was milderthan that in group M, C0 and C1, but it was clearer than that in group S1, S2 and S3. It indicatedthat the expression of PPARγprotein in the MSCs was suppressedby the packed siRNA virus.4. The determination of the amount of adipocytes and the content of TG in MSCs:On the 14th day of alcohol inducement, group M, C0 and C1 had greater amountof adipocytes and TG than Group N. The difference had statistical significance(P＜0.05).Group S1, S2 and S3 had almost the same amount of adipocytes as andhad a bit higher TG than group N. The differences had no statistical significance(P＞0.05).5. The determination of OC in the media and the ALP activity in the MSCs: On the14th day of alcohol inducement, group M, C0 and C1 had lower OC and ALPactivity than Group N. The difference had statistical significance (P＜0.05). GroupS1, S2 and S3 had almost the same amount of OC in the media as and a bit lowerALP activity values than group N. The differences had no statistical significance(P＞0.05).Conclusions:1. This study successfully constructs three target eucaryon siRNA vectors for rabbitPPARγmRNA. By means of packaging in vitro, the siRNA virus particles containhigh infectious effects on MSCs.2. The siRNA virus particles can inhibit the expression and translation of PPARγmRNA when they transfect the MSCs. The active suppression time can last for atleast 2 weeks.3. The experiment in vitro confirms that the siRNA virus particles can inhibit thedifferentiation of MSCs into adipocytes induced by alcohol and enhance thedifferentiation of MSCs into osteoblasts through the siRNA interfering theexpression of the PPARγmRNA in MSCs.4. The study sets a foundation for the research on pathogenesis and geneticprevention and treatment of alcohol-induced ONFH.