Effects Of SiRNA-STAT3-mediated Gene Silencing On The Proliferation And Apoptosis Of Fibroblast-like Synoviocytes In Patients With Rheumatoid Arthritis

ObjectiveTo explore the effects of siRNA-STAT3-mediated gene silencing on theproliferation and apoptosis of fibroblast-like synoviocytes (FLS) in patients withrheumatoid arthritis(RA).In order to provide the experimental basis for furtherclarifying the pathogenesis of RA，and provide a theoretical basis for the RAtarget therapy.Methods1、The source and the groups of synoviocytesThe rheumatoid arthritis fibroblast like synoviocytes (bought fromGuangzhou Ji Nelle biological Co. Ltd.) were divided into5groups: the controlgroup (group FLS, normal cultured), the negative control group (FLS group withblank plasmid), the RNAi-1group, the RNAi-2group and the RNAi-3group, thelast3groups are added in different small interfering RNA targeting STAT3recombinant plasmids, for3different targets respectively.2、Select the best targetThe plasmid was transfected into synoviocytes by Lifectmaine-2000. Thetransfection efficiency was observed under fluorescence microscope, after thetransfection of24h,48h,72h respectively. The RT-PCR and western blot methods were used to detect the expression levels of both STAT3mRNA and itsprotein. And from the three RNAi groups selecting the best target group as RNAigroup, the subsequent experiments were divided into three groups, namely theblank group, the negative control group and the RNAi group.3、Detection of synoviocytes proliferation activityThe proliferation of RA-FLS was examined by MTT. The optical absorbance(A) value at490nm representing the number of live cells. We recorded the Avalues at24h,48h,72h,96h,120h drawing the growth curve, to observe cellproliferation activity.4、Detection of apoptosis of synoviocytesAfter the transfection of48h, the synoviocytes were stained by AV/PI, thentheir apoptosis were detected by flow cytometry (FCM).5、Expression of apoptosis related genesThe protein expression of Bcl-xl, Bax and caspase-3were examined byWestern blot, in order to further clarifying the mechanism about the proliferationand apoptosis of synoviocytes after the RNA interference silencing of STAT3.Results1、Selection of the best targetThe expression of both STAT3mRNA and its protein inhibit rate of the blankgroup, the negative control group, the RNAi-1group,the RNAi-2group and theRNAi-3group were (27.32±2.06)%and (44.35±2.04)%,(30.61±2.47)%and(43.10±2.59)%,(76.99±2.05)%and (83.57±1.56)%,(72.75±1.74)%and(77.05±0.54)%,(66.50±2.47)%and (69.44±2.76)%, respectively, showingstatistically significant differences (F=362.92,248.08;P<0.01).There was nostatistically significant difference between the blank group and the negativecontrol group(P﹥0.05). Compared with the blank group and negative controlgroup, there were statistically significant difference in the RNAi-1group, theRNAi-2group and the RNAi-3group (P<0.05). And we can see that the RNAi-1 group both on the expression of STAT3mRNA and protein and the rate ofinhibition is the highest by the results, so this group is as RNAi group forsubsequent experiments.2、The results of MTTThe absorbance at490nm of the blank group，the negative control groupand the RNAi group (RNAi-1group) were (0.5533±0.3432),(0.5350±0.3373)and (0.1583±0.0794), respectively, showing statistically significantdifferences(F=3.761,P=0.047).And there was no statistical significance betweenthe blank group and the negative control group(P﹥0.05).Compared with theblank group and the negative control group, there was statistically significantdifference in RNAi group (P <0.05).3、The results of FCMThere was no statistically significant difference between the blank groupand the negative control group (P>0.05).Compared with the blank group andnegative control group, there were statistically significant difference in the RNAigroup (P<0.05).The apoptosis rates of the blank group, negative control groupand the RNAi group were (2.62±0.05)%,(2.95±0.03)%,(15.89±0.39)%respectively.4、Expression of apoptosis related factors detected by Western blotThe protein expression rate of Bcl-xl, Bax, caspase-3in the blank group,negative control group and RNAi group were respectively:(49.97±0.56)%(47.64±0.89)%(22.41±1.11)%;(50.28±1.74)%(48.91±2.18)%(21.88±0.88)%and (21.16±0.69)%(74.03±1.15)%(37.78±0.92)%. The three apoptosis relatedfactors expression rates: there was no significant difference between the controlgroup and the negative control group,(P=0.748,0.343,0.531, P>0.05), andcompared with the blank group and negative control group, there werestatistically significant difference in the RNAi group (P <0.01). Conclusion1、Expression of STAT3mRNA and protein can be inhibited effectively ofRA-FLS which were transfected by the target STAT3recombinant plasmid.2、 RNA interference silencing STAT3can significantly inhibit theproliferation of FLS, promote the apoptosis, and its mechanism may be throughraising the expression of apoptosis promoting gene Bax, and inhibiting theexpression of anti apoptosis gene Bcl-xl.