| ObjectiveInterferons (IFNs) are the most commonly used drugs for the treatment ofmetastatic and advanced stage renal cell carcinoma (RCC). However, IFN resistancelimits its wide clinical application. Finding molecular markers that affect sensitivity tointerferon in RCC and analysizing the resistance mechanisms will build experimentalbasis for improving the effect of IFN in the treatment of RCC.Materials and methodsIn this experiment, ACHN cell line and786-0cell line of RCC were cultured invitro. The rate of inhibition were detected using Cell Counting Kit-8(CCK-8).Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot wereused for detection of SOCS3mRNA and protein expression in ACHN cell line and786-0cell line. According to the growth inhibition rate, identified drug resistant cellline. The transfection of miR-146a mimics and inhibitors was carried out beforeadding IFN-α (1000IU/ml) in IFN-α resistant cell line. Apoptosis of these cell sampleswere detected using flow cytometry (FCM). According to the experimental results ofour own and previous results reported in the literature, systems biology approach wasused. The Janus kinase/phosphorylated signal transducer and activator of transcription(JAK/STAT) pathways of cytokine we studied include IFN-α pathway, IL-6pathwayand IFN-α and IL-6crosstalk pathway. These models were derived from BioModelsDatabase. The software COPASI4.14(Build89) was used. Model files with standsystems biology markup language (SBML）were imported into software. Through theoperation of the software follows can be achieved: browsing and modifing the mainelements involved in the model, determining the biochemical reaction equation of each molecule, browsing and adjusting the reaction parameters, determining the timeand the output types, determining the parameters and their range to be scanned, getingthe targeted curves. Analysis of the influence of dynamic characteristics ofJAK/STAT pathway and the role of suppressor of cytokine signaling-3(SOCS3)during treatment of was carried out by COPASI.ResultsCCK-8testing results showed that in IFN-α (300IU/ml) group, ACHN cells wereinhibited untill48hours, while786-0cells were inhibited untill72hours. CCK-8results showed that in24h group, when ACHN cells were inhibited the concentrationof IFN-α required above500IU/ml, while in786-0cells, that of IFN-α was above1000IU/ml. After IFN-α was admitted48hours, CCK-8results showed that when therange of concentration of IFN-α was less than1000IU/ml, the inhibition rate ofACHN and786-0cells were increased with the increasing concentration ofIFN-α(P<0.05), while the concentration of IFN-α1000IU/ml,2000IU/ml and3000IU/ml would not affect the inhibition rate of ACHN and786-0cells. Inhibition ofACHN cells were increased gradually with prolonged exposing to IFN-α, but the786-0cells were not. when the concentration of IFN-α was300~3000IU/ml in thegroup of48h, the relative growth rate of786-0cells was higher than that of ACHNcells, there were significant differencese between the two groups (P<0.05). RT-PCRresults showed that0.5h and1.5h after stimulation with IFN-a, mRNA expressionlevels of SOCS3were increased in ACHN cells compared with control group(P<0.05), but there were no difference in mRNA expression levels of SOCS3at2.5h,4h and6h after stimulation with IFN-a compared with control group (P>0.05).12hafter stimulation with IFN-a, mRNA expression levels of SOCS3were decreased inACHN cells compared with control group (P<0.05).1.5h and4h after stimulationwith IFN-a, mRNA expression levels of SOCS3were increased in786-0cellscompared with control group (P<0.05).2.5h after stimulation with IFN-a, mRNAexpression levels of SOCS3were decreased in786-0cells compared with controlgroup (P<0.05). There were no difference in mRNA expression levels of SOCS3at6hand12h after stimulation with IFN-a compared with control group (P>0.05).48h afterstimulation with IFN-a, the production of SOCS3was significantly higher in786-0than in ACHN cells by Western blot analysis. Research results of FCM showed that: the apoptosis rate of miR-146a mimics transfected group was higher than that ofnegative control (NC) group and inhibitor group, the difference was statisticallysignificant (P<0.05), and apoptosis rate of miR-146a inhibitor group was lower thanthe transfection of NC group (P<0.05). We introduced a systems biological approachto further explore the interferon resistance mechanism in renal carcinoma cell linewith high expression of SOCS3. We simulated the change of concentration of SOCS1and SOCS3by crosstalk model and IL-6simulation model under differentconcentrations of IFN-α stimulation. Effect of the concentration of IL-6, interleukin-6receptor (IL-6R), phosphorylated signal transducer and activator of transcription3(p-STAT3), tyrosine phosphatase2(PP2), or SH2domain-containing tyrosinephosphatase2(SHP2) on the expression of SOCS3were simulated, and so the numberof sensitive IL-6R. The interaction of p-STAT3.and phosphorylated signal transducerand activator of transcription1(p-STAT1) level was also simulated. We tried toinhibit and knockout SOCS3,then simulated the change of the expression level ofIL-6R, p-STAT3and the influence on inflow and outflow of STAT dipolymers causedby.the interference.Conclusion1. IFN-α can inhibit the growth of ACHN and786-0cells, the effect of inhibitioncan be detected in ACHN cells in the lower interferon concentration and shorterexposing time, but in786-0cells higher concentration and longer exposing time isneed.2. Under the conditions that IFN-α concentration less than1000IU/ml and cellsexposed to it in48h, the effect on inhibition to ACHN and786-0cells was increasedwith the increasing concentration of IFN-α. Under the conditions that within48hoursafter exposing to IFN-α, inhibition on ACHN cells increased with prolonged IFN-αexposing time; while there was no significant correlation between the inhibition effecton786-0cells and IFN-α exposing time.3. ACHN cell line is more sensitive to IFN-α,786-0cell line is less sensitive toIFN-α.4. The overexpression of SOCS3could be related to the resistance of IFN-α inthe treatment of RCC. 5. miR-146a combined with IFN-α can enhance apoptosis of786-0cells.6. Dynamic analysis was carried out for the biological pathways of IFN-α andIL-6model. These dynamic plots quantitatively interpretated drug resistance in RCCcell line by increased expression of SOCS3after IFN-α stimulation. The mechanismsinclude changes of concentration of IL-6, IL-6R, p-STAT3, PP2, SHP2and thenumber of sensitive IL-6R result in increased expression of SOCS3, and also thechange of p-STAT3level can affect the p-STAT1level. We theoreticallydemonstrated the inhibition or knockdown of SOCS3expression by RNAi method canchange concentration of IL-6R, p-STAT3and impact on STAT dipolymers, andimprove the sensitivity of RCC cells to IFN-α. |
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