SOCS3与干扰素-a治疗肾细胞癌敏感性的实验及系统生物学研究
发布时间：2018-09-04 19:51
【摘要】：目的： 干扰素（interferon，IFN）是治疗转移性及进展期肾细胞癌（Renal cellcarcinoma，RCC）最常用的免疫治疗药物。然而由于IFN的耐药性，限制了其在临床的广泛应用。寻找影响IFN敏感性的分子标志物，系统及量化的分析其作用机制，为提高IFN治疗RCC的疗效提供实验和理论依据。 材料和方法： 本实验中我们对RCC的ACHN细胞株和786-0细胞株进行体外培养，采用CCK-8（Cell Counting Kit-8）法检测干扰素α（interferon-α，IFN-α）对两种RCC细胞的抑制率，采用实时定量PCR（real-time PCR，RT-PCR）及Western Blot方法检测细胞因子信号传导抑制因子-3（suppressor of cytokine signaling-3，SOCS3）在ACHN细胞株和786-0细胞株的转录及表达情况。根据生长抑制率的不同，识别IFN-α耐药细胞系。对耐药细胞系转染miR-146a模拟物、抑制物并加入IFN-α（1000IU/ml），流式细胞仪（flow cytometry，FCM）检测细胞凋亡。在此基础上，结合我们自己的实验结果及相关文献结果，引入系统生物学方法。我们研究的关于酪氨酸激酶/信号转导和转录激活子（Janus kinase/signaltransducer and activator of transcription，JAK/STAT）通路的细胞因子途径包括IFN-α途径、白介素6（interleukin-6，IL-6）途径及IFN-α与IL-6交互途径，相关模型主要来源于BioModels Database：分别为IL-6途径模型，IFN-γ途径模型和IFN-γ与IL-6途径交互模型。分析软件选择COPASI4.14(Build89)：我们把标准格式的系统生物学标记语言（systems biology markup language，SBML）模型文件载入软件。通过操作该软件可以实现：浏览和修改模型的主要分子，确定和修改每个分子的生化反应方程式，，制定反应函数关系，调整反应参数，确定模拟的时间及输出方式，确定要扫描的参数及其范围，得到目标结果曲线。应用该软件分析了RCC细胞在IFN-α作用下JAK/STAT途径相关分子的动力学特征及SOCS3对该通路的影响。 结果： CCK-8检测结果显示，当IFN-α浓度为300IU/ml时，ACHN细胞在48h检测到明显抑制作用时，而786-0细胞在72h检测到明显抑制作用。当IFN-α作用24h时，作用于ACHN细胞的IFN-α浓度需达到500IU/ml才能检测到明显抑制作用；而作用于786-0细胞的IFN-α浓度需达到1000IU/ml才能检测到明显抑制作用。检测时间点为48h时，在IFN-α浓度小于1000IU/ml的范围内，可检测到ACHN及786-0细胞增殖抑制率均随IFN-α浓度的增加而增强（P＜0.05），而IFN-α浓度为1000IU/ml、2000IU/ml及3000IU/ml时对ACHN及786-0细胞的增殖抑制率差异无统计学意义。IFN-α作用48h内，ACHN细胞的增殖抑制率随着IFN-α作用时间的延长逐渐增加。786-0细胞未见IFN-α作用时间与增殖抑制率的相关性。干扰素作用48h，可见786-0细胞株的相对生长速率高于ACHN细胞株，两组比较差异具有统计学意义（P＜0.05）。RT-PCR结果显示：ACHN细胞系经IFN-α作用0.5h、1.5h，SOCS3mRNA水平较空白对照组明显增高，差异具有统计学意义（P＜0.05），2.5h、4h、6h SOCS3mRNA水平与空白对照组相比，差异无统计学意义（P＞0.05），12h SOCS3mRNA水平较空白对照组减低，差异具有统计学意义（P＜0.05）；786-0细胞系经IFN-α作用1.5h，SOCS3mRNA水平较空白对照组明显增高，差异具有统计学意义（P＜0.05），2.5h SOCS3mRNA水平较空白对照组减低，差异具有统计学意义（P＜0.05），4h SOCS3mRNA水平较空白对照组再次明显增高，差异具有统计学意义（P＜0.05），6h、12h SOCS3mRNA水平与空白对照组相比，差异无统计学意义（P＞0.05）。Western Bolt结果显示：786-0细胞系干扰素作用48h组SOCS3蛋白表达水平显著高于空白对照组及ACHN细胞系干扰素作用48h组。FCM检测结果显示：786-0细胞系转染miR-146a模拟物组凋亡率高于转染阴性对照(negative control，NC)组和转染miR-146a抑制物组，组间比较差异均具有统计学意义（P＜0.05），且miR-146a抑制物组凋亡率低于转染NC组（P＜0.05）。在应用系统生物学方法探讨IFN-α耐药RCC细胞株SOCS3高表达机制的研究中，采用交互模型及IL-6途径模型模拟了不同浓度IFN-α刺激下信号途径抑制因子-1（suppressor of cytokinesignaling-1，SOCS1）和SOCS3的变化，模拟了IL-6浓度、白介素6受体（interleukin-6receptor，IL-6R）敏感性或数量、磷酸化信号转导和转录激活因子3（phosphorylated signal transducer and activator of transcription3，p-STAT3）水平、酪氨酸磷酸酶2（tyrosine phosphatase2，PP2）浓度、SH2结构域酪氨酸磷酸酶2（SH2domain-containing tyrosine phosphatase2，SHP2）浓度对SOCS3表达的影响，并模拟出p-STAT3水平变化对磷酸化信号转导和转录激活因子1（phosphorylated signal transducer and activator of transcription1，p-STAT1）的影响。模拟出不同程度敲除SOCS3引起IL-6R、p-STAT3表达水平的变化及对STAT二聚体内流和外流的影响。 结论： 1.IFN-α可抑制肾癌ACHN及786-0细胞的生长，ACHN细胞在较低的IFN-α作用浓度、较短的作用时间就能被抑制，而786-0细胞需要较高的IFN-α作用浓度、较长的作用才能被抑制。 2.在一定的IFN-α作用浓度（小于1000IU/ml）和时间（48h）范围内，IFN-α对肾癌ACHN及786-0细胞的抑制作用随着IFN-α浓度的增高而增强；在一定的时间范围内（48h），IFN-α对ACHN细胞的抑制作用随着IFN-α作用时间的延长而增强；而IFN-α对786-0细胞的抑制作用与IFN-α作用时间无相关性。 3.肾癌ACHN与786-0细胞株相比，ACHN细胞株对IFN-α较敏感，786-0细胞株对IFN-α较不敏感。 4.肾癌细胞对IFN-α耐药与SOCS3过表达相关。 5.miR-146a联合IFN-α可促进786-0细胞的凋亡。 6.采用系统生物学方法模拟的动力学曲线，系统而量化地解释了IFN-α耐药RCC细胞SOCS3表达增高的机制，即IL-6浓度、IL-6R敏感性或数量、p-STAT3水平、PP2浓度、SHP2浓度变化可引起SOCS3表达异常；p-STAT3水平变化能够对p-STAT1产生影响。并从理论上论证了采用RNAi等方法抑制或敲除SOCS3表达后，可引起IL-6R、p-STAT3、STAT二聚体的变化，进而改善RCC细胞对IFN-α的敏感性。
[Abstract]:Objective:
Interferon (IFN) is the most commonly used immunotherapy drug for metastatic and advanced renal cell carcinoma (RCC). However, the drug resistance of IFN limits its wide clinical application. To find molecular markers affecting the sensitivity of IFN, systematically and quantitatively analyze their mechanism of action in order to improve the treatment of RCC by IFN. The results provide experimental and theoretical basis.
Materials and methods:
In this study, ACHN and 786-0 cell lines of RCC were cultured in vitro. CCK-8 (Cell Counting Kit-8) method was used to detect the inhibitory rate of interferon-alpha (IFN-alpha) on the two RCC cells. Real-time PCR (RT-PCR) and Western Blot were used to detect the inhibitory factor of cytokine signal transduction-3 (suppresso-3). The transcription and expression of R of cytokine signaling-3 (SOCS3) in ACHN cell line and 786-0 cell line were studied. IFN-alpha resistant cell lines were identified according to the growth inhibition rate. Mimic mimics of microarray-146a were transfected into drug resistant cell line, inhibitors were added to IFN-alpha (1000IU/ml), and apoptosis was detected by flow cytometry (FCM). The cytokine pathways of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway include the IFN-alpha pathway and interleukin-6 (IL-6). Pathway and IFN-alpha and IL-6 interaction pathway, the relevant models mainly come from BioModels Database: respectively, IL-6 pathway model, IFN-gamma pathway model and IFN-gamma and IL-6 pathway interaction model. This software can be used to browse and modify the main molecules of the model, determine and modify the biochemical reaction equation of each molecule, formulate the reaction function relationship, adjust the reaction parameters, determine the simulation time and output mode, determine the parameters to be scanned and their range, and obtain the target result curve. The kinetic characteristics of JAK/STAT pathway related molecules in RCC cells under IFN-a and the effect of SOCS 3 on the pathway were analyzed.
Result:
CCK-8 assay showed that when the concentration of IFN-a was 300 IU/ml, ACHN cells showed significant inhibition at 48 h, while 786-0 cells at 72 h. When IFN-a acted on ACHN cells for 24 h, the concentration of IFN-a needed to reach 500 IU/ml to detect significant inhibition; while the concentration of IFN-a acted on 786-0 cells needed to be detected. The inhibitory rate of ACHN and 786-0 cells increased with the increase of IFN-a concentration (P < 0.05), while the inhibitory rate of ACHN and 786-0 cells increased with the increase of IFN-a concentration (1000IU/ml, 2000IU/ml and 3000IU/ml). There was no significant correlation between IFN-alpha time and proliferation inhibition rate in 786-0 cells. The relative growth rate of 786-0 cells was higher than that of ACHN cells in 48h after IFN-alpha treatment. The results of RT-PCR showed that the levels of SOCS3 mRNA in ACHN cells treated with IFN-a for 0.5 h and 1.5 h were significantly higher than those in the blank control group (P The levels of SOCS3 mRNA in 786-0 cell lines treated with IFN-alpha for 1.5 hours were significantly higher than those in blank control group (P < 0.05). The levels of SOCS3 mRNA in 2.5 hours were significantly lower than those in blank control group (P < 0.05). The levels of SOCS3 mRNA in 4 hours were significantly higher than those in blank control group (P < 0.05). The expression of SOCS3 protein in 786-0 cell line treated with IFN for 48 hours was significantly higher than that in blank control group and ACHN cell line treated with IFN for 48 hours. The apoptosis rate of mimic group was higher than that of negative control group (NC) and mimic group (P < 0.05), and the apoptosis rate of mimic group was lower than that of transfected NC group (P < 0.05). In the study of expression mechanism, interaction model and IL-6 pathway model were used to simulate the changes of suppressor of cytokine signaling-1 (SOCS1) and SOCS3 under different concentrations of IFN-alpha stimulation. IL-6 concentration, sensitivity or quantity of interleukin-6 receptor (IL-6R), phosphorylated signal transduction and transcriptional stimulation were simulated. The effects of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), tyrosine phosphatase 2 (PP2) and SH2 domain-containing tyrosine phosphatase 2 (SHP2) on the expression of SOCS3 were simulated. The effects of phosphorylated signal transducer and activator of transcription 1 (p-STAT1) on the expression of IL-6R, p-STAT3 and its effects on the in vivo and in vitro flow of STAT dimer were simulated.
Conclusion:
1. IFN-alpha can inhibit the growth of ACHN and 786-0 cells. ACHN cells can be inhibited at a lower concentration of IFN-alpha for a shorter period of time, while 786-0 cells need a higher concentration of IFN-alpha for a longer period of time.
2. The inhibitory effect of IFN-alpha on ACHN and 786-0 cells increased with the increase of IFN-alpha concentration in a certain range of IFN-alpha concentration (less than 1000IU/ml) and time (48h), the inhibitory effect of IFN-alpha on ACHN cells increased with the increase of IFN-alpha concentration in a certain range of time (48h), and the inhibitory effect of IFN-alpha on 786-0 cells increased with the prolongation of IFN-alpha action time. There was no correlation between inhibitory effect and IFN- alpha action time.
3. Compared with 786-0 cell line, ACHN cell line is more sensitive to IFN-alpha and 786-0 cell line is less sensitive to IFN-alpha.
4. renal cell carcinoma is related to IFN- overexpression and SOCS3 overexpression.
5.miR-146a combined with IFN- alpha can promote the apoptosis of 786-0 cells.
6. Using the kinetic curves simulated by system biology method, the mechanism of increased expression of SOCS3 in IFN-alpha resistant RCC cells was explained systematically and quantitatively, i.e. the changes of IL-6 concentration, IL-6R sensitivity or quantity, p-STAT3 level, PP2 concentration and SHP2 concentration could cause abnormal expression of SOCS3, and the changes of p-STAT3 level could affect p-STAT1 theoretically. It was demonstrated that inhibition or knockout of SOCS3 expression by RNAi could induce the changes of IL-6R, p-STAT3 and STAT dimers, thereby improving the sensitivity of RCC cells to IFN-a.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.11
本文编号：2223164
[Abstract]:Objective:
Interferon (IFN) is the most commonly used immunotherapy drug for metastatic and advanced renal cell carcinoma (RCC). However, the drug resistance of IFN limits its wide clinical application. To find molecular markers affecting the sensitivity of IFN, systematically and quantitatively analyze their mechanism of action in order to improve the treatment of RCC by IFN. The results provide experimental and theoretical basis.
Materials and methods:
In this study, ACHN and 786-0 cell lines of RCC were cultured in vitro. CCK-8 (Cell Counting Kit-8) method was used to detect the inhibitory rate of interferon-alpha (IFN-alpha) on the two RCC cells. Real-time PCR (RT-PCR) and Western Blot were used to detect the inhibitory factor of cytokine signal transduction-3 (suppresso-3). The transcription and expression of R of cytokine signaling-3 (SOCS3) in ACHN cell line and 786-0 cell line were studied. IFN-alpha resistant cell lines were identified according to the growth inhibition rate. Mimic mimics of microarray-146a were transfected into drug resistant cell line, inhibitors were added to IFN-alpha (1000IU/ml), and apoptosis was detected by flow cytometry (FCM). The cytokine pathways of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway include the IFN-alpha pathway and interleukin-6 (IL-6). Pathway and IFN-alpha and IL-6 interaction pathway, the relevant models mainly come from BioModels Database: respectively, IL-6 pathway model, IFN-gamma pathway model and IFN-gamma and IL-6 pathway interaction model. This software can be used to browse and modify the main molecules of the model, determine and modify the biochemical reaction equation of each molecule, formulate the reaction function relationship, adjust the reaction parameters, determine the simulation time and output mode, determine the parameters to be scanned and their range, and obtain the target result curve. The kinetic characteristics of JAK/STAT pathway related molecules in RCC cells under IFN-a and the effect of SOCS 3 on the pathway were analyzed.
Result:
CCK-8 assay showed that when the concentration of IFN-a was 300 IU/ml, ACHN cells showed significant inhibition at 48 h, while 786-0 cells at 72 h. When IFN-a acted on ACHN cells for 24 h, the concentration of IFN-a needed to reach 500 IU/ml to detect significant inhibition; while the concentration of IFN-a acted on 786-0 cells needed to be detected. The inhibitory rate of ACHN and 786-0 cells increased with the increase of IFN-a concentration (P < 0.05), while the inhibitory rate of ACHN and 786-0 cells increased with the increase of IFN-a concentration (1000IU/ml, 2000IU/ml and 3000IU/ml). There was no significant correlation between IFN-alpha time and proliferation inhibition rate in 786-0 cells. The relative growth rate of 786-0 cells was higher than that of ACHN cells in 48h after IFN-alpha treatment. The results of RT-PCR showed that the levels of SOCS3 mRNA in ACHN cells treated with IFN-a for 0.5 h and 1.5 h were significantly higher than those in the blank control group (P The levels of SOCS3 mRNA in 786-0 cell lines treated with IFN-alpha for 1.5 hours were significantly higher than those in blank control group (P < 0.05). The levels of SOCS3 mRNA in 2.5 hours were significantly lower than those in blank control group (P < 0.05). The levels of SOCS3 mRNA in 4 hours were significantly higher than those in blank control group (P < 0.05). The expression of SOCS3 protein in 786-0 cell line treated with IFN for 48 hours was significantly higher than that in blank control group and ACHN cell line treated with IFN for 48 hours. The apoptosis rate of mimic group was higher than that of negative control group (NC) and mimic group (P < 0.05), and the apoptosis rate of mimic group was lower than that of transfected NC group (P < 0.05). In the study of expression mechanism, interaction model and IL-6 pathway model were used to simulate the changes of suppressor of cytokine signaling-1 (SOCS1) and SOCS3 under different concentrations of IFN-alpha stimulation. IL-6 concentration, sensitivity or quantity of interleukin-6 receptor (IL-6R), phosphorylated signal transduction and transcriptional stimulation were simulated. The effects of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), tyrosine phosphatase 2 (PP2) and SH2 domain-containing tyrosine phosphatase 2 (SHP2) on the expression of SOCS3 were simulated. The effects of phosphorylated signal transducer and activator of transcription 1 (p-STAT1) on the expression of IL-6R, p-STAT3 and its effects on the in vivo and in vitro flow of STAT dimer were simulated.
Conclusion:
1. IFN-alpha can inhibit the growth of ACHN and 786-0 cells. ACHN cells can be inhibited at a lower concentration of IFN-alpha for a shorter period of time, while 786-0 cells need a higher concentration of IFN-alpha for a longer period of time.
2. The inhibitory effect of IFN-alpha on ACHN and 786-0 cells increased with the increase of IFN-alpha concentration in a certain range of IFN-alpha concentration (less than 1000IU/ml) and time (48h), the inhibitory effect of IFN-alpha on ACHN cells increased with the increase of IFN-alpha concentration in a certain range of time (48h), and the inhibitory effect of IFN-alpha on 786-0 cells increased with the prolongation of IFN-alpha action time. There was no correlation between inhibitory effect and IFN- alpha action time.
3. Compared with 786-0 cell line, ACHN cell line is more sensitive to IFN-alpha and 786-0 cell line is less sensitive to IFN-alpha.
4. renal cell carcinoma is related to IFN- overexpression and SOCS3 overexpression.
5.miR-146a combined with IFN- alpha can promote the apoptosis of 786-0 cells.
6. Using the kinetic curves simulated by system biology method, the mechanism of increased expression of SOCS3 in IFN-alpha resistant RCC cells was explained systematically and quantitatively, i.e. the changes of IL-6 concentration, IL-6R sensitivity or quantity, p-STAT3 level, PP2 concentration and SHP2 concentration could cause abnormal expression of SOCS3, and the changes of p-STAT3 level could affect p-STAT1 theoretically. It was demonstrated that inhibition or knockout of SOCS3 expression by RNAi could induce the changes of IL-6R, p-STAT3 and STAT dimers, thereby improving the sensitivity of RCC cells to IFN-a.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.11
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