SOCS3與干擾素-a治療腎細胞癌敏感性的實驗及系統(tǒng)生物學研究
發(fā)布時間：2018-09-04 19:51
【摘要】：目的： 干擾素（interferon，IFN）是治療轉移性及進展期腎細胞癌（Renal cellcarcinoma，RCC）最常用的免疫治療藥物。然而由于IFN的耐藥性，限制了其在臨床的廣泛應用。尋找影響IFN敏感性的分子標志物，系統(tǒng)及量化的分析其作用機制，為提高IFN治療RCC的療效提供實驗和理論依據(jù)。 材料和方法： 本實驗中我們對RCC的ACHN細胞株和786-0細胞株進行體外培養(yǎng)，采用CCK-8（Cell Counting Kit-8）法檢測干擾素α（interferon-α，IFN-α）對兩種RCC細胞的抑制率，采用實時定量PCR（real-time PCR，RT-PCR）及Western Blot方法檢測細胞因子信號傳導抑制因子-3（suppressor of cytokine signaling-3，SOCS3）在ACHN細胞株和786-0細胞株的轉錄及表達情況。根據(jù)生長抑制率的不同，識別IFN-α耐藥細胞系。對耐藥細胞系轉染miR-146a模擬物、抑制物并加入IFN-α（1000IU/ml），流式細胞儀（flow cytometry，F(xiàn)CM）檢測細胞凋亡。在此基礎上，結合我們自己的實驗結果及相關文獻結果，引入系統(tǒng)生物學方法。我們研究的關于酪氨酸激酶/信號轉導和轉錄激活子（Janus kinase/signaltransducer and activator of transcription，JAK/STAT）通路的細胞因子途徑包括IFN-α途徑、白介素6（interleukin-6，IL-6）途徑及IFN-α與IL-6交互途徑，相關模型主要來源于BioModels Database：分別為IL-6途徑模型，IFN-γ途徑模型和IFN-γ與IL-6途徑交互模型。分析軟件選擇COPASI4.14(Build89)：我們把標準格式的系統(tǒng)生物學標記語言（systems biology markup language，SBML）模型文件載入軟件。通過操作該軟件可以實現(xiàn)：瀏覽和修改模型的主要分子，確定和修改每個分子的生化反應方程式，，制定反應函數(shù)關系，調(diào)整反應參數(shù)，確定模擬的時間及輸出方式，確定要掃描的參數(shù)及其范圍，得到目標結果曲線。應用該軟件分析了RCC細胞在IFN-α作用下JAK/STAT途徑相關分子的動力學特征及SOCS3對該通路的影響。 結果： CCK-8檢測結果顯示，當IFN-α濃度為300IU/ml時，ACHN細胞在48h檢測到明顯抑制作用時，而786-0細胞在72h檢測到明顯抑制作用。當IFN-α作用24h時，作用于ACHN細胞的IFN-α濃度需達到500IU/ml才能檢測到明顯抑制作用；而作用于786-0細胞的IFN-α濃度需達到1000IU/ml才能檢測到明顯抑制作用。檢測時間點為48h時，在IFN-α濃度小于1000IU/ml的范圍內(nèi)，可檢測到ACHN及786-0細胞增殖抑制率均隨IFN-α濃度的增加而增強（P＜0.05），而IFN-α濃度為1000IU/ml、2000IU/ml及3000IU/ml時對ACHN及786-0細胞的增殖抑制率差異無統(tǒng)計學意義。IFN-α作用48h內(nèi)，ACHN細胞的增殖抑制率隨著IFN-α作用時間的延長逐漸增加。786-0細胞未見IFN-α作用時間與增殖抑制率的相關性。干擾素作用48h，可見786-0細胞株的相對生長速率高于ACHN細胞株，兩組比較差異具有統(tǒng)計學意義（P＜0.05）。RT-PCR結果顯示：ACHN細胞系經(jīng)IFN-α作用0.5h、1.5h，SOCS3mRNA水平較空白對照組明顯增高，差異具有統(tǒng)計學意義（P＜0.05），2.5h、4h、6h SOCS3mRNA水平與空白對照組相比，差異無統(tǒng)計學意義（P＞0.05），12h SOCS3mRNA水平較空白對照組減低，差異具有統(tǒng)計學意義（P＜0.05）；786-0細胞系經(jīng)IFN-α作用1.5h，SOCS3mRNA水平較空白對照組明顯增高，差異具有統(tǒng)計學意義（P＜0.05），2.5h SOCS3mRNA水平較空白對照組減低，差異具有統(tǒng)計學意義（P＜0.05），4h SOCS3mRNA水平較空白對照組再次明顯增高，差異具有統(tǒng)計學意義（P＜0.05），6h、12h SOCS3mRNA水平與空白對照組相比，差異無統(tǒng)計學意義（P＞0.05）。Western Bolt結果顯示：786-0細胞系干擾素作用48h組SOCS3蛋白表達水平顯著高于空白對照組及ACHN細胞系干擾素作用48h組。FCM檢測結果顯示：786-0細胞系轉染miR-146a模擬物組凋亡率高于轉染陰性對照(negative control，NC)組和轉染miR-146a抑制物組，組間比較差異均具有統(tǒng)計學意義（P＜0.05），且miR-146a抑制物組凋亡率低于轉染NC組（P＜0.05）。在應用系統(tǒng)生物學方法探討IFN-α耐藥RCC細胞株SOCS3高表達機制的研究中，采用交互模型及IL-6途徑模型模擬了不同濃度IFN-α刺激下信號途徑抑制因子-1（suppressor of cytokinesignaling-1，SOCS1）和SOCS3的變化，模擬了IL-6濃度、白介素6受體（interleukin-6receptor，IL-6R）敏感性或數(shù)量、磷酸化信號轉導和轉錄激活因子3（phosphorylated signal transducer and activator of transcription3，p-STAT3）水平、酪氨酸磷酸酶2（tyrosine phosphatase2，PP2）濃度、SH2結構域酪氨酸磷酸酶2（SH2domain-containing tyrosine phosphatase2，SHP2）濃度對SOCS3表達的影響，并模擬出p-STAT3水平變化對磷酸化信號轉導和轉錄激活因子1（phosphorylated signal transducer and activator of transcription1，p-STAT1）的影響。模擬出不同程度敲除SOCS3引起IL-6R、p-STAT3表達水平的變化及對STAT二聚體內(nèi)流和外流的影響。 結論： 1.IFN-α可抑制腎癌ACHN及786-0細胞的生長，ACHN細胞在較低的IFN-α作用濃度、較短的作用時間就能被抑制，而786-0細胞需要較高的IFN-α作用濃度、較長的作用才能被抑制。 2.在一定的IFN-α作用濃度（小于1000IU/ml）和時間（48h）范圍內(nèi)，IFN-α對腎癌ACHN及786-0細胞的抑制作用隨著IFN-α濃度的增高而增強；在一定的時間范圍內(nèi)（48h），IFN-α對ACHN細胞的抑制作用隨著IFN-α作用時間的延長而增強；而IFN-α對786-0細胞的抑制作用與IFN-α作用時間無相關性。 3.腎癌ACHN與786-0細胞株相比，ACHN細胞株對IFN-α較敏感，786-0細胞株對IFN-α較不敏感。 4.腎癌細胞對IFN-α耐藥與SOCS3過表達相關。 5.miR-146a聯(lián)合IFN-α可促進786-0細胞的凋亡。 6.采用系統(tǒng)生物學方法模擬的動力學曲線，系統(tǒng)而量化地解釋了IFN-α耐藥RCC細胞SOCS3表達增高的機制，即IL-6濃度、IL-6R敏感性或數(shù)量、p-STAT3水平、PP2濃度、SHP2濃度變化可引起SOCS3表達異常；p-STAT3水平變化能夠?qū)-STAT1產(chǎn)生影響。并從理論上論證了采用RNAi等方法抑制或敲除SOCS3表達后，可引起IL-6R、p-STAT3、STAT二聚體的變化，進而改善RCC細胞對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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.11
本文編號：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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.11
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本文編號：2223164
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