AG490增強HER2陽性乳腺癌細胞對曲妥珠單抗敏感性的實驗研究
發(fā)布時間：2018-07-29 12:44
【摘要】：背景與目的乳腺癌是女性常見的惡性腫瘤之一。乳腺癌的發(fā)生與多種癌基因激活或抑癌基因失活有關。尤其是一些與細胞生長及內分泌相關的基因,如HER2、ERK、ERa等。因此,目前在臨床治療中使用了許多靶向這些特異性基因的藥物,如赫賽汀、他莫西芬等,取得了較好的療效。但隨著這些藥物的廣泛使用,耐藥性逐漸成為限制其療效的關鍵因素。赫賽汀是靶向HER2的一類臨床一線治療用藥,它可與乳腺癌細胞表面的HER2受體特異性結合,從而阻止HER2受體發(fā)生二聚化而激活,進而抑制了其介導的下游信號通路,產生抗瘤效應。但赫賽汀單獨用藥的客觀反應率并不高,只有12%-34%,且近50%的患者在使用赫賽汀的初期就會產生耐藥性,因此,深入闡明乳腺癌細胞對曲妥珠單抗耐藥的相關機制并采取針對性的措施成為目前乳腺癌治療中的重要科學問題。由于曲妥珠單抗所封閉的靶分子HER2為乳腺癌細胞表面的一類受體,該受體激活后可引起細胞內多條信號通路的活化,包括Ras/MAPK信號通路、PI3K/AKT/mTOR信號通路、JAK/STAT信號通路以及PLC-y信號通路等。這些信號通路的異常和乳腺癌的增殖、生長等密切相關。因此,曲妥珠單抗治療過程所產生的耐藥性也往往和相關信號通路異常密切相關。研究表明,HER2信號通路中任一環(huán)節(jié)發(fā)生改變,都可能導致曲妥珠單抗治療抵抗。目前已發(fā)現的抵抗機制包括：HER家族受體與胰島素樣生長因子1受體(insu-lin-like growth factor 1 receptor, IGF-1R)信號增加；HER2截短突變體p95-HER2的累積；PI3K/AKT/mTOR信號通路的異常活化。除了這些可能導致HER2陽性乳腺癌細胞對曲妥珠單抗治療抵抗的機制外,JAK/STAT通路的激活也可能是引起抵抗的另一重要機制,但目前相關報道很少。JAK是一類非受體型酪氨酸激酶,該家族包括JAK1、JAK2、JAK3和TYK2四個成員。JAK激活后可以磷酸化STAT而使后者活化,從而構成JAK/STAT信號通路。JAK/STAT信號通路在多種腫瘤組織中均存在過度激活的現象。在乳腺癌組織中,總的STAT3與磷酸化STAT3的表達水平均顯著高于正常乳腺組織,且與腫瘤的侵襲轉移程度密切相關。但STAT3的激活是否與乳腺癌細胞對曲妥珠單抗的治療抵抗有關目前尚未見報道。因此,本研究擬首先以HER2陽性的乳腺癌細胞為研究對象,觀察曲妥珠單抗處理過程中STAT3的激活情況,并利用基因沉默技術及特異性抑制劑聯合曲妥珠單抗處理來觀察細胞的反應,從而判斷STAT3的激活在乳腺癌細胞耐受曲妥珠單抗中的作用,以期為進一步的臨床治療提供理論基礎與科學依據。AG490是目前研究中常用的一種JAK/STAT通路特異性抑制劑。AG490可以競爭性結合酪氨酸激酶JAK2,從而抑制其活性。AG490抑制JAK的激活后,導致STAT3磷酸化被抑制,造成STAT3不能入核與相關DNA元件結合,從而抑制腫瘤細胞的增殖。當前,AG490已成為臨床治療多種腫瘤的抗癌藥物,但AG490能否增敏曲妥珠單抗尚不清楚。另外,由于激活的STAT3能上調抗凋亡基因Bcl-xL、Bcl-2 與 Mcl-1的表達,激活促血管生成生長因子VEGF的表達,還能上調細胞周期調節(jié)蛋白cyclins D1/D2以及促細胞增殖蛋白c-myc與Survivin的表達,并且有下調促凋亡基因p53表達的作用。尤其最近研究發(fā)現,抑癌基因SARI也可能參與了JAK/STAT信號介導的腫瘤發(fā)生。SARI又稱為BATF2,在正常人組織中表達水平較高,而在相應的腫瘤細胞中表達水平降低。外源性表達SARI可以抑制腫瘤細胞的增殖,而過表達SARI卻對正常細胞無明顯的增殖抑制效應。但SARI抗癌的分子機制還沒完全清楚,特別是調控SARI表達的上游信號還不清楚。AG490作用后是否可通過激活SARI來殺傷乳腺癌細胞也值得進一步探討。方法通過CCK-8實驗測定不同濃度的曲妥珠單抗對HER2陽性細胞SK-BR3增殖的抑制作用,通過臺盼藍染色實驗測定不同濃度的曲妥珠單抗對HER2陽性細胞SK-BR3死亡的影響,通過克隆形成實驗測定不同濃度的曲妥珠單抗對HER2陽性細胞SK-BR3克隆形成能力的影響,通過免疫印跡實驗檢測曲妥珠單抗作用后SK-BR3細胞中STAT3的激活情況,然后通過siRNA沉默SK-BR3細胞中的STAT3,免疫印跡實驗確定沉默效果,然后通過CCK-8實驗和臺盼藍實驗觀察沉默STAT3后曲妥珠單抗對SK-BR3細胞增殖與死亡的影響。再進一步使用STAT3抑制劑AG490單獨或與曲妥珠單抗聯合處理SK-BR3細胞,通過CCK-8實驗和臺盼藍實驗觀察不同處理對細胞增殖與死亡的影響。然后采用相同的策略,通過CCK-8實驗測定不同濃度的AG490分別對HER2陽性與HER2陰性細胞增殖的抑制作用,通過臺盼藍染色實驗測定不同濃度的AG490分別對HER2陽性與HER2陰性細胞死亡的影響,通過克隆形成實驗測定不同濃度的AG490分別對HER2陽性與HER2陰性細胞克隆形成能力的影響,通過免疫印跡實驗檢測AG490作用后細胞中SARI的激活情況,然后通過siRNA沉默細胞中的SARI,免疫印跡實驗確定沉默效果,然后通過CCK-8實驗和臺盼藍實驗觀察沉默SARI后AG490對細胞增殖與死亡的影響。通過生物信息方法預測SARI基因啟動子區(qū)中存在的STAT3結合位點,并采用PCR方法從乳腺癌細胞中擴增SARI基因的啟動子區(qū),并針對性的擴增包含或不包含STAT3結合位點的片段,將各片段插入報告基因載體pGL3中,然后轉染細胞,觀察AG490處理前后熒光素酶活性變化情況,從而確定AG490是否能夠通過上調SARI的轉錄來增強其表達并抗癌。結果曲妥珠單抗能以劑量依賴的方式抑制SK-BR3細胞的增殖,0.25、0.5、1.0、2.0,4.0 mg/ml曲妥珠單抗的細胞相對增殖率分別為76.22%、72.97%、61.08%、42.16%和23.78%。曲妥珠單抗也能以劑量依賴的方式促進SK-BR3細胞死亡0.25、0.5、1.0、2.0、4.0 m//ml曲妥珠單抗相對應的細胞死亡率分別為：10.35+1.66%、18.37+2.49%、31.97±3.08%、46.78±5.67%、63.77±7.81%。與對照組相比,曲妥珠單抗還能以劑量依賴的方式抑制SK-BR3細胞的克隆形成,0.25、0.5、1.0、2.0、4.0 mg/ml曲妥珠單抗相對應的細胞克隆形成率分別為：64.79+8.38%、35.21±13.29%、26.03±10.79%、16.29±10.34%、6.18±6.06%。曲妥珠單抗還可以濃度依賴性地激活SK-BR3細胞中的STAT3信號通路,體現為磷酸化STAT3水平顯著升高。用特異性siRNA沉默SK-BR3細胞中的STAT3后,CCK-8實驗分析發(fā)現可以顯著增強曲妥珠單抗對SK-BR3細胞的增殖抑制率,臺盼藍實驗分析發(fā)現可以顯著增加曲妥珠單抗對SK-BR3細胞的死亡率。CCK-8實驗結果表明,與AG490單獨處理組或曲妥珠單抗單獨處理組相比,AG490聯合曲妥珠單抗可以顯著增加SK-BR3細胞的增殖抑制率；臺盼藍實驗結果也表明,與AG490單獨處理組或曲妥珠單抗單獨處理組相比,AG490聯合曲妥珠單抗可以顯著增加SK-BR3細胞的死亡率。本研究結果進一步證明,抑制STAT3信號通路確實可以增加曲妥珠單抗對SK-BR3細胞的殺傷率。AG490能以劑量依賴的方式抑制SK-BR3細胞與MDA-MB-231細胞的增殖,AG490在兩種細胞中的IC50值分別為43.281 μM與28.327μM。 AG490也能以劑量依賴的方式促進SK-BR3細胞與MDA-MB-231細胞死亡,SK-BR3細胞的對照組死亡率為1.02+0.24%,AG490處理組按照濃度梯度升高(25、50、100 μM)關系相對應的死亡率分別為：5.78±1.67%、16.89+2.67%、31.08±4.65%；而在MDA-MB-231細胞,對照組死亡率為1.77+0.66%,AG490處理組按照濃度梯度升高(25、50、100 μM)關系相對應的死亡率分別為：8.05+2.05%、27.26±2.89%、44.18±4.98%。與對照組相比,AG490還能以劑量依賴的方式抑制SK-BR3細胞與MDA-MB-231細胞的克隆形成。在SK-BR3細胞,對照組與實驗組(依濃度升高)的細胞克隆數分別為：559±52、361+38、206±29、117±15個；在MDA-MB-231細胞,對照組與實驗組(依濃度升高)的細胞克隆數分別為：389±42、211+23、149±17、68±13個。定量PCR檢測發(fā)現,隨AG490處理濃度的升高,SK-BR3與MDA-MB-231細胞中SARI的mRNA水平均顯著升高；Western blot分析也進一步證實,AG490可以劑量依賴的方式增強SK-BR3與MDA-MB-231細胞中SARI蛋白的表達水平。沉默SARI后AG490對MDA-MB-231細胞的增殖抑制效應降低。生物信息學分析發(fā)現,在位于-1787到-1797位置存在1個經典的STAT3結合位點。將包含SARI啟動子區(qū)不同大小的兩個片段(-1到-2000bp,-1到-1700bp)分別克隆到報告質粒pGL3-basic中,并與對照質粒同時轉染MDA-MB-231細胞后進行熒光素酶報告基因檢測,發(fā)現不含STAT3結合位點的-1到-1700bp片段在AG490處理前后熒光素酶活性無明顯改變,而包含該結合位點的-1到-2000bp片段則在AG490處理后熒光素酶活性顯著增高。這一結果說明AG490作用后可通過增加SARI啟動子活性而增強SARI的轉錄來提高其表達,這一過程可能與AG490對STAT3的抑制及STAT3對SARI啟動子活性的抑制相關。結論1.曲妥珠單抗可以劑量依賴性地抑制HER2陽性乳腺癌細胞的增殖、促進其死亡、并抑制其細胞克隆的形成。2.曲妥珠單抗處理導致HER2陽性乳腺癌細胞中STAT3的激活,沉默STAT3或抑制STAT3的活性均能增強HER2陽性乳腺癌細胞對曲妥珠單抗的敏感性。3.AG490可以劑量依賴性地分別抑制HER2陽性與HER2陰性乳腺癌細胞的增殖、促進其死亡、并抑制其細胞克隆的形成。4.AG490處理導致HER2陽性與HER2陰性乳腺癌細胞中SARI表達上調,沉默SARI后能降低乳腺癌細胞對AG490的敏感性。5.SARI基因的啟動子區(qū)存在STAT3的結合位點,不含該結合位點的啟動子片段被AG490激活的能力要顯著低于野生型啟動子的受激活能力。意義1.STAT3的激活是導致HER2陽性乳腺癌細胞對曲妥珠單抗耐受的重要機制。2.AG490能有效增強HER2陽性乳腺癌細胞對曲妥珠單抗的敏感性。3.AG490殺傷乳腺癌細胞與其對抑癌基因SARI的轉錄上調作用相關。
[Abstract]:Background and objective breast cancer is one of the most common malignant tumors in women. The occurrence of breast cancer is related to a variety of oncogene activation or inactivation of tumor suppressor genes. Especially, some genes related to cell growth and endocrinology, such as HER2, ERK, and ERa, have been used in clinical therapy, such as Hertz, such as Hertz. But with the extensive use of these drugs, drug resistance has gradually become a key factor in limiting its efficacy. Herceptin is a class of clinical therapy targeted to HER2, which can specifically bind to the HER2 receptor on the surface of breast cancer cells to prevent the activation of the dimerization of HER2 receptors. It also inhibits the downstream signaling pathway and produces antitumor effects. But the objective response rate of Herceptin alone is not high, only 12%-34%, and nearly 50% of the patients will have resistance in the early stages of Herceptin. Therefore, the mechanism of breast cancer cell resistance to trastuzumab is clarified and the targeted measures are taken. Since the target molecule HER2 closed by trastuzumab is a kind of receptor on the surface of breast cancer cells, the receptor activates the activation of multiple signal pathways in the cells, including the Ras/MAPK signaling pathway, PI3K/AKT/mTOR signaling pathway, JAK/STAT signaling pathway and PLC-y signaling pathway. The abnormalities of these signaling pathways are closely related to the proliferation and growth of breast cancer. Therefore, drug resistance produced by the therapy of trastuzumab is often closely related to abnormal signal pathways. Studies have shown that any link in the HER2 signaling pathway may lead to the resistance of trastuzumab, which has been found at present. Resistance mechanisms include the increase of the HER family receptor and the insulin like growth factor 1 receptor (insu-lin-like growth factor 1 receptor, IGF-1R), the accumulation of HER2 truncated mutant p95-HER2, and the abnormal activation of the PI3K/AKT/mTOR signaling pathway. In addition to these, these may lead to the resistance of the HER2 positive breast cancer cells to the resistance to trumpet monoclonal antibodies. In addition, the activation of the JAK/STAT pathway may also be another important mechanism for resistance, but it is rarely reported that.JAK is a class of non receptor tyrosine kinase, which includes four members of JAK1, JAK2, JAK3 and TYK2 that can be activated by phosphorylated STAT after.JAK activation, thus forming the JAK/STAT signaling pathway in the.JAK/STAT signaling pathway. There is a phenomenon of excessive activation in various tumor tissues. In breast cancer, the total expression of STAT3 and phosphorylated STAT3 is significantly higher than that of normal breast tissue, and it is closely related to the degree of invasion and metastasis of the tumor. But the activation of STAT3 is not reported to be related to the resistance of breast cancer cells to the treatment of trastuzumab. Therefore, we should first study the HER2 positive breast cancer cells, observe the activation of STAT3 during the treatment of trastuzumab, and use the gene silencing technique and the specific inhibitor combined with trastuzumab to observe the cell response, so as to determine the activation of STAT3 in the tolerance of breast cancer cells to the resistance of trastuzumab. The role of.AG490 is to provide theoretical basis and scientific basis for further clinical treatment. A JAK/STAT pathway specific inhibitor,.AG490, is a competitive binding tyrosine kinase JAK2, which inhibits the activation of JAK by inhibiting its active.AG490, resulting in the inhibition of STAT3 phosphorylation, resulting in the failure of STAT3 to enter the nucleus. Related DNA elements are combined to inhibit the proliferation of tumor cells. Currently, AG490 has become an anticancer drug in the clinical treatment of various tumors. But it is not clear whether AG490 can sensitized to curfdrop monoclonal antibody. In addition, the activation of STAT3 can increase the expression of anti apoptotic gene Bcl-xL, Bcl-2 and Mcl-1, and activate the expression of angiogenic growth factor VEGF. It can increase the expression of cell cycle regulation protein cyclins D1/D2 and cell proliferation promoting protein c-myc and Survivin, and down regulation of p53 expression of apoptosis promoting gene. In particular, recent studies have found that tumor suppressor gene SARI may also participate in JAK/STAT signal mediated tumor.SARI, known as BATF2, in normal human tissues. High levels of expression in the corresponding tumor cells decrease. Exogenous expression of SARI can inhibit the proliferation of tumor cells, but overexpression of SARI has no obvious proliferation inhibition effect on normal cells. However, the molecular mechanism of SARI is not completely clear, especially if the upstream signal regulating the expression of SARI is not clear if the effect of.AG490 action is not clear. To activate SARI to kill the breast cancer cells is also worth further exploring. Methods the inhibitory effect of different concentrations of trastuzumab on the proliferation of SK-BR3 in HER2 positive cells was measured by CCK-8 experiment. The effect of different concentration of trastuzumab on the SK-BR3 death of HER2 positive cells was determined by trypan blue staining. The effect of different concentration of trastuzumab on the SK-BR3 clone formation ability of HER2 positive cells was determined. The activation of STAT3 in SK-BR3 cells after the action of trastuzumab was detected by Western blot, and then STAT3 in SK-BR3 cells was silenced by siRNA and immunoblotting was used to determine the silencing effect, and then through CCK-8 experiment and trypan blue. The effect of trastuzumab on the proliferation and death of SK-BR3 cells after STAT3 was observed. Further use of STAT3 inhibitor AG490 alone or with trastuzumab to treat SK-BR3 cells, the effects of different treatments on cell proliferation and death were observed by the CCK-8 experiment and trypan blue experiment. Then the same strategy was adopted by the CCK-8 experiment. The inhibitory effects of different concentrations of AG490 on the proliferation of HER2 positive and HER2 negative cells were measured, and the effects of AG490 on HER2 positive and HER2 negative cell death were measured by trypan blue assay, and the cloning ability of AG490 with different concentrations of AG490 to determine the ability of HER2 positive and HER2 negative cells to be cloned respectively. The activation of SARI in cells after AG490 action was detected by immunoblotting, and then the effect of silence was determined by SARI in siRNA silencing cells and Western blot test. Then the effects of AG490 on cell proliferation and death after silent SARI were observed by CCK-8 experiment and trypan blue experiment. SARI gene was predicted by biological information method. The STAT3 binding site in the promoter region was initiated and the promoter region of the SARI gene was amplified from the breast cancer cells by PCR method, and the fragments contained or not included in the STAT3 binding site were amplified and inserted into the report gene carrier pGL3, and then transfected to the cells. The changes of luciferase activity before and after AG490 treatment were observed, and the changes of the luciferase activity before and after AG490 treatment were observed. To determine whether AG490 can increase its expression and anticancer by up regulation of SARI, trastuzumab can inhibit the proliferation of SK-BR3 cells in a dose-dependent manner, and the relative proliferation rates of 0.25,0.5,1.0,2.0,4.0 mg/ml trastuzumab are 76.22%, 72.97%, 61.08%, 42.16% and 23.78%., as well as dose of trastuzumab. The relative mortality of SK-BR3 cell death 0.25,0.5,1.0,2.0,4.0 m//ml trastuzumab is: 10.35+1.66%, 18.37+2.49%, 31.97 + 3.08%, 46.78 + 5.67%, 63.77 + 7.81%., compared with the control group. The clones of SK-BR3 cells can be inhibited in a dose-dependent manner, 0.25,0.5,1.0,2.0,4.0 M The relative cell clone formation rates of g/ml trastuzumab are: 64.79+8.38%, 35.21 + 13.29%, 26.03 + 10.79%, 16.29 + 10.34%, 6.18 + 6.06%., which can also activate STAT3 signaling pathway in SK-BR3 cells in a concentration dependent manner, reflecting a significant increase in the level of phosphorylated STAT3. ST in SK-BR3 cells with specific siRNA After AT3, CCK-8 experimental analysis showed that the inhibitory rate of trastuzumab to SK-BR3 cells could be significantly enhanced. Trypan blue experimental analysis found that the death rate of trastuzumab to SK-BR3 cells could be significantly increased by.CCK-8 experimental results, and compared with AG490 alone or trastuzumab alone, AG490 combined with trastuzumab. The proliferation inhibition rate of SK-BR3 cells was significantly increased; trypan blue experimental results also showed that AG490 combined with trastuzumab could significantly increase the mortality of SK-BR3 cells compared with the single treatment group of AG490 alone or trastuzumab. The results of this study further demonstrated that the inhibition of the STAT3 signaling pathway could indeed increase the number of trastuzolones. The anti SK-BR3 cell killing rate.AG490 can inhibit the proliferation of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. The IC50 values of AG490 in two cells are respectively 43.281 M and 28.327 u M. AG490, respectively, to promote the death of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. The mortality of the control group of the SK-BR3 cells is the same. 4%, the relative mortality of AG490 treatment group was 5.78 + 1.67%, 16.89+2.67%, 31.08 + 4.65% according to the increase of concentration gradient (25,50100 M), while in MDA-MB-231 cells, the mortality of the control group was 1.77+0.66%, and the mortality rate of AG490 treatment group in accordance with the increase of concentration gradient (25,50100 u M) was 8.05+2.05%, 27.26 +. 2.89%, 44.18 + 4.98%., compared with the control group, AG490 could inhibit the clone formation of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. In SK-BR3 cells, the number of cell clones in the control group and the experimental group were 559 + 52361+38206 + 29117 + 15, respectively, in the MDA-MB-231 cells, the control group and the experimental group. The number of cell clones was 389 + 42211+23149 + 17,68 + 13 respectively. Quantitative PCR detection showed that the mRNA level of SARI in SK-BR3 and MDA-MB-231 cells increased significantly with the increase of AG490 concentration, and Western blot analysis further confirmed that AG490 could be used to enhance the SK-BR3 and protein in the cells in dose-dependent manner. The inhibitory effect of AG490 on the proliferation of MDA-MB-231 cells after SARI was reduced. Bioinformatics analysis found that there were 1 classical STAT3 binding sites in the position from -1787 to -1797. The two fragments containing the different sizes of the SARI promoter region (-1 to -2000bp, -1 to -1700bp) were cloned into the reported plasmid pGL3-basic, respectively. The luciferase reporter gene was detected after transfection of MDA-MB-231 cells with the control plasmid, and there was no significant change in the luciferase activity of the -1 to -1700bp fragments without the STAT3 binding site before and after AG490 treatment, while the -1 to -2000bp fragment containing the binding site increased significantly after the AG490 treatment. After the action of AG490, the expression of SARI can be enhanced by increasing the activity of SARI promoter, which may be related to the inhibition of AG490 to STAT3 and the inhibition of STAT3 on the activity of SARI promoter. Conclusion 1. trastuzumab can inhibit the proliferation of HER2 positive breast cancer cells in a dose-dependent manner, promote its death, and inhibit it. The formation of cell clones.2. trastuzumab leads to the activation of STAT3 in HER2 positive breast cancer cells. Silence STAT3 or inhibition of STAT3 activity can enhance the sensitivity of HER2 positive breast cancer cells to trastuzumab..3.AG490 can inhibit the proliferation of HER2 positive and HER2 negative breast cancer cells in a dose-dependent manner and promote their death. .4.AG490 treatment led to the formation of SARI expression in HER2 positive and HER2 negative breast cancer cells, and the silence SARI could reduce the binding site of STAT3 in the promoter region of the breast cancer cell to AG490 sensitivity.5.SARI gene, and the ability to activate the promoter fragment without the binding site was significant. The activation of 1.STAT3 is an important mechanism for the tolerance of HER2 positive breast cancer cells to trastuzumab,.2.AG490 can effectively enhance the sensitivity of HER2 positive breast cancer cells to trastuzumab, and the.3.AG490 killing of breast cancer cells is related to the up regulation of the tumor suppressor gene SARI.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.9
[Abstract]:Background and objective breast cancer is one of the most common malignant tumors in women. The occurrence of breast cancer is related to a variety of oncogene activation or inactivation of tumor suppressor genes. Especially, some genes related to cell growth and endocrinology, such as HER2, ERK, and ERa, have been used in clinical therapy, such as Hertz, such as Hertz. But with the extensive use of these drugs, drug resistance has gradually become a key factor in limiting its efficacy. Herceptin is a class of clinical therapy targeted to HER2, which can specifically bind to the HER2 receptor on the surface of breast cancer cells to prevent the activation of the dimerization of HER2 receptors. It also inhibits the downstream signaling pathway and produces antitumor effects. But the objective response rate of Herceptin alone is not high, only 12%-34%, and nearly 50% of the patients will have resistance in the early stages of Herceptin. Therefore, the mechanism of breast cancer cell resistance to trastuzumab is clarified and the targeted measures are taken. Since the target molecule HER2 closed by trastuzumab is a kind of receptor on the surface of breast cancer cells, the receptor activates the activation of multiple signal pathways in the cells, including the Ras/MAPK signaling pathway, PI3K/AKT/mTOR signaling pathway, JAK/STAT signaling pathway and PLC-y signaling pathway. The abnormalities of these signaling pathways are closely related to the proliferation and growth of breast cancer. Therefore, drug resistance produced by the therapy of trastuzumab is often closely related to abnormal signal pathways. Studies have shown that any link in the HER2 signaling pathway may lead to the resistance of trastuzumab, which has been found at present. Resistance mechanisms include the increase of the HER family receptor and the insulin like growth factor 1 receptor (insu-lin-like growth factor 1 receptor, IGF-1R), the accumulation of HER2 truncated mutant p95-HER2, and the abnormal activation of the PI3K/AKT/mTOR signaling pathway. In addition to these, these may lead to the resistance of the HER2 positive breast cancer cells to the resistance to trumpet monoclonal antibodies. In addition, the activation of the JAK/STAT pathway may also be another important mechanism for resistance, but it is rarely reported that.JAK is a class of non receptor tyrosine kinase, which includes four members of JAK1, JAK2, JAK3 and TYK2 that can be activated by phosphorylated STAT after.JAK activation, thus forming the JAK/STAT signaling pathway in the.JAK/STAT signaling pathway. There is a phenomenon of excessive activation in various tumor tissues. In breast cancer, the total expression of STAT3 and phosphorylated STAT3 is significantly higher than that of normal breast tissue, and it is closely related to the degree of invasion and metastasis of the tumor. But the activation of STAT3 is not reported to be related to the resistance of breast cancer cells to the treatment of trastuzumab. Therefore, we should first study the HER2 positive breast cancer cells, observe the activation of STAT3 during the treatment of trastuzumab, and use the gene silencing technique and the specific inhibitor combined with trastuzumab to observe the cell response, so as to determine the activation of STAT3 in the tolerance of breast cancer cells to the resistance of trastuzumab. The role of.AG490 is to provide theoretical basis and scientific basis for further clinical treatment. A JAK/STAT pathway specific inhibitor,.AG490, is a competitive binding tyrosine kinase JAK2, which inhibits the activation of JAK by inhibiting its active.AG490, resulting in the inhibition of STAT3 phosphorylation, resulting in the failure of STAT3 to enter the nucleus. Related DNA elements are combined to inhibit the proliferation of tumor cells. Currently, AG490 has become an anticancer drug in the clinical treatment of various tumors. But it is not clear whether AG490 can sensitized to curfdrop monoclonal antibody. In addition, the activation of STAT3 can increase the expression of anti apoptotic gene Bcl-xL, Bcl-2 and Mcl-1, and activate the expression of angiogenic growth factor VEGF. It can increase the expression of cell cycle regulation protein cyclins D1/D2 and cell proliferation promoting protein c-myc and Survivin, and down regulation of p53 expression of apoptosis promoting gene. In particular, recent studies have found that tumor suppressor gene SARI may also participate in JAK/STAT signal mediated tumor.SARI, known as BATF2, in normal human tissues. High levels of expression in the corresponding tumor cells decrease. Exogenous expression of SARI can inhibit the proliferation of tumor cells, but overexpression of SARI has no obvious proliferation inhibition effect on normal cells. However, the molecular mechanism of SARI is not completely clear, especially if the upstream signal regulating the expression of SARI is not clear if the effect of.AG490 action is not clear. To activate SARI to kill the breast cancer cells is also worth further exploring. Methods the inhibitory effect of different concentrations of trastuzumab on the proliferation of SK-BR3 in HER2 positive cells was measured by CCK-8 experiment. The effect of different concentration of trastuzumab on the SK-BR3 death of HER2 positive cells was determined by trypan blue staining. The effect of different concentration of trastuzumab on the SK-BR3 clone formation ability of HER2 positive cells was determined. The activation of STAT3 in SK-BR3 cells after the action of trastuzumab was detected by Western blot, and then STAT3 in SK-BR3 cells was silenced by siRNA and immunoblotting was used to determine the silencing effect, and then through CCK-8 experiment and trypan blue. The effect of trastuzumab on the proliferation and death of SK-BR3 cells after STAT3 was observed. Further use of STAT3 inhibitor AG490 alone or with trastuzumab to treat SK-BR3 cells, the effects of different treatments on cell proliferation and death were observed by the CCK-8 experiment and trypan blue experiment. Then the same strategy was adopted by the CCK-8 experiment. The inhibitory effects of different concentrations of AG490 on the proliferation of HER2 positive and HER2 negative cells were measured, and the effects of AG490 on HER2 positive and HER2 negative cell death were measured by trypan blue assay, and the cloning ability of AG490 with different concentrations of AG490 to determine the ability of HER2 positive and HER2 negative cells to be cloned respectively. The activation of SARI in cells after AG490 action was detected by immunoblotting, and then the effect of silence was determined by SARI in siRNA silencing cells and Western blot test. Then the effects of AG490 on cell proliferation and death after silent SARI were observed by CCK-8 experiment and trypan blue experiment. SARI gene was predicted by biological information method. The STAT3 binding site in the promoter region was initiated and the promoter region of the SARI gene was amplified from the breast cancer cells by PCR method, and the fragments contained or not included in the STAT3 binding site were amplified and inserted into the report gene carrier pGL3, and then transfected to the cells. The changes of luciferase activity before and after AG490 treatment were observed, and the changes of the luciferase activity before and after AG490 treatment were observed. To determine whether AG490 can increase its expression and anticancer by up regulation of SARI, trastuzumab can inhibit the proliferation of SK-BR3 cells in a dose-dependent manner, and the relative proliferation rates of 0.25,0.5,1.0,2.0,4.0 mg/ml trastuzumab are 76.22%, 72.97%, 61.08%, 42.16% and 23.78%., as well as dose of trastuzumab. The relative mortality of SK-BR3 cell death 0.25,0.5,1.0,2.0,4.0 m//ml trastuzumab is: 10.35+1.66%, 18.37+2.49%, 31.97 + 3.08%, 46.78 + 5.67%, 63.77 + 7.81%., compared with the control group. The clones of SK-BR3 cells can be inhibited in a dose-dependent manner, 0.25,0.5,1.0,2.0,4.0 M The relative cell clone formation rates of g/ml trastuzumab are: 64.79+8.38%, 35.21 + 13.29%, 26.03 + 10.79%, 16.29 + 10.34%, 6.18 + 6.06%., which can also activate STAT3 signaling pathway in SK-BR3 cells in a concentration dependent manner, reflecting a significant increase in the level of phosphorylated STAT3. ST in SK-BR3 cells with specific siRNA After AT3, CCK-8 experimental analysis showed that the inhibitory rate of trastuzumab to SK-BR3 cells could be significantly enhanced. Trypan blue experimental analysis found that the death rate of trastuzumab to SK-BR3 cells could be significantly increased by.CCK-8 experimental results, and compared with AG490 alone or trastuzumab alone, AG490 combined with trastuzumab. The proliferation inhibition rate of SK-BR3 cells was significantly increased; trypan blue experimental results also showed that AG490 combined with trastuzumab could significantly increase the mortality of SK-BR3 cells compared with the single treatment group of AG490 alone or trastuzumab. The results of this study further demonstrated that the inhibition of the STAT3 signaling pathway could indeed increase the number of trastuzolones. The anti SK-BR3 cell killing rate.AG490 can inhibit the proliferation of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. The IC50 values of AG490 in two cells are respectively 43.281 M and 28.327 u M. AG490, respectively, to promote the death of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. The mortality of the control group of the SK-BR3 cells is the same. 4%, the relative mortality of AG490 treatment group was 5.78 + 1.67%, 16.89+2.67%, 31.08 + 4.65% according to the increase of concentration gradient (25,50100 M), while in MDA-MB-231 cells, the mortality of the control group was 1.77+0.66%, and the mortality rate of AG490 treatment group in accordance with the increase of concentration gradient (25,50100 u M) was 8.05+2.05%, 27.26 +. 2.89%, 44.18 + 4.98%., compared with the control group, AG490 could inhibit the clone formation of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. In SK-BR3 cells, the number of cell clones in the control group and the experimental group were 559 + 52361+38206 + 29117 + 15, respectively, in the MDA-MB-231 cells, the control group and the experimental group. The number of cell clones was 389 + 42211+23149 + 17,68 + 13 respectively. Quantitative PCR detection showed that the mRNA level of SARI in SK-BR3 and MDA-MB-231 cells increased significantly with the increase of AG490 concentration, and Western blot analysis further confirmed that AG490 could be used to enhance the SK-BR3 and protein in the cells in dose-dependent manner. The inhibitory effect of AG490 on the proliferation of MDA-MB-231 cells after SARI was reduced. Bioinformatics analysis found that there were 1 classical STAT3 binding sites in the position from -1787 to -1797. The two fragments containing the different sizes of the SARI promoter region (-1 to -2000bp, -1 to -1700bp) were cloned into the reported plasmid pGL3-basic, respectively. The luciferase reporter gene was detected after transfection of MDA-MB-231 cells with the control plasmid, and there was no significant change in the luciferase activity of the -1 to -1700bp fragments without the STAT3 binding site before and after AG490 treatment, while the -1 to -2000bp fragment containing the binding site increased significantly after the AG490 treatment. After the action of AG490, the expression of SARI can be enhanced by increasing the activity of SARI promoter, which may be related to the inhibition of AG490 to STAT3 and the inhibition of STAT3 on the activity of SARI promoter. Conclusion 1. trastuzumab can inhibit the proliferation of HER2 positive breast cancer cells in a dose-dependent manner, promote its death, and inhibit it. The formation of cell clones.2. trastuzumab leads to the activation of STAT3 in HER2 positive breast cancer cells. Silence STAT3 or inhibition of STAT3 activity can enhance the sensitivity of HER2 positive breast cancer cells to trastuzumab..3.AG490 can inhibit the proliferation of HER2 positive and HER2 negative breast cancer cells in a dose-dependent manner and promote their death. .4.AG490 treatment led to the formation of SARI expression in HER2 positive and HER2 negative breast cancer cells, and the silence SARI could reduce the binding site of STAT3 in the promoter region of the breast cancer cell to AG490 sensitivity.5.SARI gene, and the ability to activate the promoter fragment without the binding site was significant. The activation of 1.STAT3 is an important mechanism for the tolerance of HER2 positive breast cancer cells to trastuzumab,.2.AG490 can effectively enhance the sensitivity of HER2 positive breast cancer cells to trastuzumab, and the.3.AG490 killing of breast cancer cells is related to the up regulation of the tumor suppressor gene SARI.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.9
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1 路釗;鄭少鵬;牛靜;賈弘y，
本文編號：2152710
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