RNA激活上调PTEN表达用于逆转EGFR突变NSCLC患者TKI耐药的研究
发布时间：2018-08-02 09:07
【摘要】：研究背景：肺癌是目前世界上最常见的恶性肿瘤,死亡率位居恶性肿瘤之首。随着基因组学的发展及靶向药物的开发和应用,晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)的一线治疗正由传统的化疗转变为个体化的分子靶向治疗。表皮生长因子受体(epidermal growth factor receptor,EGFR)酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKI)在目前的NSCLC的治疗中得到了广泛应用,尤其不吸烟的亚洲女性患者及存在EGFR突变的患者对EGFR-TKI治疗效果尤为显著。多项临床研究包括EURTAC.WJTOG340、 IPASS、NEJ0025等，，已证实EGFR-TKI作为一线药物用于治疗EGFR存在敏感突变的晚期NSCLC患者,其效果优于化疗。然而,一些NSCLC患者对EGFR-TKI存在原发性耐药现象。另外,即便是对EGFR-TKI治疗有效的患者,绝大多数也会在一年内出现获得性耐药。所以,对于接受分子靶向治疗的肺癌患者,无论对EGFR-TKI治疗是否有效,最终都难以逃脱面疾病进展及复发的厄运。EGFR-TKI的耐药问题目前已经成为NSCLC靶向治疗临床应用的主要瓶颈,只有明确了EGFR-TKI的耐药原因和机制，才能够找到克服耐药的方法,从而使更多的NSCLC患者从分子靶向治疗中获益。根据目前研究,T790M突变以及MET扩增是最为常见的两种获得性耐药机制,二者约占到所有获得性耐药的60%。另外40%的机制尚未完全阐明,其中研究相对较多的为张力蛋白同源10号染色体缺失的磷酸酶(Phosphatase and tensin homolog deleted on chromosome ten,PTEN)抑癌基因的缺失。PTEN是目前为止发现的第一个具有磷酸酶活性的抑癌基因,其表达产物在PI3K/Akt信号通路中发挥着重要的作用。它可以抑制下游信号通路中Akt的活化,控制细胞的增殖,从而起到抑制肿瘤生长的作用。研究表明PTEN在多种恶性肿瘤细胞系中(包括肺癌、食管癌、肝癌等)处于低表达状态,并对肿瘤的发生发展起重要作用。国外Kokubo等人的研究表明PTEN低表达与吉非替尼获得性耐药相关。Endoh等人的研究结果也表明了在接受吉非替尼治疗的患者中PTEN表达水平较高的患者预后较好,而EGFR突变的患者若存在PTEN缺失,EGFR-TKI治疗效果不佳。Sos等人的研究显示,PTEN可通过激活Akt导致EGFR-TKI耐药。Zhuang等人在2009年通过X线照射的方式上调了H-157肺癌细胞PTEN的表达水平,增加了癌细胞对吉非替尼的敏感性。以上研究均表明PTEN在T790M突变以及MET扩增以外的EGFR-TKI耐药机制中扮演了相当重要的角色。2006年Li等人在研究针对DNA启动子序列的dsRNA对基因表达调控的过程中发现,针对启动子区的非CpG岛序列为靶点的dsRNA可以引起某些表达水平较低的基因的表达增高,并将这种现象定义RNA激活(RNAActivation,RNAa)。他将具有RNA激活功能的小RNA分子称为“小激活RNA”(small activating RNA,saRNA)。RNAa与RNA干拢(RNA interfering,RNAi)对目标基因表达的影响,一个是增强,一个是抑制,二者均是通过向细胞内导人小片段双链RNA而发挥作用,都有高效、特异、相对容易操作的特点。RNAa现象的发现为肿瘤的基因治疗提供了新的策略。RNAa能够特异性地激活细胞内已经存在的表达水平较低的基因,在应用于治疗肿瘤时,不需要考虑肿瘤是否存在特定的致癌基因,也不需要导人新的基因。因此我们能够更加容易的使某个低表达的基因特异性增强,具有独特的优势。我们可以利用RNAa激活肿瘤抑制基因、细胞凋亡基因以及细胞周期抑制基因等来治疗肿瘤。Li等人分析了p21Waf1/Cip1(p21)和人钙粘连素E基因启动子结构,在该序列的非CpG岛序列上筛选出能激活下游基因转录的saRNA作用于靶位点。他合成了长度为21个核苷酸的saRNA,然后将该saRNA转染到前列腺癌、宫颈癌、人膀胱癌、和乳腺癌等多种体外肿瘤细胞中,结果发现转染了saRNA分子的肿瘤细胞与对照组细胞相比,钙粘连素E和p21基因的表达水平在不同的细胞出现了2～13倍的增加,肿瘤细胞的生长受到显著抑制。这些研究虽然多数是体外实验,但已经取得了较好的成果,预示RNAa技术在肿瘤基因治疗上具有极为良好的发展前景。PTEN在癌细胞中的缺失、低表达与EGFR-TKI的耐药密切相关,目前尚未有利用RNA激活技术上调肺癌PTEN表达的相关研究。鉴于此,本研究选择PTEN为靶点，设计合成针对PTEN基因启动子的saRNA来进实验,旨在探讨RNAa上调PTEN基因表达克服EGFR-TKI耐药的可行性及有效性为EGFR-TKI耐药后的治疗寻找新的方法及思路。第一部分PTEN表达与非小细胞肺癌TKI耐药关系的研究目的：通过对人非小细胞肺癌H-157、H-1355、H-1650三种细胞PTEN、 pAkt的表达强度及三种细胞对TKI的疗效进行分析，探讨PTEN低表达或表达缺失对TKI耐药的影响,并研究其导致耐药的可能机制。方法：使用逆转录PCR及Western blot对H-157、H-1355、H-1650三种肺癌细胞进行检测分别从RNA和蛋白水平评估PTEN的表达强度。绘制TKI处理前后的三种细胞的生长曲线并使用流式细胞仪检测细胞凋亡的情况，评价TKI对三种细胞的疗效。使用Western blot检测TKI处理后的三种肺癌细胞pAkt的表达情况以探讨PTEN低表达导致TKI耐药的机制。结果：H-1355细胞PTEN表达强度最高,H-157细胞表达强度较低,而H-1650细胞中PTEN蛋白表达缺失经过TKI药物处理后H-1355的生长曲线明显下移,细胞有明显的凋亡(p0.05),H-1650细胞的生长曲线略有下移,未见有明显的细胞凋亡(p0.05),H-157细胞的生长曲线没有明显变化,未见有明显的细胞凋亡(p0.05)：经过TKI处理后H-1355细胞pAkt呈现低表达，H-157及H-1650都呈现高表达状态。结论：TKI的耐药与PTEN的表达强度有一定的关系。PTEN表达水平正常的肺癌细胞对TKI治疗较为敏感,而PTEN缺失或者PTEN表达明显下降的肺癌细胞对TKI治疗效果不明显。因此PTEN的低表达或者表达缺失可能是导致肺癌对TKI耐药的机制之一。而PTEN低表达或表达缺失导致TKI耐药的原因可能与其所调控的Akt/PI3K通路的持续活化有关。第二部分针对PTEN基因的小激活RNA的构建与筛选目的：针对PTEN基因,设计并筛选出具有明显激活功能的saRNAo方法：根据现有文献报道的saRNA设计原则,设计5条dsRNA作为候选saRNA,交由上海生工公司化学合成后转染人人非小细胞肺癌H-157细胞。通过RT-PCR检测转染后细胞内PTEN的表达强度,筛选出具有激活功能的saRNAo然后将筛选出的功能性saRNA转染至H-157细胞,使用Western blot检测PTEN蛋白水平的表达强度,评估saRNA的激活作用是否适用于肺组织细胞中。结果：我们设计出的5条候选dsRNA中有3条可以使H-157细胞中的PTEN表达上调,其中的一条可以使PTEN的表达强度增强2倍以上。将该saRNA转染人H-157细胞后,可以从RNA及蛋白水平增强PTEN的表达。结论：RNA激活现象同样存在于肺组织细胞中,通过向肺癌细胞内转人针对PTEN的功能性saRNA,可以特异性的将PTEN的表达上调。第三部分saRNA上调PTEN表达对肺癌TKI耐药的影响目的：通过将筛选出的saRNA转染人H-157细胞上调PTEN的表达,观察PTEN的表达上调能否逆转TKI的耐药状态。方法：将筛选出的功能性saRNA转染至H-157细胞,使用RT-PCR及Western blot方法分别从RNA及蛋白水平检测PTEN的表达强度，评估saRNA能否使PTEN的表达上调。同时通过细胞生长曲线及细胞凋亡的检测观察转染saRNA后的H-157细胞对TKI药物的敏感性,评估PTEN的表达上调能否克服TKI耐药。结果：saRNA转染至H-157细胞后,RT-PCR及Western blot检测结果均显示PTEN的表达水平较dsControl组及mock组明显增强(p0.05),这说明saRNA在H-157细胞内发挥了RNA激活作用使得PTEN的表达上调。生长曲线显示转染saRNA 72小时后,saRNA组细胞的生长明显受抑制,与其他两组差异有显著统计学意义(p0.05)。细胞凋亡结果显示saRNA组细胞凋亡率为17.82±2.37%,dsControl组细胞凋亡率为1.74+0.17%,mock组细胞凋亡率为0.33+0.14%(p0.05)。结论:PTEN的低表达是造成TKI耐药的机制之一,导致耐药的原因是PTEN的低表达造成Akt的过度磷酸化,从而使Akt/PI3K通路活化,引起耐药。通过筛选出的功能性saRNA可以上调PTEN的表达水平,从而增加肺癌细胞对TKI的敏感性,逆转TKI的耐药状态。
[Abstract]:Background: lung cancer is the most common malignant tumor in the world and the death rate is the first in malignant tumor. With the development of genomics and the development and application of targeted drugs, the first-line treatment of advanced non small cell lung cancer (non-small cell lung cancer, NSCLC) is changing from traditional chemotherapy to individualized molecular targeting therapy. The growth factor receptor (epidermal growth factor receptor, EGFR) tyrosine kinase inhibitor (tyrosine kinase inhibitors, TKI) has been widely used in the current treatment of NSCLC, especially in non smoking Asian women and those with EGFR mutations, which are particularly effective for EGFR-TKI treatment. G340, IPASS, NEJ0025, and so on, have proved EGFR-TKI as a first-line drug for the treatment of EGFR with sensitive mutations in late NSCLC patients, the effect is better than chemotherapy. However, some NSCLC patients have primary drug resistance to EGFR-TKI. In addition, the overwhelming majority of patients who are effective for EGFR-TKI treatment will also have acquired resistance within one year. Therefore, for patients with lung cancer receiving molecular targeted therapy, whether the treatment of EGFR-TKI is effective or not, it is difficult to escape the problem of resistance to the progression of the face disease and the relapse of the drug-resistant.EGFR-TKI, which has become the main bottleneck in the clinical application of the target therapy of NSCLC. Only the reason and mechanism of the drug resistance of EGFR-TKI can be found to be overcome. The drug resistance method makes more NSCLC patients benefit from molecular targeting therapy. According to current research, T790M mutation and MET amplification are the two most common mechanisms of acquired resistance. The two other 40% mechanisms for all acquired resistance to 60%. have not been fully elucidated, and more of them are tenmifying protein homologous 10. The deletion of the phosphatase (Phosphatase and tensin homolog deleted on chromosome ten, PTEN) the deletion of the tumor suppressor gene is the first tumor suppressor gene found with the activity of phosphatase so far. Its expression product plays an important role in the PI3K/Akt signaling pathway. It can inhibit the downstream signal pathway in the Akt. The study shows that PTEN is in a low expression state in a variety of malignant tumor cell lines (including lung, esophageal and liver cancer), and plays an important role in the development of tumor. The study of foreign Kokubo et al. Shows that the low expression of PTEN is associated with the acquired resistance of gefitinib. .Endoh et al's results also suggest that patients with high PTEN expression in gefitinib patients have a better prognosis, while EGFR mutations in patients with PTEN deletion and EGFR-TKI treatment are not effective in.Sos et al. Studies show that PTEN can lead to EGFR-TKI - resistant.Zhuang and others through X - ray exposure in 2009 by activating Akt The expression level of PTEN in H-157 lung cancer cells increased and the sensitivity of cancer cells to gefitinib was increased. All of these studies showed that PTEN played a significant role in the mechanism of T790M mutation and EGFR-TKI resistance other than MET amplification,.2006 Li et al. In the study of the gene expression regulation of dsRNA for the DNA promoter sequence. It was found that dsRNA, the target of the non CpG Island sequence of the promoter region, could cause a higher expression of some genes with lower levels of expression, and defined the phenomenon as RNA activation (RNAActivation, RNAa). He called the small RNA molecule with the RNA activation function as "small activated RNA" (small activating RNA, saRNA) and dried together. The effects of interfering, RNAi) on the expression of target genes, one is enhancement, one is inhibition, and the two are both effective, specific, and relatively easy to operate by the small fragment of double stranded RNA. The discovery of the.RNAa phenomenon provides a new strategy for the treatment of tumor gene therapy,.RNAa can specifically activate the cells. In the treatment of tumors, a gene with a lower level of expression is not required to consider whether a tumor has a specific oncogene or a new gene. Therefore, we can make a particular gene specific enhancement of a low expression more unique. We can use RNAa to activate tumor inhibition. Gene, apoptosis gene, and cell cycle inhibition gene were used to treat tumor.Li and others to analyze the promoter structure of p21Waf1/Cip1 (p21) and human cadherin E gene, and to screen out saRNA that can activate downstream gene transcription on the target site on the non CpG Island sequence of this sequence. He synthesized the saRNA of 21 nucleotides, and then The saRNA was transfected into a variety of extracorporeal tumor cells, such as prostate cancer, cervical cancer, human bladder cancer, and breast cancer. The results showed that the expression level of saRNA and the expression level of calcin E and p21 gene increased 2~13 times in different cells compared with the control group, and the growth of tumor cells was significantly inhibited. Although most of the studies are in vitro experiments, good results have been achieved, which indicates that RNAa technology has a very good development prospect in tumor gene therapy, the lack of.PTEN in cancer cells, low expression is closely related to the drug resistance of EGFR-TKI. At present, there is no related research on the use of RNA activation technology to increase the expression of PTEN in lung cancer. In this study, we selected PTEN as the target to design and synthesize the saRNA for the PTEN gene promoter, aiming at exploring the feasibility and effectiveness of RNAa up regulation of PTEN gene expression to overcome EGFR-TKI resistance and finding new ways and ideas for the treatment of EGFR-TKI after drug resistance. The first part of the study of the relationship between PTEN expression and TKI resistance in non-small cell lung cancer By analyzing the expression intensity of three cells of human non-small cell lung cancer (H-157, H-1355, H-1650), the expression intensity of pAkt and the effect of three kinds of cells on TKI, the effect of PTEN low expression or expression loss on the drug resistance of TKI was investigated and the possible mechanism of drug resistance was studied. Method: to make use of reverse transcriptase PCR and Western blot to H-157, three The expression intensity of PTEN was evaluated from RNA and protein levels. The growth curve of three cells before and after TKI treatment was plotted and the apoptosis was detected by flow cytometry. The effect of TKI on three kinds of cells was evaluated. The expression of pAkt in three lung cancer cells treated with TKI was detected by Western blot The mechanism of low expression of PTEN led to TKI resistance. Results: the expression of PTEN in H-1355 cells was the highest, and the expression intensity of H-157 cells was low, while the growth curve of PTEN protein expression in H-1650 cells was obviously down after TKI drug treatment, and the cells had obvious apoptosis (P0.05), and the growth curve of H-1650 cells was slightly down, and there was no obvious change in the growth curve of H-1650 cells. There was no obvious change in the growth curve of H-157 cells and no obvious cell apoptosis (P0.05): pAkt showed low expression in H-1355 cells after TKI treatment, and both H-157 and H-1650 showed high expression state. Conclusion: the resistance of TKI to PTEN expression intensity has certain relation to the normal lung cancer cells with.PTEN expression level. It is more sensitive to TKI therapy, but the effect of PTEN deletion or the decreased expression of PTEN on TKI is not obvious. Therefore, the low expression of PTEN or the lack of expression may be one of the mechanisms that lead to the resistance of lung cancer to TKI. The reason that the low expression of PTEN or the deletion of the expression causes the drug resistance of TKI may be related to the holding of the Akt/PI3K pathway controlled by the PTEN. The second part is aimed at the construction and screening of the small activation RNA of PTEN gene: designed and screened the saRNAo method with obvious activation function for the PTEN gene: according to the saRNA design principle reported in the existing literature, the 5 dsRNA is designed as the candidate saRNA, and the transfection of the Shanghai raw material company after chemical synthesis is not small. Cell lung cancer H-157 cells. The expression intensity of PTEN in the cells after transfection was detected by RT-PCR. The activation function was screened and then the functional saRNA was transfected into H-157 cells. The expression intensity of PTEN protein level was detected by Western blot, and the activation of saRNA was evaluated in lung tissue cells. 3 of the 5 candidate dsRNA we designed can increase the expression of PTEN in H-157 cells, one of which can increase the expression intensity of PTEN by more than 2 times. After transfection of the saRNA to human H-157 cells, the expression of PTEN can be enhanced from RNA and protein levels. Conclusion: RNA activation is also present in lung tissue cells, through the lung. The tumor cells transferred to the functional saRNA of PTEN, the expression of PTEN can be specifically up-regulated. Third part saRNA up-regulated the effect of PTEN expression on the drug resistance of lung cancer. By using the selected saRNA transfected human H-157 cells to up-regulation the expression of PTEN, whether the up regulation of PTEN expression can reverse the TKI drug resistance state. The functional saRNA was transfected into H-157 cells, and the expression intensity of PTEN was detected from RNA and protein levels by RT-PCR and Western blot methods. The expression of PTEN was up regulated by saRNA, and the sensitivity of H-157 cell to TKI drugs was evaluated through the detection of cell growth curve and apoptosis. TKI resistance was overcome. Results: after saRNA transfection to H-157 cells, the results of RT-PCR and Western blot showed that the expression level of PTEN was significantly higher than that of dsControl and mock groups (P0.05), which indicated that saRNA in the H-157 cells was activated to make the expression up. The growth curve showed that the transfection was 72 hours after transfection. The growth of the group cells was obviously inhibited, and there was significant difference with the other two groups (P0.05). Apoptosis results showed that the apoptosis rate of group saRNA was 17.82 + 2.37%, the apoptosis rate of group dsControl was 1.74+0.17%, and the apoptosis rate of mock group was 0.33+0.14% (P0.05). Conclusion: the low expression of PTEN is one of the mechanisms causing TKI resistance. The reason for the resistance is that the low expression of PTEN causes excessive phosphorylation of Akt, which causes the activation of Akt/PI3K pathway and causes resistance. Through the screening of functional saRNA, the expression level of PTEN can be up-regulated, thus increasing the sensitivity of lung cancer cells to TKI and reversing the drug resistance of TKI.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
本文编号：2158944
[Abstract]:Background: lung cancer is the most common malignant tumor in the world and the death rate is the first in malignant tumor. With the development of genomics and the development and application of targeted drugs, the first-line treatment of advanced non small cell lung cancer (non-small cell lung cancer, NSCLC) is changing from traditional chemotherapy to individualized molecular targeting therapy. The growth factor receptor (epidermal growth factor receptor, EGFR) tyrosine kinase inhibitor (tyrosine kinase inhibitors, TKI) has been widely used in the current treatment of NSCLC, especially in non smoking Asian women and those with EGFR mutations, which are particularly effective for EGFR-TKI treatment. G340, IPASS, NEJ0025, and so on, have proved EGFR-TKI as a first-line drug for the treatment of EGFR with sensitive mutations in late NSCLC patients, the effect is better than chemotherapy. However, some NSCLC patients have primary drug resistance to EGFR-TKI. In addition, the overwhelming majority of patients who are effective for EGFR-TKI treatment will also have acquired resistance within one year. Therefore, for patients with lung cancer receiving molecular targeted therapy, whether the treatment of EGFR-TKI is effective or not, it is difficult to escape the problem of resistance to the progression of the face disease and the relapse of the drug-resistant.EGFR-TKI, which has become the main bottleneck in the clinical application of the target therapy of NSCLC. Only the reason and mechanism of the drug resistance of EGFR-TKI can be found to be overcome. The drug resistance method makes more NSCLC patients benefit from molecular targeting therapy. According to current research, T790M mutation and MET amplification are the two most common mechanisms of acquired resistance. The two other 40% mechanisms for all acquired resistance to 60%. have not been fully elucidated, and more of them are tenmifying protein homologous 10. The deletion of the phosphatase (Phosphatase and tensin homolog deleted on chromosome ten, PTEN) the deletion of the tumor suppressor gene is the first tumor suppressor gene found with the activity of phosphatase so far. Its expression product plays an important role in the PI3K/Akt signaling pathway. It can inhibit the downstream signal pathway in the Akt. The study shows that PTEN is in a low expression state in a variety of malignant tumor cell lines (including lung, esophageal and liver cancer), and plays an important role in the development of tumor. The study of foreign Kokubo et al. Shows that the low expression of PTEN is associated with the acquired resistance of gefitinib. .Endoh et al's results also suggest that patients with high PTEN expression in gefitinib patients have a better prognosis, while EGFR mutations in patients with PTEN deletion and EGFR-TKI treatment are not effective in.Sos et al. Studies show that PTEN can lead to EGFR-TKI - resistant.Zhuang and others through X - ray exposure in 2009 by activating Akt The expression level of PTEN in H-157 lung cancer cells increased and the sensitivity of cancer cells to gefitinib was increased. All of these studies showed that PTEN played a significant role in the mechanism of T790M mutation and EGFR-TKI resistance other than MET amplification,.2006 Li et al. In the study of the gene expression regulation of dsRNA for the DNA promoter sequence. It was found that dsRNA, the target of the non CpG Island sequence of the promoter region, could cause a higher expression of some genes with lower levels of expression, and defined the phenomenon as RNA activation (RNAActivation, RNAa). He called the small RNA molecule with the RNA activation function as "small activated RNA" (small activating RNA, saRNA) and dried together. The effects of interfering, RNAi) on the expression of target genes, one is enhancement, one is inhibition, and the two are both effective, specific, and relatively easy to operate by the small fragment of double stranded RNA. The discovery of the.RNAa phenomenon provides a new strategy for the treatment of tumor gene therapy,.RNAa can specifically activate the cells. In the treatment of tumors, a gene with a lower level of expression is not required to consider whether a tumor has a specific oncogene or a new gene. Therefore, we can make a particular gene specific enhancement of a low expression more unique. We can use RNAa to activate tumor inhibition. Gene, apoptosis gene, and cell cycle inhibition gene were used to treat tumor.Li and others to analyze the promoter structure of p21Waf1/Cip1 (p21) and human cadherin E gene, and to screen out saRNA that can activate downstream gene transcription on the target site on the non CpG Island sequence of this sequence. He synthesized the saRNA of 21 nucleotides, and then The saRNA was transfected into a variety of extracorporeal tumor cells, such as prostate cancer, cervical cancer, human bladder cancer, and breast cancer. The results showed that the expression level of saRNA and the expression level of calcin E and p21 gene increased 2~13 times in different cells compared with the control group, and the growth of tumor cells was significantly inhibited. Although most of the studies are in vitro experiments, good results have been achieved, which indicates that RNAa technology has a very good development prospect in tumor gene therapy, the lack of.PTEN in cancer cells, low expression is closely related to the drug resistance of EGFR-TKI. At present, there is no related research on the use of RNA activation technology to increase the expression of PTEN in lung cancer. In this study, we selected PTEN as the target to design and synthesize the saRNA for the PTEN gene promoter, aiming at exploring the feasibility and effectiveness of RNAa up regulation of PTEN gene expression to overcome EGFR-TKI resistance and finding new ways and ideas for the treatment of EGFR-TKI after drug resistance. The first part of the study of the relationship between PTEN expression and TKI resistance in non-small cell lung cancer By analyzing the expression intensity of three cells of human non-small cell lung cancer (H-157, H-1355, H-1650), the expression intensity of pAkt and the effect of three kinds of cells on TKI, the effect of PTEN low expression or expression loss on the drug resistance of TKI was investigated and the possible mechanism of drug resistance was studied. Method: to make use of reverse transcriptase PCR and Western blot to H-157, three The expression intensity of PTEN was evaluated from RNA and protein levels. The growth curve of three cells before and after TKI treatment was plotted and the apoptosis was detected by flow cytometry. The effect of TKI on three kinds of cells was evaluated. The expression of pAkt in three lung cancer cells treated with TKI was detected by Western blot The mechanism of low expression of PTEN led to TKI resistance. Results: the expression of PTEN in H-1355 cells was the highest, and the expression intensity of H-157 cells was low, while the growth curve of PTEN protein expression in H-1650 cells was obviously down after TKI drug treatment, and the cells had obvious apoptosis (P0.05), and the growth curve of H-1650 cells was slightly down, and there was no obvious change in the growth curve of H-1650 cells. There was no obvious change in the growth curve of H-157 cells and no obvious cell apoptosis (P0.05): pAkt showed low expression in H-1355 cells after TKI treatment, and both H-157 and H-1650 showed high expression state. Conclusion: the resistance of TKI to PTEN expression intensity has certain relation to the normal lung cancer cells with.PTEN expression level. It is more sensitive to TKI therapy, but the effect of PTEN deletion or the decreased expression of PTEN on TKI is not obvious. Therefore, the low expression of PTEN or the lack of expression may be one of the mechanisms that lead to the resistance of lung cancer to TKI. The reason that the low expression of PTEN or the deletion of the expression causes the drug resistance of TKI may be related to the holding of the Akt/PI3K pathway controlled by the PTEN. The second part is aimed at the construction and screening of the small activation RNA of PTEN gene: designed and screened the saRNAo method with obvious activation function for the PTEN gene: according to the saRNA design principle reported in the existing literature, the 5 dsRNA is designed as the candidate saRNA, and the transfection of the Shanghai raw material company after chemical synthesis is not small. Cell lung cancer H-157 cells. The expression intensity of PTEN in the cells after transfection was detected by RT-PCR. The activation function was screened and then the functional saRNA was transfected into H-157 cells. The expression intensity of PTEN protein level was detected by Western blot, and the activation of saRNA was evaluated in lung tissue cells. 3 of the 5 candidate dsRNA we designed can increase the expression of PTEN in H-157 cells, one of which can increase the expression intensity of PTEN by more than 2 times. After transfection of the saRNA to human H-157 cells, the expression of PTEN can be enhanced from RNA and protein levels. Conclusion: RNA activation is also present in lung tissue cells, through the lung. The tumor cells transferred to the functional saRNA of PTEN, the expression of PTEN can be specifically up-regulated. Third part saRNA up-regulated the effect of PTEN expression on the drug resistance of lung cancer. By using the selected saRNA transfected human H-157 cells to up-regulation the expression of PTEN, whether the up regulation of PTEN expression can reverse the TKI drug resistance state. The functional saRNA was transfected into H-157 cells, and the expression intensity of PTEN was detected from RNA and protein levels by RT-PCR and Western blot methods. The expression of PTEN was up regulated by saRNA, and the sensitivity of H-157 cell to TKI drugs was evaluated through the detection of cell growth curve and apoptosis. TKI resistance was overcome. Results: after saRNA transfection to H-157 cells, the results of RT-PCR and Western blot showed that the expression level of PTEN was significantly higher than that of dsControl and mock groups (P0.05), which indicated that saRNA in the H-157 cells was activated to make the expression up. The growth curve showed that the transfection was 72 hours after transfection. The growth of the group cells was obviously inhibited, and there was significant difference with the other two groups (P0.05). Apoptosis results showed that the apoptosis rate of group saRNA was 17.82 + 2.37%, the apoptosis rate of group dsControl was 1.74+0.17%, and the apoptosis rate of mock group was 0.33+0.14% (P0.05). Conclusion: the low expression of PTEN is one of the mechanisms causing TKI resistance. The reason for the resistance is that the low expression of PTEN causes excessive phosphorylation of Akt, which causes the activation of Akt/PI3K pathway and causes resistance. Through the screening of functional saRNA, the expression level of PTEN can be up-regulated, thus increasing the sensitivity of lung cancer cells to TKI and reversing the drug resistance of TKI.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
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