兰州大学机构库 >第二临床医学院
利用超声靶向微泡破坏技术沉默 PDLIM5 探索非小细胞性 肺癌自噬与耐药关系的实验研究
Alternative TitleExploring the Relationship Between Autophagy and Drug Resistance in NSCLC Cells by Silencing PDLIM5 Using Ultrasound-targeted Microbubbles Destruction Technology
张瑶
Subtype博士
Thesis Advisor聂芳
2023-05-31
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学博士
Degree Discipline影像医学与核医学
Keyword非小细胞性肺癌 Non-small-cell lung cancer (NSCLC) 肿瘤细胞耐药 Drug resistance of tumor cell 超声微泡靶向破坏技术 Ultrasound targeted microbubble destruction (UTMD) 纳米材料 nanomaterials PDLIM5 PDLIM5 细胞自噬 autophagy 基因沉默 gene silencing
Abstract

背景及目的:

人类肺癌是目前人类肿瘤中恶性程度最高、侵袭性最强的一种,是全球癌症相关死亡的主要原因,其中NSCLC是最常见的组织学类别。近年来,随着基因靶向治疗的发展,它逐渐成为包括NSCLC在内的人类癌症抗肿瘤治疗的主要方法。在所有的NSCLC基因靶向药物中,表皮生长因子酪氨酸激酶抑制剂(EGFR-TKIs)已被广泛研究,并逐渐成为最具代表性的药物之一。然而,大多数伴有EGFR突变的NSCLC患者在接受EGFR-TKIs治疗后的总生存率并没有得到改善。其主要原因是他们在治疗过程中不可避免地会出现耐药性。因此,耐药性已成为目前包括NSCLC患者在内的许多肿瘤患者化疗和靶向治疗失败的最重要原因之一。

PDLIM5作为一种细胞骨架相关蛋白,参与膜相关蛋白、细胞骨架蛋白和各种信号分子的信号调控过程,并参与多种肿瘤的进展。研究表明,PDLIM5在肿瘤细胞的增殖、侵袭和迁移中起着至关重要的作用。有研究表明,PDLIM5蛋白的高表达与NSCLC的增殖、侵袭和凋亡密切相关。然而,PDLIM5是否与NSCLC患者的耐药性有关尚不清楚,需要我们进一步探索。

近年来,超声靶向微泡破坏(UTMD)技术作为一种新的无创方法,可通过在局部靶向爆破微泡从而破坏细胞膜,并增加毛细血管通透性,因此,被广泛用于各种肿瘤放化疗及基因或药物的递送,从而提高外源性基因或药物到靶组织及器官的转染效率。现UTMD技术已成为近年来肿瘤及其它类型疾病治疗研究的重点,特别是通过增加不同种类siRNA的递送来提高基因治疗的效率。

随着纳米技术,特别是纳米材料的发展,其在药物传递领域具有很好的应用前景。由于其具有更高的稳定性和更长的循环时间,可通过增强通透性和保留(EPR)效应在肿瘤组织中有效积累,被广泛用于超声靶向成像和治疗。由于其高通透性、滞留效应和胞吞作用(即纳米颗粒通过内皮细胞运输进入肿瘤)等特点,使其能够顺利通过肿瘤毛细血管并在实体肿瘤中积累,从而可以提高对肿瘤细胞的治疗效果。

其中,聚合物纳米颗粒,如:聚合物如聚乳酸-共乙醇酸(PLGA)、聚乳酸(PLA)、壳聚糖和聚己内酯等已被广泛用于siRNA及药物递送。聚合物-siRNA偶联可作为最有效的siRNA传递聚合物之一。因此,它们可以被视为有价值的工具,并提供有效的开发策略。

基于以上基础,我们拟将具有PDLIM5靶向性的siRNA与具有超声响应性的聚合物无机纳米粒子连接起来,构成一个复合纳米平台,以通过高效靶向递送siRNA增加基因传递的作用,从而提高NSCLC肿瘤治疗效能。在超声、纳米科技和生物医学的协同下,我们研发设计了一种新型复合纳米制剂,即以PLGA@PEI为加载平台,负载特异性靶向PDLIM5的siRNA的超声响应性纳米制剂。该纳米颗粒具有高分散性、无毒性、生物相容性好、易于制备等特点。PEI修饰的磷脂膜包覆纳米颗粒有助于提高复合纳米制剂的生物相容性和安全性,增加治疗的疗效、降低免疫原性。我们拟制备携载特异性靶向PDLIM5 siRNA的纳米平台,对其进行表征分析,并验证其生物安全性、超声敏感性、体外抗肿瘤细胞耐药作用及机制探索,以期为治疗NSCLC耐药性探索一种新型、高效的治疗方案。

材料与方法:

1.    PDLIM5基因在PC9及PC9GR细胞中的表达

1.1 PDLIM5的生物信息学分析

分别从癌症基因组图谱(TCGA)数据集和GTEx数据库获得NSCLC的RNAseq数据(level3)和相应的临床信息,得到PDLIM5生物信息学分析结果。

1.2 PC9及PC9GR细胞系对吉非替尼耐药性的鉴定

实验将初始PC9及PC9GR细胞分别分为:阴性对照组,空白对照组及实验组(吉非替尼药物浓度分别为2、4、8、16、32、64、128μg/L)。通过细胞计数活性检测试剂盒(Cell counting kit-8, CCK-8)测得各组细胞系初始对吉非替尼药物的耐药性(用IC50表示)。

1.3 PDLIM5在PC9和PC9GR细胞中的差异性表达

分别通过PCR及Western blot方法测定PC9及PC9GR细胞系中PDLIM5 mRNA及PDLIM5蛋白表达含量。

1.4 PDLIM5 siRNA序列的筛选

用Lipo6000与备选的3个靶向PDLIM5的siRNA同时作用于PC9GR细胞,通过Western blot的方法筛选出基因沉默效果最好的PDLIM5基因序列。

2.    PDLIM5靶向的纳米粒制备及体外相关性研究

2.1 PLGA@PEI纳米粒的制备

分别制备空的PLGA@PEI纳米颗粒及携载FAM标记的特异性靶向PDLIM5的siRNA的纳米颗粒PLGA@PEI,进行形态学观测并通过分光光度计定量测定纳米颗粒对siRNA的连接效率。

2.2 纳米颗粒的基本理化性质及稳定性观察

1)纳米粒径电位分析仪莫尔文纳米ZS探测器检测纳米颗粒的粒径、Zeta 电位及PDI;2)采用透射电镜及扫描电镜观察纳米颗粒形貌、大小及分散度。

2.3 纳米颗粒的稳定性和超声敏感性测定

1)连续30天观察纳米颗粒粒径以评估其在体外稳定性;2)不同纳米颗粒的体外超声感应性测定:将实验分为阴性对照组:PBS溶液,SonoVue MBs组、NBs组以及siRNA-NBs组,通过临床超声扫描仪系统对比各组纳米颗粒在体外40min的超声造影成像效果,并通过Image J软件进行定量分析。

2.4 纳米粒的体外安全性研究

细胞毒性实验:将不同浓度的纳米颗粒与PC9GR细胞共孵育,通过CCK-8检测两种纳米颗粒的体外生物安全性。

2.5 超声联合纳米粒介导的细胞转染最佳条件的筛选

分别通过荧光显微镜下观察及流式细胞术定量分析法筛选超声联合纳米粒介导的细胞转染的最佳条件,包括:超声强度、超声频率、超声占空比及超声暴露时间。

3.    超声联合纳米泡介导的PDLIM5基因沉默对PC9GR细胞自噬及相关机制的研究

3.1 UTMD介导PDLIM5靶向的纳米粒抑制PDLIM5基因表达的体外研究

将PC9GR细胞悬液分为对照组、siRNA组、siRNA+US组、siRNA-NBs组、siRNA-NBs+US组,固定US辐照条件进行干预,通过3种方法检测干预后各组细胞内PDLIM5的表达量。方法一,在激光共聚焦显微镜下观察法;方法二,PCR技术定量测定转染后细胞内PDLIM5 mRNA表达量;方法三,Western blot法定量测定转染后细胞内PDLIM5蛋白表达量。

3.2 抑制PDLIM5基因的表达诱导PC9GR细胞自噬的相关研究

通过2种方法研究PC9GR细胞内自噬相关研究。方法一,Western blot法测定转染后细胞内自噬相关蛋白LC3II/I及p62表达量;方法二,通过电镜下观察各组细胞内自噬小体表达量。

3.3 抑制PDLIM5基因的表达诱导PC9GR细胞自噬相关机制的研究

通过Western blot方法研究PC9GR细胞内自噬相关通路PI3K/AKT/mTOR信号通路中相关蛋白表达量的研究。

结果:

1.    PDLIM5基因在PC9及PC9GR细胞中的表达

1.1 PDLIM5的生物信息学分析

与正常组织相比,NSCLC组织中PDLIM5的表达显著增加(P<0.001)。随着该基因的表达量增高,NSCLC风险性也在不断升高,且PDLIM5基因高表达的样本5年以上生存率大幅降低。此外,PDLIM5的表达水平与生存时间成反比,PDLIM5基因的3年生存率预测能力最强。

1.2 PC9及PC9GR细胞系对吉非替尼耐药性的鉴定

通过CCK-8法测得初始PC9细胞对吉非替尼的耐药性IC50=3.622μg/L。初始PC9GR细胞对吉非替尼的耐药性IC50=10.300μg/L。

1.3 PDLIM5在PC9和PC9GR细胞中的差异性表达

与PC9细胞相比,PC9GR细胞中PDLIM5/GAPDH蛋白水平升高(n=3),差异具有统计学意义(0.85±0.11 vs 0.63±0.05,P<0.0001)。

1.4 PDLIM5 siRNA序列的筛选

筛选得到最佳PDLIM5 siRNA基因序列:

Sense:5’-CUGGGACUGAACAUUUGAATT-3’

Antisense:5’-UUCAAAUGUUCAGUCCCAGTT-3’。

2.    PDLIM5靶向的纳米颗粒制备及体外相关性研究

2.1 PLGA@PEI纳米粒的制备

1)成功制备裸纳米颗粒及携载PDLIM5 siRNA的纳米颗粒;2)NBs对 siRNA的封装率EE=(94.08±0.28)%(n=3)。

2.2 纳米颗粒的基本理化性质及稳定性观察

NBs和siRNA-NBs两种纳米颗粒平均尺寸分别为216.30±1.54nm和223.17±2.23nm(n=3)。二者平均Zeta电位分别为22.63±0.55mV和−1.94±3.72mV(n=3)。

2.3 纳米颗粒的稳定性和超声敏感性测定

1)NBs与siRNA-NBs在15内稳定性较好,此后粒径逐渐增大。

2)从开始至实验结束 40min 时,对照组PBS 对比增强模式下始终未显影,且相对稳定。SonoVueMBs 对比增强超声造影图像灰度强度减少比 NBs 和 siRNA-NBs 更明显,三者图像灰度值分别为(22.53±0.68 与 80.50±0.81 94.32±3.26,P<0.0001,n=3)。NBs 与siRNA-NBs 组之间并无明显差异统计学差异(80.50±0.81 vs 94.32±3.26,P>0.05,n=3)。

2.4 纳米粒的体外安全性研究

对照组、NBs组和siRNA-NBs组PC9GR细胞存活率分别为(98.82±1.19)%、(94.39±2.88)%和(96.77±1.18)%,各组间差异无统计学意义(P>0.05,n=3),且三组细胞存活率均在95%以上。当各组NPs溶液浓度在0.1~2.0μg/L之间变化时,三组间的细胞存活率均无显著差异(P>0.05)。当各组溶液浓度增加至5μg/L时,与对照组相比,NBs组和siRNA-NBs组的细胞活力开始显著降低,各组间差异有统计学意义(P<0.05),但两组细胞整体细胞存活率仍超过75%。

2.5 超声联合纳米粒介导的细胞转染最佳条件的筛选

荧光显微镜下观察及流式细胞术定量分析得出超声靶向爆破纳米粒条件为:超声强度500mW/dm2,占空比20%,脉冲频率1000Hz,暴露时间90s。

3.    超声联合纳米泡介导的PDLIM5基因沉默对PC9GR细胞自噬及相关机制的研究

3.1 UTMD介导PDLIM5靶向的纳米粒抑制PDLIM5基因表达的体外研究

1)定量分析导致荧光显微镜各组的平均荧光强度比(绿/蓝),与对照组相比,siRNA组、siRNA+US组、siRNA-NBs和siRNA-NBs+US组的细胞的荧光强度分别为0.98±0.17,0.98±0.17,5.05±0.68和9.48±0.68,其中,对照组、siRNA组及siRNA+US组各组荧光强度并无统计学差异(P>0.05,n=3),当给予NBs携载的siRNA后,与其余各组相比,siRNA-NBs和siRNA-NBs+US组siRNA转染效率明显增高(P<0.01,n=3),且二者相比,siRNA-NBs+US组荧光强度更高,差异有统计学意义(P<0.001,n=3)。

2)PCR技术在mRNA水平上检测定量分析各组PC9GR细胞中PDLIM5的表达,结果示:与siRNA组和siRNA+US组相比,siRNA-NBs组和siRNA-NBs+US组PDLIM5 mRNA表达量明显下降(0.60±0.04,0.35±0.04 vs 1.00±0.09,0.94±0.10,P<0.001,n=3);且与siRNA-NBs组相比,siRNA-NBs+US组 PDLIM5 mRNA表达量下降更多(P<0.001)。

3)Western blot 在蛋白水平上分析PC9GR细胞中PDLIM5的表达量,结果表明:与siRNA组和siRNA+US组相比,PDLIM5/GAPDH水平在siRNA-NBs组和siRNA-NBs+US组表达量明显下降(0.69±0.10和0.31±0.13 vs 0.97±0.06和0.98±0.03,P<0.001,n=3);且与siRNA-NBs组相比,siRNA-NBs+US组PDLIM5/GAPDH表达水平下降更多,差异具有统计学意义(P<0.001)

3.2 抑制PDLIM5基因的表达诱导PC9GR细胞自噬的相关研究

1)Western blot法测定转染后细胞内自噬相关蛋白LC3II/I及p62表达量;结果表明:与其他组相比,siRNA-NBs和siRNA-NBs+US组中LC3-II/I和p62的表达增加。其中siRNA-NBs和siRNA-NBs+US组中p62/GAPDH的蛋白水平相比于siRNA和siRNA+US组而言显著增高,差异有统计学意义(1.34±0.06,1.64±0.23 vs 1.04±0.021.04±0.02,P<0.05,n=3),且siRNA-NBs+US组比siRNA-NBs组升高更多,差异有统计学意义(P<0.05),蛋白LC3-II/I水平在siRNA-NBs和siRNA-NBs+US组中表达量显著比siRNA组和siRNA+US组表达量升高(1.25±0.04,1.48±0.07 vs 1.00±0.02,1.00±0.05,P<0.001,n=3),其中,siRNA-NBs+US组比siRNA-NBs组表达量升高更多,差异具有统计学意义(P<0.001)。

2)透射电镜观察各组细胞内自噬小体,结果显示:对照组、siRNA组及siRNA+US组细胞中仅存在数量较少的自噬小体。siRNA-NBs及siRNA-NBs+US组细胞中自噬小体数量明显增多,后者出现了大量自噬溶酶体,可见自噬溶酶体内大量胞浆降解,呈现单层膜结构。

3.3 抑制PDLIM5基因的表达诱导PC9GR细胞自噬相关机制的研究

Western blot法测定转染后细胞内自噬相关通路蛋白p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR表达量;siRNA-NBs和siRNA-NBs+US组中p-PI3K/PI3K的表达水平相比于siRNA和siRNA+US组显著增高,差异有统计学意义(1.24±0.01,1.36±0.02 vs 1.00±0.06,1.06±0.01,P<0.05,n=3),且siRNA-NBs+US组比siRNA-NBs组升高更多,差异有统计学意义(P<0.05)。此外,siRNA-NBs和siRNA-NBs+US组中p-AKT/AKT的表达水平相比于siRNA和siRNA+US组显著增高,差异有统计学意义(1.31±0.01,1.53±0.02 vs 1.06±0.08,1.22±0.15,P<0.05,n=3),且siRNA-NBs+US组比siRNA-NBs组升高更多,差异有统计学意义(P<0.05)。同样地,siRNA-NBs和siRNA-NBs+US组中p-mTOR/mTOR的表达水平相比于siRNA和siRNA+US组显著增高,差异有统计学意义(1.96±0.93,2.56±0.12 vs 1.10±0.14,1.36±0.30,P<0.05,n=3),且siRNA-NBs+US组比siRNA-NBs组升高更多,差异有统计学意义(P<0.05)。

结论:

1.   与正常组织相比,NSCLC组织中PDLIM5的表达显著增加,PDLIM5的表达水平与患者生存时间成反比,且PDLIM5基因可预测患者3年及5年生存率,其中3年生存率预测能力最强,且PC9GR细胞中PDLIM5表达量高于PC9细胞,提示PDLIM5表达量升高或促进PC9GR细胞耐药性。

2.   本实验制备的NPs不仅具有粒径小,生物安全性及稳定性好的特点,且具有较好的体外超声造影效果及稳定性,筛选得到的体外超声靶向爆破最佳条件为:超声强度500mW/dm2、占空比20%、脉冲频率1000Hz、暴露时间90s,可明显提高PC9GR细胞的siRNA传递效率,成功提高体外细胞PDLIM5基因沉默效率。

3.   在PC9GR细胞中,PDLIM5被沉默后细胞自噬作用增强,提示抗耐药机制与增强细胞自噬有关,且在众多信号通路及介质参与的自噬调控机制中,PI3K/AKT/mTOR信号通路在调控PC9细胞自噬,增加对EGFR-TKIs药物敏感性的过程中发挥了重要的作用。

Other Abstract

Background and Purpose:

Human lung cancer is the most malignant and aggressive form of human tumors and a leading cause of cancer-related death worldwide at present, among which NSCLC is the most common histological category. In recent years, with the development of gene-targeted therapy, it has gradually become the main method of anti-tumor therapy in human cancers, including NSCLC. Among all of the gene-targeted drugs for NSCLC, epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs) have been widely researched and gradually become one of the most representatives. However, the overall survival rate of majority of NSCLC patients with EGFR mutations haven’t been improved after receiving EGFR-TKIs therapy. The main reason is that they developed drug resistance inevitably during the process of treatment. Therefore, drug resistance has become one of the most important reasons for the failure of chemotherapy and targeted therapy in many tumors now, including NSCLC patients as mentioned above.

As a cytoskeleton-related protein, PDLIM5 is involved in the process of signal regulation of membrane-associated proteins, cytoskeletal proteins and various signaling molecules, and is involved in the progression of various tumors as well. Studies have shown that PDLIM5 plays a crucial role in proliferation, invasion, and migration of tumor cells. In addition, some studies suggest that the high expression of PDLIM5 protein is closely related to the proliferation, invasion and apoptosis of NSCLC. However, whether PDLIM5 is related to drug resistance among the patients of NSCLC is not clear and needs our further exploration.

In recent years, ultrasonic targeted microbubble destruction (UTMD) technology, as a new non-invasive method, can be always widely used in the applications of radiotherapy and chemotherapy in the treatment of  various cancers and the delivery of gene or drug as well, to improve the transfection efficiency of exogenous genes or drugs to the target tissues and organs by the way of blasting the microbubbles locally to destroy the cell membrane and increasing capillary permeability, which makes UTMD technology has become the focus of therapeutic research on cancer and other diseases in recent years, especially in the improvement of the efficiency of gene therapy by increasing the delivery of different kinds of siRNAs.

With the development of nanotechnology, especially nanomaterials, they are promising in the field of application of drug and gene delivery. Due to their higher stability and longer circulation time, they can be effectively accumulated in tumor tissues through enhanced permeability and retention (EPR) effect for ultrasound-targeted imaging and therapy. Besides, because of their high permeability, retention effect and endocytosis (i. e., nanoparticles are transported into tumors through endothelial cells), they can pass smoothly through tumor capillaries and be accumulated in solid tumors, thus improving the therapeutic effect on tumor cells.

Among those nanometerials, polymer nanoparticles, such as polylactic acid-glycolic acid (PLGA), polylactic acid (PLA), chitosan and polycaprolactone, have been widely used in siRNA and drug delivery. Polymer-siRNA coupling, as the most effective siRNA delivery polymer, they can be regarded as valuable tools and provide strategies for effective delivery of siRNA.

Based on the above basis, we plan to combine the siRNA targeted to PDLIM5 with ultrasound-responsive polymer inorganic nanoparticles to form a composite nanoplatform to improve the effect of gene delivery through increase the efficiency of delivery of siRNA targeted to PDLIM5, which therefore improves the therapeutic efficacy of treatment to NSCLC. With the combination and coordination of ultrasound, nanotechnology and biomedicine, we have designed and developed a novel kind of composite nanoparticles successfully, that is a kind of ultrasound-responsive PLGA@PEI nanoparticles loaded with siRNA specifically targeted PDLIM5 as a loading platform. The nanoparticles are characterized by high dispersion, non-toxicity, good biocompatibility and easy preparation. Besides, the nanoparticles modified by PEI can help to improve the biocompatibility and safety of composite nanopreparations, increase the efficacy of therapy and reduce immunogenicity. Therefore, we plan to prepare a novel kind of nanoplatform with the loading of specifically-targeted PDLIM5 siRNA, and characterize and analyze it. Besides, its characteristics of biological safety and ultrasound sensitivity will be verified and the drug resistance and related mechanism of NSCLC tumor cells will be explored in vitro, in order to explore a new and efficient treatment scheme for the drug resistance of NSCLC.

Materials and Methods:

1. Expression of the PDLIM5 in PC9 and PC9GR cell lines.

1.1 Bioinformatics analysis of the PDLIM5.

The RNAseq data (level3) and corresponding clinical information of NSCLC were obtained from the Cancer Genome Atlas (TCGA) database and GTEx database respectively to obtain the PDLIM5 bioinformatics analysis results.

1.2 Identification of gefitinib resistance in the PC9 and PC9GR cell lines.

The initial PC9 and PC9GR cell lines were divided into negative control group, blank control group and experimental group (gefitinib concentration were 2,4,8,16,32,64,128μg/L, respectively). Initial resistance to gefitinib (IC50) was measured by the Cell count activity asskit (Cell counting kit-8, CCK-8).

1.3 Differential expression of PDLIM5 in PC9 and PC9GR cell lines.

The expression of PDLIM5 mRNA and PDLIM5 protein in PC9 and PC9GR cell lines were determined by method of PCR and Western blot, respectively.

1.4 Screening of the PDLIM5 siRNA sequences.

By combining Lipo6000 with the alternative three siRNAs targeting PDLIM5 to work together on PC9GR cells, the best PDLIM5 gene sequence of gene silencing was selected by Western blot.

2. The preparation of PLGA@PEI nanoparticles targeting PDLIM5 and the correlation studies in vitro.

2.1 Preparation of the PLGA@PEI nanoparticles.

Empty PLGA@PEI nanoparticles and PLGA@PEI nanoparticles with FAM labeled siRNA specifically targeting PDLIM5 were prepared. The morphology of the nanoparticles were both observed and the ligation efficiency of nanoparticles with siRNA were quantitatively determined by spectrophotometer.

2.2 Observation of the basic physicochemical properties and stability of nanoparticles.

1) Molvern Nanometer ZS detector of nanometer size potential analyzer detects the size, Zeta potential and PDI of nanoparticles; 2) Observation of the morphology, size and dispersion of nanoparticles by TEM and SEM.

2.3 Determination of stability and ultrasonic sensitivity of nanoparticles.

1) Evaluation of the stability of nanoparticles by the observation of size of nanoparticles for 30 consecutive days in vitro; 2) The determination of ultrasonic sensibility of different nanoparticles in vitro: all the nanoparticles were divided into different groups as follows: the negative control groups (NBs+PBS solution), the SonoVue MBs group, the NBs group, as well as the siRNA-NBs group. The ultrasound imaging effects of different nanoparticles were detected, recorded and compared within 40mins in vitro through a clinical ultrasound scanner system. Quantitative analysis of all the results were analyzed with multiple Image J software.

2.4 Determination the biosafety of nanoparticles in vitro.

Cytotoxicity experiment: Different concentrations of nanoparticles were incubated with PC9GR cells, and the biosafety in vitro of the two nanoparticles was measured by CCK-8.

2.5 The screening of optimal conditions for cell transfection mediated by the combination of ultrasound with nanoparticles.

The optimal conditions for cell transfection mediated by the combination of ultrasound with nanoparticles were screened by using fluorescence microscope observation and quantitative flow cytometry. They are including intensity, frequency, duty cycle and exposure time.

3. The researches of autophagy and relevant mechanism in PC9GR cell line caused by the inhibition of PDLIM5 gene expression by UTMD combined with PDLIM5-targeted nanoparticles.

3.1 The researches of PDLIM5 gene expression in PC9GR cell line caused by the inhibition of PDLIM5 with UTMD combined with PDLIM5-targeted nanoparticles in vitro.

PC9GR cell suspension were divided into groups as follows: control group, siRNA group, siRNA + US group, siRNA-NBs group and siRNA-NBs + US group. With fixed US irradiation conditions, the expression of PDLIM5 in each group were detected through three methods. Method 1, The expression of PDLIM5 were observed under laser confocal microscope ; Method 2, The expression of PDLIM5 mRNA were quantitatively analyzed with the method of PCR after cell transfection; Method 3, The expression of PDLIM5 protein were quantitatively analyzed with the method of Western blot.

3.2 The research of autophagy induced by the inhibition of expression of PDLIM5 gene in PC9GR cells.

Autophagy was studied with two methods. Method 1, The expression of autophagy related proteins (LC3II/I and p62) were quantitatively analyzed by Western blot after cell transfection; Method 2, The expression of autophagosomes in each group were observed by electron microscope.

3.3 The autophagy-related mechanisms were researched induced by the inhibition of expression of PDLIM5 gene in PC9GR cells.

The autophagy-related mechanisms were researched by Western blot.

Results:

1 Expression of the PDLIM5 in PC9 and PC9GR cell lines.

1.1 Bioinformatics analysis of the PDLIM5.

The expression of PDLIM5 was significantly increased in NSCLC tissues compared to normal tissues (P <0.001). As the expression of this gene increases, the risk of NSCLC is also increasing, and the survival rate of samples with high expression of PDLIM5 gene decreases greatly over 5 years. Moreover, the expression level of PDLIM5 was inversely proportional to survival time, with the strongest predictive 3-year survival..

1.2 Identification of gefitinib resistance in the PC9 and PC9GR cell lines.

Resistance to gefitinib of initial PC9 cells and PC9GR cells were measured by the CCK-8 method and the IC50 of each of them were 3.622 μg/L and 10.300 μg/L separately.

1.3 Differential expression of PDLIM5 in PC9 and PC9GR cells.

Compared with the PC9 group (n=3), PDLIM5/GAPDH protein levels were increased significantly, and the difference was statistically significant (0.85 ± 0.11 vs 0.63 ± 0.05, P <0.0001),

1.4 Screening of the PDLIM5 siRNA sequences.

The best PDLIM5 siRNA gene sequence was screened:

Sense:5’-CUGGGACUGAACAUUUGAATT-3’

Antisense:5’-UUCAAAUGUUCAGUCCCAGTT-3’.

2. Ultrasound combined with nanobubbles mediated the preparation and characterization of PDLIM5 targeted PLGA@PEI nanoparticles, safety, targeting and efficacy in vitro

2.1 Fabrication of different PLGA@PEI nanoparticles.

1) Bare nanoparticles and nanoparticles carrying PDLIM5 siRNA were fabricated successfully; 2) The encapsulation efficiency (EE) of NBs to siRNA is (94.08 ± 0.28) % (n=3).

2.2 Observation on the basic physicochemical properties and stability of nanoparticles.

The average sizes of NBs and siRNA-NBs were 216.30±1.54nm and 223.17±2.23nm, respectively (n=3). The mean Zeta potential of them were 22.63±0.55mV and 1.94±3.72mV, respectively (n=3).

2.3 Stability and ultrasonic sensitivity determination of nanoparticles.

1) The size of NBs and siRNA-NBs were stable within 15 days and they increased gradually after that.

2) At 40min from the beginning to the end of the experiment, the control group in PBS contrast enhancement mode remained unchanged and was relatively stable. The gray intensity of SonoVueMBs group decreased was more pronounced than NBs and siRNA-NBs groups, the gray values of the three groups were (22.53 ± 0.68 and 80.50 ± 0.81 94.32 ± 3.26, P <0.0001, n=3). There was no significant statistical difference between NBs and siRNA-NBs groups (80.50 ± 0.81 vs 94.32 ± 3.26, P> 0.05, n=3).

2.4 Determination the biosafety of nanoparticles in vitro.

 The results showed that the cell viability of PC9GR cells in control, NBs and siRNA-NBs groups were (98.82 ± 1.19) %, (94.39 ± 2.88) % and (96.77 ± 1.18) % respectively and there were no statistical differences among these groups (P >0.05, n = 3). At the same time, the survival rates of all groups were above 95%. There were no significant differences of cell viability of PC9GR cells among these groups when the solution concentration of NPs varied between 0.1-2.0μg/mL (P> 0.05). While cell viability of PC9GR cells in the NBs group and the siRNA-NBs group began to be significantly reduced compared with the control group when the solution concentration of each group increased to 5 u g/ml, and there were significant differences among them (P <0.05), and the overall cell survival rate of both groups were still over 75%.

2.5 The screening of optimal conditions for cell transfection mediated by the combination of ultrasound with nanoparticles.

The optimal conditions for cell transfection mediated by the combination of ultrasound with nanoparticles were observed by the fluorescence microscopy and quantitatively analyzed by flow cytometry. The results showed that the optimal conditions were: ultrasonic intensity of 500mW/dm2, duty cycle of 20%, pulse frequency of 1000Hz, and exposure time of 90s.

3. The researches of autophagy and relevant mechanism in PC9GR cell line caused by the inhibition of PDLIM5 gene expression by UTMD combined with PDLIM5-targeted nanoparticles.

3.1 The researches of PDLIM5 gene expression in PC9GR cell line caused by the inhibition of PDLIM5 with UTMD combined with PDLIM5-targeted nanoparticles in vitro.

1) The average fluorescence intensity ratio of each group (green / blue) was quantitatively analyzed. The results showed that: Compared with the control group, the fluorescence intensity of the cells in the siRNA, siRNA + US, siRNA-NBs, and siRNA-NBs + US groups was 0.98 ± 0.17, 0.98±0.17, 5.05 ± 0.68 and 9.48 ± 0.68 respectively and there were no statistical differences among the control, siRNA, and siRNA + US groups (P> 0.05, n=3). While the siRNA transfection efficiency was significantly higher in the siRNA-NBs and siRNA-NBs + US groups with the comparison to the other groups (P <0.01, n=3). In particularly, the fluorescence intensity was much higher in the siRNA-NBs + US group and difference was statistically significant (P <0.001, n=3).

2) The expression of PDLIM5 mRNA level in PC9GR cells were quantified by PCR technology. The results showed that the expression of PDLIM5 mRNA level decreased significantly in siRNA-NBs group and siRNA-NBs + US group when compared to the siRNA and siRNA + US groups (0.60 ± 0.04, 0.35±0.04 vs 1.00±0.09, 0.94±0.10, P<0.001, n=3); When compared with siRNA-NBs group, the expression of PDLIM5 mRNA level decreased much more in siRNA-NBs + US group, and there was statistical significance (P <0.001).

3) The expression of PDLIM5 protein in PC9GR cells were detected and analyzed by Western blot at the protein level. The results show that the expression of PDLIM5 / GAPDH was significantly decreased in the siRNA-NBs and siRNA-NBs + US groups when compared with siRNA and siRNA + US groups (0.69 ± 0.10 and 0.31 ± 0.13 vs 0.97 ± 0.06 and 0.98 ± 0.03), and there were statistical differences (P<0.001, n=3). Especially, the expression of PDLIM5 / GAPDH decreased much more in the siRNA-NBs + US group compared to the siRNA-NBs group, and the difference was statistically significant (P <0.01).

3.2 The research of autophagy induced by the inhibition of expression of PDLIM5 gene in PC9GR cells.

1) The expression of autophagy-related proteins LC3 II/I and p62 were measured by Western blot after cell transfection. The expression of LC3-II/I and p62 increased significantly in siRNA-NBs and siRNA-NBs + US groups compared with other groups, among which the expression of p62/GAPDH in siRNA-NBs and siRNA-NBs + US groups were much higher than other groups, and the difference was statistically significant (1.34 ± 0.06, 1.64±0.23 vs 1.04±0.02, 1.04±0.02, P<0.05, n=3). Moreover, the expression of P62 were much more than siRNA-NBs group and there was statistical significance (P <0.05). The expression of protein LC3-II/I levels was significantly increased in siRNA-NBs and siRNA-NBs + US groups as compared to that in the siRNA and siRNA + US groups (1.25 ± 0.04, 1.48±0.07 vs 1.00±0.02, 1.00±0.05, P<0.001, n=3). Especially, the expression of LC3-II/I was much higher in siRNA-NBs + US group than it in siRNA-NBs group and the difference was statistically significant (P <0.001).

2) Autophagosomes were observed by transmission electron microscopic in each group. The results showed that there were only a small number of autophagosomes in PC9GR cells in control, siRNA and siRNA + US groups. What’s more, there was a tendency to wrap the cytoplasmic component. Besides, the number of mitochondria increased with a clear structure and a normal morphology in PC9GR cells among these groups mentioned above. On the contrary, the number of autophagosomes with a clear bilayer membrane structure in PC9GR cells in siRNA-NBs and siRNA-NBs + US group increased significantly, and a large number of cytoplasmic degradations in autophagosomes appeared with a monolayer membrane structure.

3.3 The autophagy-related mechanisms were researched induced by the inhibition of expression of PDLIM5 gene in PC9GR cells.

The expression of proteins about autophagy-related mechanisms were measured by Western blot after cell transfection, they were p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR. The results showed that the expression of p-PI3K/PI3K in siRNA-NBs and siRNA-NBs + US group were significantly higher compared to the siRNA and siRNA + US group, and there was statistical significance (1.24 ± 0.01, 1.36±0.02 vs 1.00±0.06, 1.06±0.01, P<0.05, n=3). Especially, it increased much more in siRNA-NBs + US group than that in siRNA-NBs group and the difference was statistically significant (P <0.05). In addition, the expression of p-AKT/AKT in siRNA-NBs and siRNA-NBs + US groups increased significantly compared to the siRNA and siRNA + US group, and there were statistical significances (1.31 ± 0.01,1.53 ± 0.02, vs 1.06 ± 0.08,1.22 ± 0.15, P <0.05, n=3). Especially, it increased much more in siRNA-NBs + US group than that in siRNA-NBs group and the difference was statistically significant (P <0.05). Similarly, the expression of p-mTOR/mTOR in siRNA-NBs and siRNA-NBs + US groups also increased significantly compared to siRNA and siRNA + US groups (1.96 ± 0.93,2.56 ± 0.12 vs 1.10 ± 0.14,1.36 ± 0.30, P <0.05, n=3), and there were statistical significances (1.24 ± 0.01, 1.36±0.02 vs 1.00±0.06, 1.06±0.01, P<0.05, n=3). Especially, it increased much more in siRNA-NBs + US group than that in siRNA-NBs group and the difference was statistically significant (P <0.05).

Conclusion:

1. The expression of PDLIM5 was significantly increased in NSCLC tissues compared with normal tissues, which was inversely proportional to survival time of patients. Besides, the PDLIM5 gene could predict survival time of patients in 3 years and 5 years, among which the prediction of survival time of patients in 3 years was the strongest. What’s more, the overexpression of PDLIM5 in PC9GR cells than in PC9 cells suggested that the increasement of PDLIM5 expression could promote drug resistance of PC9GR cells.

2. The NPs fabricated in this experiment not only had the characteristics of small particle size, good biosafety and stability, but also had the good ultrasound contrast effect and stability in vitro. The optimal conditions were: ultrasonic intensity of 500mW/dm2, duty cycle of 20%, pulse frequency of 1000Hz, and exposure time of 90s, which can significantly improve the siRNA transmission efficiency of PC9GR cells, and successfully improve the PDLIM5 gene silencing efficiency of in vitro cells.

3. Autophagy could be enhanced in PC9GR cells after PDLIM5 gene silencing, which suggested that the drug resistance mechanism was probably related to the enhancement of autophagy. What’s more, PI3K / AKT / mTOR signaling pathway might play an important role in regulating autophagy and increasing drug sensitivity to EGFR-TKIs.

MOST Discipline Catalogue医学 - 临床医学 - 影像医学与核医学
URL查看原文
Language中文
Other Code262010_120190901540
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/537489
Collection第二临床医学院
Affiliation
兰州大学第二临床医学院
Recommended Citation
GB/T 7714
张瑶. 利用超声靶向微泡破坏技术沉默 PDLIM5 探索非小细胞性 肺癌自噬与耐药关系的实验研究[D]. 兰州. 兰州大学,2023.
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