| 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 si RNAs.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 si RNA and drug delivery.Polymer-si RNA coupling,as the most effective si RNA delivery polymer,they can be regarded as valuable tools and provide strategies for effective delivery of si RNA.Based on the above basis,we plan to combine the si RNA targeted to PDLIM5with ultrasound-responsive polymer inorganic nanoparticles to form a composite nanoplatform to improve the effect of gene delivery through increase the efficiency of delivery of si RNA 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 si RNA 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 si RNA,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 were2,4,8,16,32,64,128μg/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 m RNA 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 si RNA sequences.By combining Lipo6000 with the alternative three si RNAs 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 si RNA specifically targeting PDLIM5 were prepared.The morphology of the nanoparticles were both observed and the ligation efficiency of nanoparticles with si RNA 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 Sono Vue MBs group,the NBs group,as well as the si RNA-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,si RNA group,si RNA+US group,si RNA-NBs group and si RNA-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 m RNA 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 3.622μg/L and 10.300μg/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 vs0.63±0.05,P<0.0001),1.4 Screening of the PDLIM5 si RNA sequences.The best PDLIM5 si RNA 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 vitro2.1 Fabrication of different PLGA@PEI nanoparticles.1)Bare nanoparticles and nanoparticles carrying PDLIM5 si RNA were fabricated successfully;2)The encapsulation efficiency(EE)of NBs to si RNA 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 si RNA-NBs were 216.30±1.54nm and 223.17±2.23nm,respectively(n=3).The mean Zeta potential of them were 22.63±0.55m V and 1.94±3.72m V,respectively(n=3).2.3 Stability and ultrasonic sensitivity determination of nanoparticles.1)The size of NBs and si RNA-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 Sono Vue MBs group decreased was more pronounced than NBs and si RNA-NBs groups,the gray values of the three groups were(22.53±0.68 and80.50±0.81 94.32±3.26,P<0.0001,n=3).There was no significant statistical difference between NBs and si RNA-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 si RNA-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/m L(P>0.05).While cell viability of PC9GR cells in the NBs group and the si RNA-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 500m W/dm~2,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 si RNA,si RNA+US,si RNA-NBs,and si RNA-NBs+US groups was 0.98±0.17,0.98±0.17,5.05±0.68 and 9.48±0.68respectively and there were no statistical differences among the control,si RNA,and si RNA+US groups(P>0.05,n=3).While the si RNA transfection efficiency was significantly higher in the si RNA-NBs and si RNA-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 si RNA-NBs+US group and difference was statistically significant(P<0.001,n=3).2)The expression of PDLIM5 m RNA level in PC9GR cells were quantified by PCR technology.The results showed that the expression of PDLIM5 m RNA level decreased significantly in si RNA-NBs group and si RNA-NBs+US group when compared to the si RNA and si RNA+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 si RNA-NBs group,the expression of PDLIM5 m RNA level decreased much more in si RNA-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 si RNA-NBs and si RNA-NBs+US groups when compared with si RNA and si RNA+US groups(0.69±0.10 and0.31±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 si RNA-NBs+US group compared to the si RNA-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 si RNA-NBs and si RNA-NBs+US groups compared with other groups,among which the expression of p62/GAPDH in si RNA-NBs and si RNA-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 si RNA-NBs group and there was statistical significance(P<0.05).The expression of protein LC3-II/I levels was significantly increased in si RNA-NBs and si RNA-NBs+US groups as compared to that in the si RNA and si RNA+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 si RNA-NBs+US group than it in si RNA-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,si RNA and si RNA+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 si RNA-NBs and si RNA-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-m TOR/m TOR.The results showed that the expression of p-PI3K/PI3K in si RNA-NBs and si RNA-NBs+US group were significantly higher compared to the si RNA and si RNA+US group,and there was statistical significance(1.24±0.01,1.36±0.02 vs1.00±0.06,1.06±0.01,P<0.05,n=3).Especially,it increased much more in si RNA-NBs+US group than that in si RNA-NBs group and the difference was statistically significant(P<0.05).In addition,the expression of p-AKT/AKT in si RNA-NBs and si RNA-NBs+US groups increased significantly compared to the si RNA and si RNA+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 si RNA-NBs+US group than that in si RNA-NBs group and the difference was statistically significant(P<0.05).Similarly,the expression of p-m TOR/m TOR in si RNA-NBs and si RNA-NBs+US groups also increased significantly compared to si RNA and si RNA+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 si RNA-NBs+US group than that in si RNA-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 of500m W/dm~2,duty cycle of 20%,pulse frequency of 1000Hz,and exposure time of90s,which can significantly improve the si RNA 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/m TOR signaling pathway might play an important role in regulating autophagy and increasing drug sensitivity to EGFR-TKIs. |
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