Study On The Lentiviral-Vector-Mediated SiRNA Of Cyclophilin A (CyPA) Inhibits Non-Small Cell Lung Cancer Cell Growth In Vitro And In Vivo

Lung cancer is considered as one of the most lethal cancers which has posed a threat to the patient's health and life.About 1,200,000 new cases were predicted every year in the world.In China,600,000 cases were died of lung cancer annually and the overall 5-year survival remains at 8.9%.About 85%of patients with lung cancer were non-small cell lung cancer(NCSLC) and among those 85%were in advanced stage and have no opportunity to have surgical treatment.Carcinogenesis of lung is cooperated with the environmental and genetic factors.Many genes play an important roles in tumorigenesis during the progression of non-small cell lung cancer.With the development of proteomics some overor low- expression protein were found in non-small cell lung cancer tissues,One of these proteins,Cyclophilin A(CyPA),was found to have overexpression in lung tumor tissues by two-dimension electrophoresis.CyPA is a member of the peptidyl-prolyl isomerases.CyPA also catalyzes protein folding and conformational changes which depends on its capacity as chaperon proteins. Recently,correlations of CypA with tumor pathogenesis have been studied.A possible role for CyPA in tumor cell growth has been demonstrated by the overexpression of CyPA in human pancreatic adenocarcinoma tissues and addition of exogenous CypA significantly stimulated pancreatic cancer cell proliferation in a dose-dependent manner. The technique of RNAi is rapid,cost effective and can be easily adapted to study homo -logous gene function in a wide variety of organisms.Lentiviral vector hold great promise for gene therapeutic applications because it can mediated stable gene transfer both in dividing and non-dividing cells.In addition,the lentiviral products are integrated into the host genome ensuring siRNA stable long-term expression.Therefore Lentiviral vector is adapted to study in vivo.Objectives1.We proposed that it is a potential pathway to inhibite the growth of NSCLC by silencing CyPA gene.So,CyPA siRNA mediated by lentiviral vector was firstly constructed and screened.2.To investigate the biological functions of CyPA among the development in non-small cell lung cancer,and to seek new treatment targets.Lentiviral-vector-mediated siRNA of CyPA was used to study its inhition of A549 and H1299 in vitro and in vivo,respectively.Materials and Methods1.The expression of CyPA mRNA and protein in NSCLC cell and lung tissues1.1 Total RNA was extracted from cells using TRIZOL reagent.RT and cDNA amplification were performed according to SYBR Greenâ… real-time PCR kit.Primer pairs were designed for CyPA andβ-actin.The products amplified were purified and serial diluted ranging from 10 to 10⁹ as a standard curve,respectively.β-actin as an internal control used to normalized the signal value of each sample.The relative quantitation of CyPA mRNA was presented as unit values of 2^{-ΔΔCt}.To confirm amplification specificity the PCR products were subjected to a melting curve analysis.1.2 The expressions of CyPA protein were examined in 45 lung cancer tissues,22 paired tumor-adjacent tissues and 33 normal pulmonary tissues using tissue microarray immuno-histochemistry method.The relationship of CyPA expression in NSCLC with clinic pathological characteristic was analyzed.2.Construction and screening of CyPA siRNA mediated by lentiviral vector2.1 Four target sequences were selected and the complementary DNA contained both sense and antisense oligonucleotides were designed,synthesized and cloned into pGCL-GFP vector.The recombinant lentiviral vector containing CyPA shRNA was confirmed by PCR and sequencing.2.2 The positive recombinant lentiviral vector was cotransfected with pHelper1.0 and pHelper 2.0 into 293T cells to pack lentivirus particles which was named after Lv-shCyPA. At the same time,the packed virus infected A549,H1299,the expression level of CyPA protein after infection was detected by Western Blot in order to screen the effective target of CyPA siRNA.2.3 A549 and H1299 were infected with specific or negative control packed virus (Lv-shCyPA,NC) using multiplenty of infection of 5,10,20,respectively.The infected cells were maintained in culture medium for 4-5 passages and selected the single colony with GFP strongly expressed to establish stable silencing lines.The MOI of A549 and H1299 were optimized according to GFP.3.The growth of A549 and H1299 was suppressed by lentiviral mediated CyPA siRNA in vitro3.1 A549 and H1299 cells were infected by Lv-shCyPA or NC according to the MOI of A549/20 and H1299/5,respectively.Cultured cells were collected at 5^th day,and total RNA was extracted from cultured cells.The quantity of CyPA mRNA was quantited by real-time RT-PCR.3.2 Add stable silencing lines A549/Lv-shCyPA and H1299/Lv-shCyPA 100μl into 96 well (approximately 5×10³cells/well).After incubation for 1d,2d,3d,4d,5d,6d,respectively. MTT assay were performed at the end of the cultivation period.The growth curve of cells and proliferation rate of cells were analyzed,respectively.3.3 A549 and H1299 cells were infected by Lv-shCyPA or NC according to the MOI of A549/20 and H1299/5,respectively.At 5^th day after infected,cells were harvested and fixed in 70%ethanol.Cell cycle and apoptosis rate were analyzed using the flow cytometer.3.4 Uninfected cells of A549 and H1299 were as control groups in the study,respectively.3.5 Data from real-time PCR,Western Blot,MTT and FCM are expressed as(?)±s from at three independent groups.Significant differences were determined by paired Student t-test(two tails,paired) using SPSS12.0 software for Window XP.4.The growth of xenografts were inhibited by lentiviral mediated CyPA siRNA in vivoApproximately 1×10⁶ A549/Lv-shCyPA,A549/NC,H1299/Lv-shCyPA,H1299/NC and their parental cells were injected subcutaneously into the right flank of the mouse.Tumor growth and animal weight were monitored every 4 days starting from the 4^th day to 38^th after injection.Tumor dimensions were measured with calipers and the volume was calculated according to the equationâ…¤(mm³)=ab²/2(a:length,b:width,b≤a).RESULTS1.The expression of CyPA mRNA and protein in NSCLC cell and lung tissues1.1 The overexpression of CyPA mRNA in A549 and H1299 were significant difference compared to that in BEAS-2B by fluorescent quantitative PCR(P＜0.05),and so,we can take A549 and H1299 as the target cell for CyPA gene silencing.1.2 The expressions of CyPA protein in NSCLC tissues and paired tumor adjacent tissues were significantly higher than that in the normal lung tissues(P＜0.05).The positive rates of CyPA protein in the normal lung tissues,paired tumor adjacent tissues and NSCLC tissues were 9.09%(3/33),53.62%(12/22) and 82.22%(37/45),respectively.We can see the level expressions were increased in turn.No significant correlations were found in histological types(P＞0.05) and TNM stage(P＞0.05).2.Construction and screening of CyPA siRNA mediated by lentiviral vectorThe effective sequence of CyPA siRNA was 5'-GTGAAAGAAGGCATGAATA-3'. Pseudotyped virus were packed and concentrated.The titer of pseudotyped virus was 2×10⁹TU/ml.3.The expression of CyPA mRNA and protein were downregulated by lentiviral mediated CyPA siRNA3.1 GFP was strongly expressed in infected cell lines at 5^th day.The relative expression of CyPA mRNA in A549/Lv-shCyPA and H1299/Lv-shCyPA were 0.048 and 0.1452, respectively.The CyPA mRNA was downregulated by 95.2%and 85.48%compared to the parent cells,and the relative expression of CyPA protein were 14.78%and 13.78%compared to control groups.CyPA protein was downregulated by 86.52%and 85.3%compared to the parent cells,respectively(P＜0.05).So,it is suggested that these results confirm consistency.3.2 A549/Lv-shCyPA and H1299/Lv-shCyPA cells grew more slowly than control groups. Proliferation rate of A549 and H1299 infected by Lv-shCyPA was significant declined at 5^th day compared to control group(P＜0.05).Flow cytometric analysis demonstrated no significant cell cycle arrested in G₀-G₁ phase and S phase,while G₂-M phase was decreased relatively in A549/Lv-shCyPA and H1299/Lv-shCyPA compared to control groups (P＜0.05).The apoptosis rate of A549/Lv-shCyPA and H1299/Lv-shCyPA was higher than control groups(P＜0.05).No significant difference was found both in distribution of cell cycle and in apoptosis rate of A549/NC and H1299/NC compared to control groups (P＞0.05).4.Xenografts growth were inhibited by lentiviral mediated CyPA siRNA in vivoVisible xenograft tumors in the parent cell groups and NC groups were readily detectable at 4^th day after implantation and grew rapidly in the next days.In contrast, visible tumors were only detectable at 6^th day after inoculated by A549/Lv-shCyPA and H1299/Lv-shCyPA.The xenograft tumors of A549/Lv-shCyPA and H1299/Lv-shCyPA cells remarkably delayed tumor growth and remained at a similarly small average size at 38^th days after inoculation compared to the control group(P＜0.05),the mass of xenograft tumors inoculated by A549/Lv-shCyPA and H1299/Lv-shCyPA were decreased at 77.76% and 78.85%,respectively.No significant difference was found in H1299/NC and A549/NC compared to the control group(P＜0.05).Conclusion1.It is consistent with the results performed by 2-DE method that CyPA is overexpressed in NSCLC tissues,suggesting that CyPA might serve as one of the biomarkers involved the development and progression of NSCLC.2.The sequence of CyPA siRNA was 5'-GTGAAAGAAGGCATGAATA-3'.Pseudotyped lentivirus particles containg CyPA siRNA can dowrtregulate the expression of CyPA mRNA in A549 and H1299,and also can suppress the cell growth of A549 and H1299 in our study.3.The results show that CyPA siRNA mediated by lentiviral vector can inhibite the xenograft tumor growth of A549 and H1299 in vivo.This data suggested that CyPA may have a good performance in therapies for NSCLC.Lentiviral vector system could be a potential therapeutic approach.