| BackgroundLung cancer is a common malignant tumor with high morbidity and mortality[1].According to the biological characteristics,it could be divided into Non-Small Cell Lung Cancer?NSCLC?and Small Cell Lung Cancer?SCLC?,wherein NSCLC accounts for 80-85%of lung cancer cases.75%patients with NSCLC could not have operatations due to extensive metastasis of the lesions[2].The 5-year survival rate is only 15%for NSCLC according to the literature[3].Therefore,exploring the molecular mechanism of NSCLC is important to improve the survival rate of lung cancer and improve patient's quality of life.miRNAs are a class of non-coding single-stranded RNAs that contain 18-25 nucleotides typically.Deregulation of miRNA expression is associated with tumor cell proliferation,invasion,apoptosis and metastasis.Studies have found that[4]:miRNA-200 family,as an epithelial cell marker,have obvious differences expression compared with tumors and normal tissues.miRNA-200a?miR-200a?is a member of the miRNA-200 family.It has been reported that miR-200a is involved during the proliferation in difference tumors including pancreatic cancer,kidney cancer,ovarian cancer,breast cancer,and lung cancer in the literature[5-9].The Cx43 gene is a kind of tumor suppressor gene[10,11].Both miR-200a and Cx43 are important regulators during tumor proliferation and metastasis,and whether miR-200a promotes the proliferation and metastasis of lung cancer by regulating the expression of Cx43 has not been reported.Ming et al[12]found that miR-200a inhibits the expression of Cx43 gene by directly acting on the 3'-UTR of Cx43 gene in breast cancer,thereby promoting the proliferation and metastasis of breast cancer cells.The aim of this study was to evaluate whether miR-200a promotes the proliferation of non-small cell lung cancer by inhibiting the expression of the target gene Cx43 in NSCLC cells,thereby providing a molecular therapeutic target for the treatment of non-small cell lung cancer in the future.Erlotinib is a small molecule that inhibits the signaling pathway of human epidermal growth factor receptor by inhibiting the activity of tyrosine kinase and be used for EGFR mutation-positive,advanced and metastatic non-small cell lung cancer[13].Ordinary liposomes have natural passive targeting,while long-circulating liposomes modified with polyethylene glycol?PEG?or ganglioside?GM1?can escape the attack of the immune system and prolong the drug efficacy.The time of action has become one of the hot spots in the field of liposome research[14].Whether the long-circulating liposome erlotinib is superior to the free liposomal erlotinib in the clinical treatment of NSCLC requires further study.Because GM1 is expensive and difficult to synthesize and extract.This study used PEG liposome erlotinib,liposomal erlotinib and free erlotinib combined with A549 cell,NSCLC tumor cells or normal rats,compare the stability of three erlotinib preparations,drug release rate,pharmacokinetic characteristics,and drug toxicity to lung cancer cells,in order to provide guidance for PEG-erlotinib liposome application in the future.MethodsPart ?:To show the expression levels of miR-200a and Cx43 in NSCLC tissue and their correlation.Gross specimens of NSCLC patients were collected and real-time quantitative PCR and Western blotting were used to detect the expression levels of miR-200a and Cx43 in NSCLC tumor tissues and adjacent normal tissues.Part ? Expression and correlation of miR-200a and Cx43 in non-small cell lung cancer cell lines.qRT-PCR was used to analyze the expression of microRNA-200a and Cx43 in human lung cancer cell line A549 and normal human lung epithelial cell line BEAS-2B.miR-200a mimics and inhibitors were transfected into NSCLC cell line A549 by LipofectamineTM 2000 Reagent reagent.The mRNA expression of miR-200a and Cx43 was determined by qRT-PCR.The expression of Cx43 was detected by Western blotting.The miR-200a was detected by CCK8 method.Effects of mimics and inhibitors on the activation and proliferation of NSCLC cell line A549cells.Part ? Study of long-circulating liposomes of erlotinib.PEG liposomes and normal liposomes were made to compare the stability of the two liposomes.The cytotoxicity of liposome free erlotinib,common liposome erlotinib and PEG liposome erlotinib on human lung cancer A549 cell line was detected by MTT assay.The pharmacokinetics of three drugs were compared.Characteristics and bioavailability.The degree of pathological damage of various erlotinib preparations on various tissues and organs of rats was compared by HE staining.ResultsPart ?:To show the expression levels of miR-200a and Cx43 in NSCLC tissue and their correlation.Specimens came from 40 NSCLC patients were collected.Using qRT-PCR technique,expression level of miR-200a in NSCLC tumor tissues was significantly higher than that in adjacent tissues?P<0.05?,while the expression level of Cx43 in NSCLC tumor tissues was significantly lower in the adjacent tissues?P<0.05?.And the results showed that they were negatively correlated.Part ? Expression and correlation of miR-200a and Cx43 in non-small cell lung cancer cell lines.Using qRT-PCR technique,the expression level of miR-200a in A549 cells was significantly higher than that in BEAS-2B cells?P<0.05?.The expression level of Cx43 in A549 cells was significantly lower than that in BEAS-2B cells?P<0.05?.The mRNA level and protein level of Cx43 were significantly decreased and the cell proliferation ability was significantly enhanced when over expressing miR-200a in A549 cells;the mRNA level and protein level of Cx43 were significantly increased and the proliferative capacity was significantly reduced when inhibiting the expression of miR-200a in A549 cells?all P<0.05?.Part ? Study of long-circulating liposomes of erlotinib.Storage at 4°C for 3months,both common liposomal erlotinib and PEG liposome erlotinib had similar particle size,polydispersity index?PDI?,and drug content compared with that three month earlier before storage.Release kinetics studies in vitro showed that the common liposomal erlotinib and the PEG liposome erlotinib had similar sustained slow release compared to the rapid release of liposomal free erlotinib.No initial burst phenomenon was observed among the three medicine.A549 lung cancer cells were incubated with the same concentration of liposome free erlotinib,common liposomal erlotinib and PEG liposome erlotinib for 48 hours.The cell viability was detected by MTT assay.The PEG liposome erlotinib had the strongest inhibitory effect on A549lung cancer cells,while the liposome-free erlotinib had the weakest inhibitory effect on A549 lung cancer.The pharmacokinetic characteristics of the three formulations of liposome in rats were compared.Drug clearance rate,average drug retention time,drug distribution volume,and the area under the ROC curve in liposome-free erlotinib group were significantly reduced.Compared with the common liposomal erlotinib,PEG liposomes erlotinib showed a longer half-life?PEG liposomal erlotinib 96.8±11.2 hours,common liposomes erlotinib 92.7±10.1 hours?,lower clearance?PEG liposomal erlotinib 0.07±0.03 mL/kg,common liposomal erlotinib 0.09±0.05 mL/kg?and larger area under the ROC curve?PEG liposome erlotinib 898.5±82.4mmol/L*h,common liposome erlotinib 625.5±72.7mmol/L*h?;PEG liposome erlotide due to prolonged circulation time of the drug The bioavailability of erlotinib is nearly2-fold higher than that of common liposomes.Studies also showed that after anatomical and 10%formalin-fixed rat tissues?including the lung,heart,liver,kidney and spleen tissues of the rats?that had been injected the medicine for liposomal free erlotinib,common liposomal erlotinib or PEG liposomes erlotinib and complete stained with hematoxylin and eosin,no pathological changes like cell ischemia or necrosis had been found in the target organs when observed under 5000 times light microscope.Conclusions1.The expression level of miR-200a in NSCLC tumor tissues was significantly higher than that in adjacent tissues,while the expression level of Cx43 in NSCLC tumor tissues was significantly lower than that in adjacent tissues.Intervention the expression level of miR-200a in A549 lung cancer cell line may have some effect on mRNA and protein expression levels of Cx43,which in turn affected A549 lung cancer cell proliferation.Negative correlation between the expression of miR-200a and Cx43 also be found in this study.2.After interfering the expression of miR-200a,the proliferative capacity of lung cancer cells had been affected:overexpression of miR-200a may enhanced the proliferation of lung cancer cells,while inhibition of miR-200a expression may decreased proliferation of lung cancer cells.3.Liposomal erlotinib has good encapsulation efficiency and stability whether PEGylation?PEG?or not.Liposomal free erlotinib,common liposomal erlotinib and PEG liposome erlotinib are safe for major organs in rats?such as lung,heart,liver,kidney and spleen?).4.Compared with liposomal free erlotinib and common liposomal erlotinib,PEG liposome erlotinib had the highest bioavailability,and it also has the strongest inhibitory effect on the cell viability of A549 lung cancer cell lines. |
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