| Background and ObjectiveSmall cell lung cancer(SCLC),accounts for about 15% of all lung cancer,with high degree of malignancy and diagnosis and more difficulties in diagnosis and treatment.Compared to non-small cell lung cancer(NSCLC),the clinical treatment is still based on traditional chemotherapy and radiotherapy,because of indistinct of the potential mechanism and lack of targeted driving-genes.Although SCLC is sensitive to initial chemotherapy,but recurrence and metastasis leading to treatment failure and death will soon occur,due to the short doubling time.Therefore,the generation of resistance to chemotherapy is the primary obstacle to the clinical efficacy of small cell lung cancer patients.Previous studies have shown that drug-resistant SCLC cell lines Letp/EP and H446/EP had multidrug resistance.Compared with the parental cell line Letp and H446,the levels of autophagy were significantly increased in the resistant cell lines and related to the drug resistance.This study aimed to identify the molecular targets involved in the formation of SCLC resistance phenotype and expression and clinical significance of Beclin-1,a key factor of initiation of autophagosome formation in SCLC.U sing bioinformatics methods combining with miRNA array,we screened out mi R30a-5p,the pivotal miRNA regulating the expression of Beclin-1 and verified the result with luciferase report gene,for further study on its effect in drug-resistance of SCLC cells.Thus we provided the experimental basis for the treatment of chemoresistance in SCLC.Materials and Methods1.Human small cell lung cancer chemoresistant cell lines Letp/EP and H446/EP were established and cultured in our laboratory.Real-time PCR(q-PCR)and Western Blot(WB)were conducted to detect the expression of Beclin-1.The concentration-dependent induction assay was performed to treat Letp and H446 cells with anticancer drugs in different concentration(VP16 0,1,3 μg/ml + DDP 0,0.3,0.6 μg/ml)and time-dependent induction assays was conducted to treat Letp and H446 cells with 1μg/ml VP16 combined with 0.3 μg/ml DDP for diverse time(0,3,5days).q-PCR and WB were employed to validate Beclin-1 expression in various conditions.2.A number of 92 cases diagnosed with SCLC,of which 54 cases received treatment of etoposide combined with cisplatin chemotherapy,were collected to detected Beclin-1 expression using immunohistochemistry staining.All patients were closely followed up.The correlation between Beclin-1 and clinicopathological characters or chemosensitivity was assessed by statistical analysis.3.Beclin-1 was significantly overexpressed in Letp/EP and H446/EP cells,compared with tha in their parental cell lines.siRNA of Beclin-1(si Beclin-1)was introduced and transfected into drug-resistant cell lines to artificially regulate the expression of Beclin-1.The effect of Beclin-1 in chemoresistant SCLC cells in vitro was assessed via MTT,WB,colony formation and flow cytometric analysis to further estimate chemo-sensitivity,autophagic activity,apoptosis rate,proliferation ability as well as cell cycle distribution.4.Three open access online miRNA databases(miRTarBase,TargetScan and Pictar)were exploited to forecast regulators of Beclin-1.Combined with our previous research and miRNA array,five down-regulated miRNAs(miR-27a-3p,miR-5096,miR-30a-5p,miR-103a-3p and miR-24-3p)were certified to be related to chemoresistance of SCLC cells.5.The crosstalk of miR-30a-5p and Beclin-1 was further confirmed by Luciferase activity assay.After transfected with miR-30a-5p mimics,the expressions of Beclin-1 in Letp/EP and H446/EP were calculated by q-PCR and WB.The expressions of Beclin-1 in H446 and Letp were calculated while transfected with miR-30a-5p inhibitor.6.The function of mi R-30a-5p was estimated through MTT,WB,colony formation and flow cytometric analysis.Further rescue experiment was performed after co-transfection with siBeclin-1 and miR-30 a mimics or-inhibitor.Resluts1.Beclin-1 was strikingly overexpressed in drug-resistant cells in comparation with that in parental ones.Besides,Beclin-1 gradually heightened in pace with drug concentration augment or time extension.2.In patients diagnosed with SCLC,the expression level of Beclin-1 was reversely related to TNM stage,lymph node metastasis and vascular invasion.In patients treated with VP16/DDP(EP)chemotherapy,Beclin-1 expression was upregulated in lower sensitivity group.3.Beclin-1 expression sharply decreased in Letp/EP and H446/EP after transfected with siBeclin-1.The IC50 values of VP16 and DDP in siBeclin-1-transfected chemoresistant SCLC cells significantly reduced when compared with control.Inhibition of Beclin-1 resulted in a dramatic decline of autophagy and increase of apoptosis in drug-resistant SCLC cells.Colony formation assays revealed that Beclin-1 could enhance proliferation ability.Furthermore,inhibition of Beclin-1 mediated a remarkable reduction in S phase and a raise in G2/M phase of cell cycle.4.MiRNAs which could combine to the 3’-untraslated regions of Beclin-1 were predicted by online miRNA databases.Combined with previous research,mi R-30a-5p was chosen for further study.5.As a regulator of Beclin-1,miR-30a-5p was lower expressed in drug-resistant cells compared to parental ones.Increase of miR-30a-5p in drug-resistant cells could inhibit the expression of Beclin-1,while decrease of miR-30a-5p in drug-sensitive cells could enhance the expression of Beclin-1.The result revealed that miR-30a-5p inversely regulated the Beclin-1 expression.6.After transfected with miR-30a-5p mimics,Letp/EP and H446/EP cells exhibited decrease of IC50 value,inhibition of autophagy,increase of apoptosis rate,reduction of proliferation capacity and G0/G1 arrest.Letp and H446 cells exhibited the opposite reactions after transfected with miR-30a-5p inhibitor.In addition,suppression of Beclin-1 could partly reverse the effect induced by miR-30a-5p inhibition in drug-sensitive cells.But in drug-resistant cells,inhibition of Becclin-1 had no further enhancing effect to the transfection with miR-30a-5p mimics.Conclusions1.Beclin-1 played a vital role in chemoresistant phenotype and could function as a potential biomarker in human small cell lung cancer.Suppression of Beclin-1 mediated an obviously IC50 reduction,autophagy inhibition,apoptosis induction,proliferation attenuation and G2/M phase arrest in Letp/EP and H446/EP cells.2.Beclin-1 was negatively regulated by miR-30a-5p,which was related with chemoresistance,autophagy,apoptosis,proliferation and cell cycle redistribution,acting as an onco-suppressor.3.Beclin-1 was a direct target of miR-30a-5p.Dysregulation of miR-30a-5p/Beclin-1 axis contributed to chemoresistant phenotype in SCLC cells.4.In this study,the expression of Beclin-1 in SCLC and its relationship with clinicopathological features and prognosis were analyzed,the role of abnormal regulation of miR-30a-5p/Beclin-1 signaling axis in the chemotherapy resistance of SCLC cells was investigated.It would provide a novel data and strategies for reversing the drug resistance and clinical individualized treatment of small cell lung cancer. |
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