| Background: Lung cancer is the leading cause of cancer mortality worldwide.Non-small cell lung cancer(NSCLC)accounts for about 80 % of all lung cancers,in which brain metastases(BM)occur in 50 % of all NSCLC cases and represent a major pattern of treatment failure and cause of mortality.Unfortunately,due to lack of understanding the mechanism,the treatment options for NSCLC BM patients,the prognosis of Which was very poor,have lagged behind.Thus,looking for the biomarkers able to accurately detect early tumor changes and characterizes BM is the principle warrant in NSCLC management.Micro RNAs(mi RNAs)are noncoding endogenous RNA species,which regulate the expression of gene post-transcriotionally.The stability of mi RNAs in tissues and fluids makes them attractive candidates for use in predictive and prognostic markers.Micro RNA-375(mi R-375)was found to be differentially expressed in brain metastases in previous studies,which revealed that mi R-375 may be associated with brain metastasis.However,there have been no reports about the study of mi R-375 in NSCLC brain metastasis.Recently,mi R-375 has been found significantly dysregulated in many human cancers,such as esophageal carcinoma,gastic carcinoma,pancreatic carcinoma,prostatic cancer,head and neck carcinoma,et al.A study has showed that mi R-375 was down-regulated in NSCLC,but the underlying mechanism has not been reported.In our study,mi R-375 expression level was detected in NSCLC tissues with and without BM,and statistically analyzed for the purpose of understanding its role in BM.Mi R-375-EGFP,anti-mi R-375-EGFP and EGFP plasmids packed with HIV and HEK-293 T cells,was constructed and transfected into A549 cell line stably,so as to detect the effects of mi R-375 in the cell line,and to understand the mechanism of BM.This is the first study to evaluate mi R-375 expression in NSCLC patients with brain metastasis,and may provide a predictive marker and strategy for blocking brain metastasis.Objectives:(1)The aim was to detected the different expression of mi R-375 in NSCLC patients with and without brain metastasis,clarify the role in NSCLC brain metastasis and its prognostic significance.(2)Mi R-375 lentiviral expression vector was constructed and transfected into NSCLC cells stably,so as to detect the influence of mi R-375 in the NSCLC cell line and explore the down-stream markers,to understand the possible mechanism of mi R-375 in brain metastasis.Methods:(1)Quatitive Real-time PCR(q RT-PCR)was used to detect the expression of mi R-375 in NSCLC patients with BM,as compared to those without BM.EGFR and K-ras mutation were tested by polymerase chain reaction(PCR),and the expression of VEGF and MMP-9 by immunohistochemistry(IHC).(2)The differences of mi R-375 expression level were assessed using Student's t-test.ANOVA was used to evaluated the the relationship between the expression of mi R-375 and the clinicopathological characteristics.(Stage,Region of lymph node metastasis,and number of brain metastase).The prognostic significance was analyzed using the log-rank test,Kaplan-Meier method and Cox regression.In addition,the Chi-square test and t-test were used to explore the mechanism of BM clinically.(3)Three plasmids including mi R-375-EGFP,anti-mi R-375-EGFP,and EGFP,were got by Eco RI and Bam HI enzyme-cuting and re-connection,packed by HIV and HEK-293 T cells,were stably transfected to A549 cell line,and formed 4 cell colons consisted of A549-mi R-375-EGFP,A549-anti-mi R-375-EGFP,A549-EGFP and A549.The effects of mi R-375 on the biological characteristics of NSCLC cells were studied by MTT assay,Transwell invasion and migration array,as well as Scratch test.(4)To understand the mechanism of mi R-375 in BM,tube formation array was used to examine A549 cell tube formation in vitro,and the down-stream markers was tested by western Blot.Results:(1)Mi R-375 expression was significantly down-regulated in NSCLC patients with BM compared with NSCLC without BM(P<0.001).The relative expressions of NSCLC BM+ brain tissues,NSCLC BM+ lung tissues,NSCLC BM-lung tissues and adjacent noncancerous tissues were 0.28±0.05,0.47±0.06,0.71±0.13,1.00±0.17,respectively.The differences between the every group were statistically significant(P<0.05).The expression of mi R-375 was down-regulated in brain metastases and primary lung cancer tissues in NSCLC patients with BM,as compared with primary lung cancer tissues in NSCLC without BM(P<0.05),as well as that in adjacent normal tissues(P<0.01).Mi R-375 expression in the brain metastases was significantly decreased,comparing with the matched primary lung cancer tissues in NSCLC brain metastasis(P<0.05).(2)Statistical analysis indicated low mi R-375 expression was linked to advanced stage(P<0.001)and BM(P<0.001)in NSCLC patient.Survival analysis suggested that low-expression group had shorter overall survival than high-expression group in NSCLC patients with BM(P <0.05)and total NSCLC patients(P <0.01).Cox regression indicated that low mi R-375 expression was independently linked to poor survival of patients with NSCLC(HR = 5.48,P= 0.001).In addition,we found VEGF and MMP-9 were over-expressed in down-regulated mi R-375 cases.(3)Three plasmids including mi R-375-EGFP,anti-mi R-375-EGFP,and EGFP,packed by HIV and HEK-293 T cells,were successfully transfected to A549 cell line,and formed 4 cell colons consisted of A549-mi R-375-EGFP,A549-anti-mi R-375-EGFP,A549-EGFP and A549.MTT assay showed that the proliferation rate of A549-mi R-375-EGFP group was significantly lower than that of A549-EGFP and A549 groups(P<0.05).However,the proliferation rate in A549-anti-mi R-375-EGFP group was higher than that in A549-EGFP and A549 groups(P<0.05).There were no significant difference in A549-EGFP group and A549 group(P>0.05).After 72 h,the proliferation of A549-mi R-375-EGFP,A549-anti-mi R-375-EGFP,and A549-EGFP was observed by fluorescence microscope,showed that the proliferation rate in A549-anti-mi R-375-EGFP group was high,but low in A549-mi R-375-EGFP group,as compared to the control.(4)Transwell incasion assay showed that A549 cell could travel through the matrix gel.The numbers of A549 cells passed the matix gel in the A549-mi R-375-EGFP group was 75.12±6.21,significantly less than those in A549(125±10.27)and A549-EGFP(129.51±8.23)(P<0.05).However,the cell numbers was largest in A549-anti-mi R-375-EGFP group(199.67±9.23).In addition,There were no significant difference in A549-EGFP group and A549 group(P>0.05).(5)Transwell migration assay showed that the numbers of A549 cells over the matix gel in the A549-mi R-375-EGFP group was 50.33±9.32,significantly less than those in A549(108±9.38)and A549-EGFP(110±7.82)(P<0.05).However,the cell numbers was largest in A549-anti-mi R-375-EGFP group(178±10.89).In addition,There were no significant difference in A549-EGFP group and A549 group(P>0.05).(6)Under fluorescence microscope,scratch test showed that,after 24 h,the scratch area of A549-anti-mi R-375-EGFP group was minium,as compared with A549-EGFP group,but the growth of A549-mi R-375-EGFP cells obviously slowed down and the wound area was wide.(7)Tuber formation test:3D-tube was found in A549-anti-mi R375-GFP group,but not in other cell groups in vitro.(8)VEGF?MMP-2?MMP-9 were over-expressed in PC14/B which is the specific cell line of brain metastasis,as compared to PC14.VEGF?MMP-2?MMP-9 were significantly over-expressed in A549-anti-mi R375-GFP group as compared to the control and blank group.While VEGF?MMP-2?MMP-9 were significantly low-expressed in A549-mi R375-GFP group.There was no differences about the expression of EGFR mutation in PC14/B and PC14,as well as A549 subtype groups.In addition,,MMP-2 was different in A549-EGFP group from that in A549.Conclusions: 1)Down-regulated mi R-375 was obviously associated with brain metastasis in NSCLC.Down-regulation of mi R-375 may be a potential biomarker for characterizing NSCLC brain metastasis and satratifying the high-risk patients.Mi R-375 may play an important role as a prognostic factor and/or biomarker for the prediction of survival in NSCLC.Over expression of VEGF and MMP-9 may partly explained the reason of poor prognosis in down-regulated mi R-375 expression patients.Moreover,our data recommend that mi R-375 may serve as a new therapeutic target for NSCLC.2)Down-regulated mi R-375 was associated with NSCLC brain metastasis by promoting cell proliferation,invasion,migration,and angiogenesis.EGFR and K-ras mutation was probably not the predictive biomarker in NSCLC brain metastasis.Mi R-375 may be a new target in mitigating BM development. |
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