Expression And Effect Of XLOC₀08466 On The Proliferation,Invasion And Apoptosis In Non-Small Cell Lung Cancer

Background Lung cancer has been one of the most prevalent cancers in the world.According to the results from epidemiological investigation,smoking and environmental pollution are the main inducing factors.Non-small cell lung cancer?NSCLC?and small cell lung cancer are the two main histological types of lung cancer.The former,NSCLC,is the predominant form of lung cancer,accounting for approximately 80%.Concerning the therapy for lung cancer,targeted therapy to NSCLC especially adenocarcinoma is attracted attention besides surgery,chemotherapy,and radiation therapy.However,the efficacy and prognosis of most patients who are not suitable for targeted therapy are unsatisfactory.Therefore,it is conducive to provide new and potential target for therapy.And administrative interference measures to target can be adopted for controlling the development of cancer,which may be help to improve the survival rate and reduce the death rate.With the development of microarray technology,researchers found that part of human genome cannot encode proteins but play a role on the physiological and pathological processes of cells.It is non-coding RNA?nc RNA?.Types of nc RNA are long non-coding RNA?lnc RNA?,micro RNA?mi RNA?,si RNA,pi RNA,r RNA,t RNA,sn RNA,sno RNA and so on.Among these nc RNAs,mi RNA,lnc RNA,si RNA and pi RNA have been reported to play regulatory roles.Mi RNA has been verified to play an important role by regulating the 3'UTR of target gene at the transcription and post-translation levels.Long non-coding RNA?lnc RNA?is a diverse class of nc RNA with 200¹00000 nucleotides in length.Several lnc RNAs have been reported to participate in the regulation of gene expression at the transcription,pre-translation and post-translation levels,and play a regulatory role by interacting with proteins or RNA.Recent studies have found that lnc RNA and mi RNA have mutual regulation and control,which plays an important role in the biological function of tumor cells.Based on the mutual relationships among lnc RNA and mi RNA,mi RNA and m RNA,we infer that there is a tight regulatory network among lnc RNA,mi RNA and m RNA,which will provide more important directions for studying the pathogenesis of disease.In our preliminary research,we detected the expression profiles of lnc RNAs in non-small cell lung cancer tissues and paired normal tissues by microarray technology.With further screening,identification and analysis of clinical and pathological data,we found one of lnc RNA,XLOC₀08466,has high expression level in non-small cell lung cancer tissues and related to lymph node metastasis and tumor stage.In combination with preliminary bioinformatic analysis,we infer that XLOC₀08466plays some molecular regulatory mechanisms in the development of non-small cell lung cancer.Therefore,our study systematically analysed the expression and function of XLOC₀08466 in non-small cell lung cancer and discussed its potential mechanism.Methods1.Detection of XLOC₀08466 expression in non-small cell lung cancer tissues Primers of PCR amplification were designed according to XLOC₀08466sequence.The expression levels of XLOC₀08466 in 63 NSCLC patients were detected by RNA extraction,reverse transcription and real-time PCR.2.Analysis of the correlation between XLOC₀08466 expression and clinicopathological features of NSCLC patients Correlation analysis was used to verify the correlation between XLOC₀08466expression and gender,age,smoking status,differentiation status,lymph node metastases and TNM stage.3.Small interferring RNA?si RNA?technology was used to downregulate XLOC₀08466 expression in A549 and H460 cells si RNA against XLOC₀08466,and control oligonucleotides?negative control,NC?were synthesized and transfected into A549 and H460 cells using lipofectamineTM2000.Experiments were divided into three groups: Blank,NC and si RNA Lnc group.q RT-PCR was used to detected XLOC₀08466 expression for screening the satisfying si RNA sequence.4.Cell proliferation detection after XLOC₀08466 down-regulation in different transfection groups A cell proliferation assay was performed using cell counting kit-8?CCK-8?solution to detect the proliferation abilities of A549 and H460 cells at 0h,24 h,48 h and 72 h.5.Nude mice tumor growth detection after XLOC₀08466down-regulation in A549 xenograft model Xenograft model of nude mice was established by using A549 cell line stably expressing lnc RNA XLOC₀08466 and luciferase?A549-Luc cells infected with sh RNA-Lnc?or its negative control cells?A549-Luc cells infected with sh RNA-NC?Fluorescence signal intensity of transplanted tumor in nude mice was observed weekly by small animal live imaging system and the growth curve was drawn.6.Cell apoptosis detection after XLOC₀08466 down-regulation in different transfection groups A cell apoptosis assay was performed using flow cytometry with propidium iodide?PI?and Annexin V-FITC double staining to detect the apoptosis abilities of A549 and H460 cells.7.Cell invasion detection after XLOC₀08466 down-regulation in different transfection groups A cell invasion assay was performed using the transwell chamber?8?m?covered with matrigel to detect the invasion abilities of A549 and H460 cells.8.UCSC data to analyze the potential interaction sequence with XLOC₀084669.Luciferase reporter assays to investigate the potential interaction of mi R-874 with XLOC₀08466The primers sequences were designed according to the interaction sequence between XLOC₀08466 and mi R-874.Overlap PCR methods were used to amplify the wild-type and mutant fragments of XLOC₀08466,which were cloned into a pmir GLO vector as pmir GLO XLOC₀08466-wt/ pmir GLO-XLOC₀08466-mut.And then the recombinant plasmids were transiently co-transfected with a mi R-874 mimic and scramble oligonucleotides into 293 T cells using Lipofectamine?2000.10.SPR assay to further investigate the potential interaction of mi R-874 with XLOC₀08466SPR assay was performed using a carboxymethylated dextran coated SA chip mi R-874 or scramble oligonucleotides of different concentrations?325.5n M,625 n M,1250 n M,2500 n M,5000 n M,1000 n M,325.5n M?were injected to detect the signal.11.RIP assay to further investigate the potential interaction of mi R-874 with XLOC₀08466The RIP assay is an antibody-based technique that is used to map XLOC₀08466 and mi R-874 interactions.The Ig G antibody was used as control. 12.Analysis of the relationship between XLOC₀08466 and mi R-874expression Correlation analysis was used to verify the correlation between XLOC₀08466and mi R-874 expression.13.Bioinformatics analysis about the potential targets of mi R-87414.Luciferase reporter assays to investigate the potential regulative function among XLOC₀08466,mi R-874,MMP2 and XIAP The primers sequences were designed according to the interaction sequence between MMP2 and XIAP 3'UTR and mi R-874.Overlap PCR methods were used to amplify the wild-type and mutant fragments of MMP2 and XIAP 3'UTR,which were cloned into a pmir GLO vector as pmir GLO-MMP2-Wt/pmir GLO-MMP2-Mut and pmir GLO-XIAP-Wt/pmir GLO-XIAP-Mut.And then the recombinant plasmids were transiently co-transfected with a mi R-874 mimic or scramble into 293 T cells using lipofectamine?2000.15.mi R-874,MMP2 and XIAP expression after XLOC₀08466 downregulation in A549 and H460 cells In A549 and H460 cells,q RT-PCR was used to detect the expression of mi R-874,MMP2 and XIAP m RNA.Western blot was used to detect the expression of MMP2 and XIAP after XLOC₀08466 down-regulation and mi R-874 up-regulation.16.CCK-8 array to detect the proliferation abilities of A549 and H460 cells after XLOC₀08466 down-regulation and mi R-874 up-regulation17.Anncxin V-FITC/PI double staining to detect the apoptosis abilities of A549 and H460 cells after XLOC₀08466 down-regulation and mi R-874 up-regulation18.Transwell invasion assay to detect the invasion abilities of A549 and H460 cells after XLOC₀08466 down-regulation and mi R-874up-regulation 19.Statistical analysis All experiments were performed in five independent replications.SPSS 21.0software was used for all statistical analyses.All of the results are expressed as the mean ±SD.T-test and a one-way analysis of variance?ANOVA?were used for the comparison of means from different samples.Correlation analysis was used to detect the correlation of two variables.P < 0.05 was considered to be statistically significant.P < 0.01 was considered to be distinct difference.Results1.Expression of XLOC₀08466 in NSCLC tissues q RT-PCR showed that XLOC₀08466 was significantly up-regulated in NSCLC tissues compared to matched adjacent normal tissues?P <0.01?.We also observed that XLOC₀08466 expression was significantly related to lymph node metastasis and the TNM stage and was higher in the lymph node positive group or TNM III stage than negative group or TNM I and II stage?P <0.01?.2.XLOC₀08466 expression after XLOC₀08466 related si RNA transfection in A549 and H460 cells Compared to NHBE cells,blank and NC groups,XLOC₀08466 expression in A549 cells had significant down-regulation in si RNA Lnc1 and si RNA Lnc2 group?P<0.01?;In H460 cells,XLOC₀08466 expression had significant down-regulation in si RNA Lnc2 group?P<0.01?.So the si RNA sequence of si RNA Lnc2 group was chosen as the target sequence for the following experiments.3.Effects of XLOC₀08466 down-regulation on cell proliferation CCK-8 assay showed that down-regulation of XLOC₀08466 in the si RNA Lnc group led to slower proliferation 24 h after transfection than the blank group or scramble group,and the proliferation-suppressing trend became more obvious 48-72 h after transfection?P < 0.05?.4.Effects of XLOC₀08466 down-regulation on A549 nude mice tumor growth Compared with NC group,down-regulation of lnc RNA XLOC₀08466expression in vivo could decrease the fluorescence intensity of xenografts?P <0.05?.The result showed that down-regulation XLOC₀08466 expression inhibited the proliferation of A549 nude mice in vivo.5.Effects of XLOC₀08466 down-regulation on cell apoptosis Flow cytometry demonstrated that the apoptotic population was notably up-regulated in the si RNA Lnc group of A549 and H460 cells?P < 0.05?6.Effects of XLOC₀08466 down-regulation on cell invasion Transwell invasion assay demonstrated that the number of invading cells was significantly lower in the si RNA group than in the two control groups in A549 and H460 cells?P<0.05?,which indicated that down-regulation of XLOC₀08466inhibited cell invasion.7.UCSC analysis and luciferase reporter assays validated that XLOC₀08466 had a specific binding site with mi R-874 Sequence analysis from UCSC data showed the putative binding sites between XLOC₀08466 and mi R-874.Luciferase reporter assays was proceeded and we measured luciferase activities 24 h posttransfection,and the results showed that the mi R-874 mimic reduced the luciferase activity of pmir GLO-XLOC₀08466-wt but not of pmir GLO-XLOC₀08466-mut?P<0.01?,which indicated that XLOC₀08466had a specific binding site with mi R-874.8.SPR assay validated XLOC₀08466 had a specific binding site with mi R-874 SPR assay showed appositive binding between wild-type XLOC₀08466 and miR-874 mimic with dissociation constants?KD?2.922 ± 0.906 ?M,which had statistical difference compared with the other three control groups?P < 0.05?.9.RIP assay validated XLOC₀08466 had a specific binding site with mi R-874 RIP assay was performed using the Ago2 antibody.The q RT-PCR results showed that XLOC₀08466 and mi R-874 were preferentially enriched in Ago2-containing beads compared to the input group?P < 0.01?,which indicated that XLOC₀08466 had a specific binding site with mi R-874.10.The relationship between XLOC₀08466 and mi R-874 expression in NSCLC tissues The inverse relationship between XLOC₀08466 and mi R-874 expression?R2 =0.612?.11.Bioinformatics analysis,luciferase reporter assays and q RT-PCR validated that XLOC₀08466 improves MMP2 and XIAP expression by down-regulating the mi R-874 level Bioinformatics analysis indicated that MMP2 and XIAP were potential targets of mi R-874.The luciferase activities were measured and showed that the mi R-874 mimic reduced the luciferase activity of pmir GLOMMP2-wt,but not of pmir GLOMMP2-mut,and the down-regulation of XLOC₀08466 had the same effect?P <0.05?.Similar results were obtained when the recombinant plasmids were pmir GLOXIAP-wt/pmir GLO-XIAP-mut?P<0.05?.q RT-PCR showed that up-regulation of mi R-874?P < 0.01?,down-regulation of MMP2 and XIAP m RNA?P < 0.05?in the si RNA Lnc group of A549 and H460 cells.12.Expression of MMP2 and XIAP proteins after XLOC₀08466down-regulation and mi R-874 up-regulation in A549 and H460 cells Western blotting showed the down-regulation of MMP2 and XIAP expression in the mi R-874 group and si RNA Lnc group cells?P <0.05?. 13.Comparison to the cell proliferation changes after XLOC₀08466down-regulation and mi R-874 up-regulation in A549 and H460 cells CCK-8 assay showed that A549 and H460 cells transfected with mi R-874 or si RNA of XLOC₀08466 showed a trend of lower proliferation rates?P <0.05?.There was no significant difference between the si RNA Lnc group and the mi R-874group?P> 0.05?.14.Comparison to the cell apoptosis changes after XLOC₀08466down-regulation and mi R-874 up-regulation in A549 and H460 cells Anncxin V-FITC/PI double staining showed that cell apoptosis rate in the mi R-874 group and si RNA Lnc group was higher than that of the control group?P<0.05?.There was no significant difference between the si RNA Lnc group and the mi R-874 group?P> 0.05?.15.Comparison to the cell invasion changes after XLOC₀08466down-regulation and mi R-874 up-regulation in A549 and H460 cells Transwell invasion assay showed revealed that the cell invasion of the mi R-874 group and si RNA Lnc group were significant decreased compared to that of the control group?P <0.05?.There was no significant difference between the si RNA Lnc group and the mi R-874 group?P> 0.05?.Conclusions1.Lnc RNA XLOC₀08466 expression is significantly increased in NSCLC tissues and was associated lymph node metastases and TNM stage.2.Down-regulation of XLOC₀08466 in A549 and H460 cells can significantly inhibit cell proliferation and invasion ability,and promote cells apoptosis.3.XLOC₀08466 functions as ce RNA directly binding to mi R-874 and regulates MMP2 and XIAP expression4.XLOC₀08466 can play a role in cancer promotion and is expected to become a new target for NSCLC therapy.