TCF3-Activated LINC00152 Exerts Oncogenic Role In Osteosarcoma Through Regulating MiR-1182/CDK14 Axis

It has been reported that long non-coding RNA is involved in tumorigenesis and various cellular processes in osteosarcoma;however,the role of lncRNA LINC00152 in OS remains elusive.In this study,LINC00152 was significantly more highly expressed in osteosarcoma tissues and cell lines than normal adjacent tissues and normal osteoblast lines.Moreover,MTT and colony formation assays showed that knockdown of LINC00152 significantly inhibited cell proliferation;transwell assay analysis showed that knockdown of LINC00152 expression in tumor cells,tumor cell migration and invasion ability decreased.In addition,chromatin immunoprecipitation(ChIP)and luciferase reporter assays indicated that LINC00152 is transcriptionally activated by the transcription factor TCF3.Subcellular separation experiments showed that LINC00152 is mainly located in the cytoplasm of OS cells.Further experiments demonstrated that LINC00152 plays a regulatory role as a competitive endogenous RNA(ceRNA),and regulates the biological behavior of osteosarcoma cells by regulating miR-1182/CDK14 axis.Overall,LINC00152 was activated by TCF3 to promote cell proliferation and migration in osteosarcoma via the miR-1182-CDK14 axis.Purpose1 Real-time quantitative PCR was used to detect the expression level of LINC00152 in osteosarcoma.The tumor cells were transfected with sh-RNA to knock down the expression of LINC00152.The function of the transfected tumor cells was tested to determine the effect of LINC00152 knockdown on the cell process in OS,such as proliferation.,invasion and migration.2 Further analysis of the upstream and downstream molecular mechanisms of LINC00152 regulating the biological behavior of osteosarcoma revealed the mechanism and function of LINC00152 in the progression of osteosarcoma.Materials and MethodsMaterials1 Tissue samples 84 osteosarcoma tissues and adjacent normal tissues(tumor distance>5 cm)were received from osteosarcoma patients at the Second Affiliated Hospital of Suzhou University.2 Four osteosarcoma cell lines(U20S,MG-63,HOS,143B)and a normal human fetal osteoblast cell line(hFOB).Plasmid transfection was performed in MG-63 and HOS cells using Lipofectamine 2000.To knock down the expression of LINC00152,a short hairpin RNA(shRNA)and a negative control(sh-NC)against LINC00152 were designed.MiR-1182 mimetic/inhibitor and pcDNA-CDK14 and their negative controls.Methods1 RNA isolation and qRT-PCR:The expression level of LINC00152 was obtained from each tumor cell line and normal osteoblasts,tumor tissues and normal adjacent tissues.2 Cell viability was measured by MTT assay:After incubating the tumor cells for 24 hours at 37 ℃,5%CO 2,10 μl of MTT solution(5 mg/mL)was added to each well at different time points(24,48,72,96 hours);150 μl of DMSO was applied to dissolve MTT Crystal;then,the absorbance of the plate at 570 nm was analyzed by a microplate reader.3 Colony formation assay:The transfected tumor cells were cultured in DMEM containing 10%FBS at 37℃,5%CO 2;the medium was changed every three days;two weeks later,the colonies were washed three times in phosphate buffered saline and used 10%formaldehyde was fixed for half an hour;fixed cells were stained with 0.1%crystal violet in PBS for 15 minutes;the number of colony cells was manually calculated.4 Transwell analysis:Cell migration and invasion ability were analyzed by microwell counts in the lower chamber of Transwell chamber.5 Subcellular fractionation:The localization of LINC00152 in the cytoplasm or nucleus was detected using a cytoplasmic and nuclear RNA purification kit;then,expression levels of LINC00152 in the cytoplasm and nucleus were detected by qRT-PCR;GAPDH was considered to be a cytoplasmic control,and U6 was used as a nuclear control.6 Dual luciferase report analysis:Construction of wild-type LINC00152(LINC00152-WT)and mutant LINC00152(LINC00152-MUT);co-transfection of pmirGLO-LINC00152 or pmirGLO-LINC00152-MUT with miR-1182 mimic and miR-NC into MG-63 or HOS cells After 48 hours,luciferase activity was measured.7 RIP determination:The tumor cell line is lysed in RNA lysis buffer,the cell lysate is cultured in RNA immunoprecipitation buffer,and then these samples are digested with proteinase K;the immunoprecipitated RNA complex is isolated;the spectrophotometer and bioanalyzer are used for measurement RNA concentration and quality;quantitative real-time PCR detection of purified RNA.8 Northern blot analysis:The tumor cell RNA was isolated and purified,and the RNA sample was size fractionated by electrophoresis.The biotinylated oligonucleotide probe specific for LINC00152 was hybridized with the blot,and the signal intensity of the hybridized band was quantified by densitometry.9 Western blot analysis:Measurement of CDK14 expression in tumor cells.Statistical AnalysisStatistical differences were analyzed using SPSS 18.0.Data are expressed as mean±standard deviation(SD).Differences were analyzed using Student’s t test and one-way analysis of variance(ANOVA)with Tukey’s post hoc test.Pearson correlation analysis was performed to measure the expression relationship between LINC00152,miR-1182 and CDK14.Each experiment was performed at least three times.Statistical significance is expressed as a p-value less than 0.05,p<0.01 means significant difference.Result1 Compared with adjacent normal tissues,LINC00152 is overexpressed in osteosarcoma tissues.The expression of LINC00152 is closely related to tumor size.The overall survival rate of patients with high LINC00152 expression is lower than that of patients with low expression of LINC00152.2 The expression level of osteosarcoma cell line LINC00152 was significantly higher than that of normal osteoblast cell line,with MG-63 and HOS.3 By transfection with sh-LINC00152,LINC00152 was down-regulated in MG-63 and HOS cells.MTT assay and Traswell assay showed that knockdown of LINC00152 significantly inhibited cell proliferation and migration in MG-63 and HOS cell lines.And invasion.4 TCF3 is a potential transcription factor for LINC00152,which is up-regulated in OS tissues and positively correlated with TCF3 of LINC00152.Overexpression of TCF3 significantly increased the level of TCF3 in MG-63 and HOS cells.The results of qRT-PCR and northern blot analysis showed that TCF3 overexpression significantly increased the expression level of LINC00152 in MG-63 and HOS cells,and the results indicated that LINC00152 was activated by TCF3 transcription in OS.5 LINC00152 acts as a molecular sponge of miR-1182 in OS,LINC00152 upregulates CDK14 expression by competitive binding to miR-1182,and LINC00152 promotes osteosarcoma progression by modulating the miR-1182/CDK14 axis.Conclusion1 LINC00152 was highly expressed in osteosarcoma and osteosarcoma cell lines,knocking down LINC00152 to inhibit proliferation,migration and invasion of osteosarcoma cells.2 LINC00152 is activated by TCF3 and plays a carcinogenic role in OS by regulating the miR-1182/CDK14 axis.Our results may be helpful in the study of new OS therapeutic targets.