| Part One Study of the expression and prognosis of linc00921 in triple-negative breast cancerObjectives:1.To analyze the differential lnc RNA between triple-negative breast cancer(TNBC)and normal breast tissue in the bioinformatics database,analyze the overlapping results,and look for the differential lnc RNA.2.To detect the expression of the top 5 overlapping linc RNAs down-regulated in TNBC tissue specimens,and look for the lowest expression of linc RNA.3.To detect and analyze the relationship between the expression level of linc00921 and clinical parameters in the tissues of TNBC patients,and to analyze the possible risk factors affecting the prognosis of TNBC patients and relationship between the expression level of linc00921 and to analyze the disease-free survival and overall survival of TNBC patients,and to provide a theoretical basis for judging the impact of linc00921 on the prognosis of TNBC patients.Methods:1.Analysis of differential lnc RNAs between TNBC tissue and normal breast tissue in two public GEO datasets,GSE119233 and GSE115275.Differentially expressed linc RNAs are shown by overlapping results.2.qRT-PCR was used to detect the expression levels of the top five significantly downregulated lnc RNAs in TNBC tissues,and the linc00921 with the lowest expression level was screened for subsequent functional experiments.3.To analyze the relationship between the expression level of linc00921 in TNBC tissues and the patient’s age,tumor size,lymph node metastasis and TNM stage.4.The Kaplan-Meier method was used to analyze the correlation between the expression level of linc00921 and disease-free survival(DFS)and overall survival(OS)in TNBC patients.5.To evaluate the clinical significance of linc00921 as a biomarker in TNBC to predict DFS and OS was evaluated by applying receiver operating curve(ROC).6.Cox proportional risk regression model was used to analyze the factors affecting DFS and OS in TNBC patientsResults:1.The overlapping results from GSE119233 and GSE115275 databases showed that 27 lnc RNAs were down-regulated and 29 lnc RNAs were up-regulated in TNBC tissues compared with normal breast tissues.2.The expression level of linc00921 in TNBC tissues was the lowest,and the difference was statistically significant(P<0.001).3.The expression level of linc00921 was significantly correlated with large tumor,positive lymph node metastasis and high TMN stage(P<0.05),but not with age(P>0.05).4.The DFS and OS of TNBC patients with low expression of linc00921 were shorter than those with high expression of linc00921(P<0.05).5.Receiver operating curve(ROC)was used to evaluate the predictive value of linc00921 for DFS and OS in TNBC patients.The area under the ROC curve for disease-free survival was 86.59%,and the overall survival was79.33%..6.The results of multivariate analysis showed that the TNM stage and the expression level of linc00921 were significantly correlated with the DFS and OS of TNBC patients(P<0.05).Conclusions : The expression level of linc00921 decreased in TNBC tissue,and the expression level of linc00921 in TNBC tissue was correlated with tumor size,lymph node metastasis,and TMN stage.The overall survival time is shorter than that of high expressors,and linc00921 can be used as an independent biomarker to predict the prognosis of TNBC patients.Part Two linc00921 suppresses tumorigenesis and epithelial-to-mesenchymal transition of triple-negative breast cancer via targeting miR-9-5p/LZTS2 axisObjective:To detect the expression of linc00921 in triple-negative breast cancer cell lines and normal breast epithelial cell lines,and to explore investigate its effects on the proliferation,migration and invasion of triple-negative breast cancer(TNBC)cells.The subcellular localization characteristics of Linc00921 in triple-negative breast cancer cell lines were detected,the ce RNA regulatory network of linc00921 was predicted by bioinformatics tools,and the molecular biology experiments were used to verify the effect and potential mechanism of linc00921 on the malignant biological behavior of TNBC.Methods:1.Expression levels of linc00921 in 5 triple-negative breast cancer cell lines and normal breast epithelial cell lines were detected by qRT-PCR.2.A vector overexpressing linc00921(referred to as pCDH-linc00921)was constructed using pCDH plasmids,transfection was performed using Lipofectamine TM2000 in TNBC cell lines MDA-MB-231 and HCC-1937,and transfection efficiency was detected using pCDH plasmid empty carrier as the control group(EV group),the transfection efficiency was detected by qRT-PCR.3.The effects of overexpression of linc00921 on the proliferation,invasion and migration of TNBC cells were detected by CCK-8,Transwell and Scratch Healing Experiments.4.Fluorescence in situ hybridization(FISH)was used to detect the localization characteristics of Linc00921 in TNBC cell lines.5.Bioinformatics tools predicted the expression levels of candidate miRNAs in linc00921 and used qRT-PCR to detect the expression levels of candidate miRNAs in TNBC cell lines and analyzed their correlation with the expression levels of linc00921.6.The targeted regulatory relationship between linc00921 and miR-9-5p was verified by the double luciferase reporter gene.RNA immunoprecipitation(RIP)experiments using Ago2 antibody confirmed that linc00921 and miR-9-5p can co-form RNA-induced silencing complex(RISC)in TNBC cell lines.7.Prediction of target gene of miR-9-5p using bioinformatics tools and analysis of target gene LZTS2 expression in the TCGA database TNBC tissue cohort.The targeted regulatory relationship between miR-9-5p and LZTS2 was verified using the biluciferase reporter gene.8.miRNA rescue experiments using miR-9-5p mimics Validation of the linc00921/miR-9-5p/LZTS2 axis in TNBC cells.9.Immunohistochemistry showed the staining of LZTS2 in TNBC tissues,and the expression level of LZTS2 positive and LZTS2-negative TNBC tissues was detected by qRT-PCR method.Correlation between linc00921 and LZTS2 was analyzed using a TNBC cohort from the TCGGA database.10.GSEA was applied to GSE115725 for gene enrichment analysis to further predict the potential downstream pathways of the linc00921/miR-9-5p/LZTS2 axis,the separation of nucleus and cytoplasmic components,and the effect of the linc00921/miR-9-5p/LZTS2 axis on the localization and phosphorylation levels of β-catenin subcellular cells.11.The effect of linc00921 on the expression of EMT-related proteins in HCC-1937 and MDA-MB-231 cells was detected by Western blotting.The effects of linc00921/miR-9-5p/LZTS2 axis on the proliferation,invasion and migration of HCC-1937 and MDA-MB-231 cells were detected by CCK-8,Transwell,and wound healing assays.Results:1.qRT-PCR results showed that linc00921 was expressed lower in TNBC cells compared with normal breast epithelial cells(P<0.001),of which HCC-1937 and MDA-MB-231 cells had the lowest relative expression,so these two cell lines were selected for the next step of the study.2.qRT-PCR results showed a significant increase in the expression of linc00921 in the pCDH-linc00921 group compared with the negative control group(EV group).(P<0.01)3.The results of CCK-8,Transwell,and scratch healing experiments showed that the proliferation,invasion and migration ability of TNBC cell lines(MDA-MB-231,HCC-1937)in pCDH-linc00921 group Significantly increased significantly compared with EV group(P<0.001).This indicates that linc00921 can inhibit the proliferation,invasion and migration of triple-negative breast cancer cells.4.FISH detected linc00921 was mainly located in the cytoplasm of TNBC cell lines.5.Bioinformatics algorithms predict that miR-330-5p,miR-326,miR-30a-5p and miR-9-5p may be the miRNAs that linc00921 can adsorb.qRT-PCR results showed that only miR-9-5p was significantly negatively correlated with linc00921 expression in TNBC tissues(Spearman r =-0.484,P< 0.001),miR-330-5p,miR-326,miR-30a-5p were not significantly correlated with linc00921 expression(P >0.05)6.Double luciferase reporter gene test results show that linc00921 can bind to miR-9-5p,and linc00921 regulates the expression of miR-9-5p by acting as a ce RNA,and the expressions of the two are negatively correlated(P< 0.001).7.Immunoprecipitation experiments(RIP)show that the expression level of linc00921 and miR-9-5p in the Ago2 pellets of HCC-1937 and MDA-MB-231 cells is significantly higher than that of Ig G pellets(P<0.01),indicating that linc00921 co-forms RISC with miR-9-5p.8.bioinformatic algorithms analyzed the target genes of miR-9-5p,found a total of 5 candidate genes,and selected LZTS2 as the research object.Triple-negative breast cancer cohort results from the TCGA database showed that LZTS2 expression in tumor tissue was significantly lower than in normal breast tissue(P=0.033)9.Luciferase reporter gene results showed that miR-9-5p mimics were able to reduce luciferase activity in HCC-1937 and MDA-MB-231 cells transfected with LZTS2 m RNA 3’-UTR-WT(P<0.001),indicating that miR-9-5p specifically targets the binding site in the 3′-UTR of LZTS2 m RNA.Meanwhile,tthe western blot detection results showed that after co-transfection of miR-9-5p inhibitors,the expression level of LZTS2 in HCC-1937 and MDA-MB-231 cells increased(P<0.001).It was shown that LZTS2 is a target gene of miR-9-5p in TNBC.10.miRNA rescue experiments confirmed that miR-9-5p mimics can antagonize the up-regulation of LZTS2 by overexpression of linc00921 in triple-negative breast cancer cells,indicating that there is a linc00921/miR-9-5p/LZTS2 axis in triple-negative breast cancer cells.11.Immunohistochemical staining of LZTS2 was mainly located in the cytoplasm of TNBC cells,with 51 cases(53.68%)of LZTS2 negative expression and 44 cases(46.62%)positive expression.qRT-PCR results showed that the expression of linc00921 in the LZTS2-positive group was significantly higher than that in the LZTS2-negative group(P< 0.001),and the TCGA data of the TNBC cohort showed that linc00921 and LZTS2 were positively correlated(Spearman r=0.235,P=0.01),indicating that LZTS2 was positively correlated with linc00921 expression levels.12.Gene set enrichment analysis GSEA results showed that LZTS2 enrichment is in the Wnt/βcatenin pathway genome,which plays a key role in epithelial-mesenchymal transition(EMT).Experimental results of nucleus and cytoplasmic component separation experiments showed that overexpression of linc00921 significantly reduced the nuclear-cytoplasmic ratio ofβ-catenin.Western blotting results showed that overexpression of linc00921down-regulated the phosphorylation level of β-catenin,increased the expression of E-cadherin and decreased the expression of N-cadherin in HCC-1937 and MDA-MB-231 cells,while co-transfected miR-9-5p mimetics were able to reverse this phenomenon.The results of CCK-8,Transwell,and wound healing experiments showed that overexpression of linc00921 could significantly inhibit the proliferation of HCC-1937 and MDA-MB-231 cells,while co-transfection of miR9-5p mimics and co-transfection of miR-9-5p mimics can reverse this phenomenon.It shows that the linc00921/miR-9-5p/LZTS2 axis can inhibit the proliferation,invasion,migration and epithelial mesenchymal transformation of triple-negative breast cancer cells.Conclusions:1.The expression of linc00921 was low in TNBC cells.2.Overexpression of 2.linc00921 promotes the proliferation,migration and invasion of TNBC cells.3.Linc00921 is mainly located in the cytoplasm of triple-negative breast cancer cell lines(MDA-MB-231,HCC-1937),and miR-9-5p is the target miRNA of Linc00921,which is highly expressed in TNBC cells.4.LZTS2 is a target gene of miR-9-5p,and the Linc00921/miR-9-5p/LZTS2 axis inhibits the EMT process of HCC-1937 and MDA-MB-231 cells.Part Three In vivo study of linc00921/miR-9-5p/LZTS2 axis affecting the occurrence and development of triple-negative breast cancerObjective: To explore the carcinogenic effect of the linc00921/miR-9-5p/LZTS2 axis in nude mice,and to lay a theoretical foundation for the reversal treatment of TNBC.Methods:1.The three-impression breast cancer MDA-MB-231 cells were transfected with Lipofectamine TM2000 by lipofection method,and the cells were divided into control group(EV transfection group)and linc00921 overexpression group(pCDH-linc00921 transfection group)).2.Two groups of MDA-MB-231 cells were subcutaneously injected into the groin of 4-week-old female BALB/c nude mice,5 in each group,to construct a nude mouse xenograft model.3.Weigh 10 nude mice every 7 days;the tumor volume of the transplanted tumor in nude mice was measured in vitro with a ruler every 1week,and the tumor growth curve was drawn;after 4 weeks,all nude mice were sacrificed by cervical dislocation,and the primary tumor was removed and weighed.4.qRT-PCR method was used to detect the expression level of miR-9-5p in tumor tissue in mice.5.The expression level of LZTS2 protein in tumor tissue of mice was detected by immunohistochemistry.Results:1.Tumor growth curve showed that the growth rate of the transplanted tumor in the pCDH-linc00921 group was significantly lower than that in the EV group(P<0.01).The transplanted tumor was taken out and weighed.As shown in the figure,the tumor weight was significantly reduced in the pCDH-linc00921 group and EV group(P<0.01).It shows that overexpression of linc00921 can significantly inhibit the growth of transplanted tumors in nude mice,and has an anti-tumor effect.2.The results of qRT-PCR showed that the expression of miR-9-5p in pCDH-linc00921 group was significantly lower than that in EV group(P<0.05).It shows that overexpression of linc00921 in nude mice can significantly reduce the expression of miR-9-5p.3.Immunohistochemical results showed that the expression of LZTS2 in the tumor tissue of the pCDH-linc00921 group was positive,while the expression of LZTS2 in the tumor tissue of the EV group was negative,indicating that overexpression of linc00921 in mice can significantly increase the expression of LZTS2.Conclusion:1.Overexpression of linc00921 significantly inhibited the growth of xenografts.2.linc00921 exerts a tumor suppressor effect in nude mice through the miR-9-5p/LZTS2 axis,and inhibits the occurrence and development of triple-negative breast cancer in vivo. |
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