| Endometrial cancer is one of the three most common malignancies originated from the female reproductive system,and the specific pathogenesis of endometrial cancer is not clear.In recent years,the morbidity and mortality of endometrial cancer remain stubbornly high.Surgery is the primary treatment for women with localized endometrial cancer,and adjuvant therapy is required for patients depending on the stage of disease and other risk factors.Patients with early-stage endometrial cancer would have good prognosis.However,the current treatment methods have not satisfactorily reduced the mortality of advanced(above stage IB)and recurrent endometrial cancer cases.Therefore,it is particularly important to further explore the mechanisms involved in pathogenesis of endometrial cancer and find new therapeutic targets.Long non-coding RNAs(lncRNAs)are a class of non-coding RNAs with lengths more than 200 nucleotides,which can regulate genes expression at multiple levels.LncRNAs are widely involved in pathogenesis and metastasis of a variety of malignant tumors,which indicates that lncRNA can be used as tumor diagnostic markers and potential therapeutic targets.There are relatively few studies focus on lncRNAs in endometrial cancer.In this research,we analyzed the RNA transcripts of endometrial cancer tissues from The Cancer Genome Atlas(TCGA)and first reported a novel lncRNA,BMPR1B-AS1,that was more highly expressed in endometrial cancer tissues than in adjacent tissues.In order to further investigate the potential functions of BMPR1B-AS1 in endometrial cancer,this study focused on the following four parts:Part I Expression of lncRNA BMPR1B-AS1 in endometrial cancer and its effect on malignant phenotype of endometrial cancer cell;Part II BMPR1B-AS1 promotes proliferation,migration and invasion of endometrial cancer cells,and inhibits apoptosis through endogenous competitive inhibition of miR-7-2-3p;Part Ⅲ BMPR1B-AS1/miR-7-2-3p can modulate PI3K/Akt/NF-κB signaling pathway by promoting the expression of DCLK1;Part Ⅳ Effects of BMPR1B-AS1/miR-7-2-3p/DCLK1 on endometrial cancer growth in an animal model of endometrial cancer.Part I Expression of lncRNA BMPR1B-AS1 in endometrial cancer and its effect on malignant phenotype of endometrial cancer cellBackgroundThere still lacks of effective treatment for patients with advanced and recurrent endometrial cancer,because the molecular mechanism of the disease is not fully studied.LncRNAs are widely involved in the carcinogeneses and development of tumors.TCGA data analysis suggested that the expression of lncRNA BMPR1B-AS1 was increased in endometrial cancer,but its role needs to be further clarified.ObjectivesTo study the expression difference of BMPR1B-AS1 between endometrial cancer tissues and corresponding paracancerous tissues,and investigate the effect of BMPR1B-AS1 on malignant phenotype of endometrial cancer cell.MethodsThe RNA transcription data from endometrial carcinoma samples were obtained from TCGA.The para-cancerous tissues and carcinoma tissues were distinguished according to the rules for naming TCGA samples,and the differential expression patterns were analyzed with R[the mean values of FPKM in the two groups were both greater than 1,|log2fold change(FC)|>1.0,False Discovery Rate(FDR)<0.05].28 cancer tissues and paired adjacent nontumor tissues were obtained from patients who were pathologically diagnosed with endometrial cancer during surgery at the department of gynecology,First Affiliated Hospital of Zhengzhou University from October 2018 to April 2020.RT-qPCR was used to verify the differential expression of BMPR1B-AS1 in tumor tissue and paracancerous tissue.Hec-1b and Ishikawa cell lines were selected for overexpression or knockdown of BMPR1B-AS1 using lentivirus.CCK8 assay was used to study the effect of BMPR1B-AS1 on cell proliferation.Scratching assay and transwell experiments were carried to study the effect of BMPR1B-AS1 on cell migration and invasion ability.The effect of BMPR1B-AS1 on cell cycle was detected by flow cytometry.The apoptosis rate was detected by flow cytometry to find the effect of BMPR1B-AS1 on apoptosis.ResultsBioinformatics results showed that the log2 Fold Change value of lncRNA BMPR1B-AS1 was 3.504,which was the most significantly increased in endometrial cancer(P<0.0001).In the collected 28 endometrial cancer specimens,the expression of BMPR1B-AS1 in the tumor tissues was significantly higher than that in the adjacent tissues(P<0.01).BMPR1B-AS1-overexpressing cell line(Lv-BMPR1B-AS1)was established using Hec-lb cells.The proliferation of Lv-BMPR1B-AS1 cells lines was significantly increased(P<0.05).Compared with the control group,the accumulation of cells in S phase of Lv-BMPR1B-AS1 group was increased(P<0.0001),and the accumulation of cells in G0/G1 phase was decreased(P<0.001).The expression of Cyclin D1 and CDK4 protein were both increased in Lv-BMPR1B-AS1 group(P<0.05,P<0.05).The scratch confluence rates of Lv-BMPR1B-AS1 group at 24 h and 48 h were higher than those in the control group(P<0.0001,P<0.001).Transwell assay showed more cells migrated and invaded into the lower chamber in the Lv-BMPR1B-AS1 group than those in the control group(P<0.001,P<0.0001).The expression of E-cadherin protein was decreased(P<0.05),and the expression of N-cadherin protein was increased(P<0.05)in the Lv-BMPR1B-AS1 group.The apoptosis rate was decreased in Lv-BMPR1B-AS1 group(P<0.0001).BMPR1B-AS1 knockdown cell line(Lv-sh-BMPR1B-AS1)was established using Ishikawa cells.The proliferation of Lv-sh-BMPR1B-AS1 cell line was significantly reduced(P<0.01).Compared with the control group,the accumulation of cells in S phase of Lv-sh-BMPR1B-AS1 group was decreased(P<0.0001),and the accumulation of cells in G0/G1 phase was increased(P<0.0001).The expression level of Cyclin D1 and CDK4 protein were both decreased(P<0.05,P<0.05)in Lv-sh-BMPR1B-AS1 group.The scratch confluence rates of Lv-sh-BMPR1B-AS1 group at 24 h and 48 h were lower than those in the control group(P<0.0001,P<0.001).Transwell assay showed that less cells migrated and invaded into the lower chamber in the Lv-sh-BMPR1B-AS1 group than those in the control group(P<0.01,P<0.0001).The expression of E-cadherin protein was increased(P<0.05),and the expression of N-cadherin protein was decreased(P<0.05)in the Lv-sh-BMPR1B-AS1 group.The apoptosis rate was increased in Lv-sh-BMPR1B-AS1 group(P<0.0001).ConclusionsThe expression of BMPR1B-AS1 was upregulated in endometrial cancer tissues.In vitro experiments confirmed that BMPR1B-AS1 can promote the proliferation,migration and invasion of endometrial cancer cells,and inhibit apoptosis.Part Ⅱ BMPR1B-AS1 promotes proliferation,migration and invasion of endometrial cancer cells,and inhibits apoptosis through endogenous competitive inhibition of miR-7-2-3pBackgroundRNA transcripts containing miRNA-binding sites can act as competing endogenous RNA(ceRNA)with the help of miRNA-binding seed sequences,also known as miRNA sponges.The software predicts that BMPR1B-AS1 has a binding site of miR-7-2-3p.ObjectivesTo explore whether BMPR1B-AS1 influences the carcinogeneses and progression of endometrial carcinoma by sponging miR-7-2-3p.MethodsImmunofluorescence in situ hybridization(FISH)was conducted to detect the subcellular localization of BMPR1B-AS1.Bioinformatics prediction and dual luciferase reporter system were used to verify BMPR1B-AS1 can target bind to miR-7-2-3p.The expression levels of miR-7-2-3p in endometrial cancer tissues and cell lines were detected by RT-qPCR,and the correlation with the expression levels of BMPR1B-AS1 was analyzed.To confirm that BMPR1B-AS1 could act as an oncogene by directly targeting miR-7-2-3p,the following experiments were conducted:Lv-BMPR1B-AS1 cell line was transfected with miR-7-2-3p mimic,Lv-sh-BMPR1B-AS1 cell line was transfected with miR-7-2-3p inhibitor.The above experimental methods were used to detect the differences of cell proliferation,migration,invasion and apoptosis capability.ResultsFISH assays showed that BMPR1B-AS1 was mainly located in the cytoplasm,and bioinformatics predicted that it might target bind to miR-7-2-3p.The dual-luciferase assay result showed that transfection of miR-7-2-3p mimic could significantly reduce the luciferase activity of WT-pmirGlo-BMPR1B-AS1 plasmid(P<0.0001),so BMPR1B-AS1 could target bind to miR-7-2-3p.When the expression of BMPR1B-AS1 was up-regulated in endometrial cancer cell lines,the expression of miR-7-2-3p was significantly down-regulated(P<0.01),whereas the expression of miR-7-2-3p was significantly up-regulated after knockdown of BMPR1B-AS1(P<0.001).BMPR1B-AS1 expression and miR-7-2-3p expression were negatively correlated in endometrial cancer tissues(r=-.549,P<0.01).Cotransfection with the miR-7-2-3p mimic inhibited the proliferation,migration and invasion of the Lv-BMPR1B-AS1 cell line which were induced by overexpression of BMPR1B-AS1,while partially reversed the decrease of cell apoptosis.Cotransfection of miR-7-2-3p inhibitor promoted the proliferation,migration and invasion of the Lv-sh-BMPR1B-AS1,while inhibited apoptosis,and these effects were reversed by knockdown of BMPR1B-AS1 expression in Ishikawa cells.ConclusionsBMPR1B-AS1 located in the cytoplasm could facilitate the proliferation,migration,and invasion,and inhibit apoptosis of endometrial cancer cells by competitively binding miR-7-2-3p.Part Ⅲ BMPR1B-AS1/miR-7-2-3p can modulate PI3K/Akt/NF-κB signaling pathway by promoting the expression of DCLK1BackgroundThere are extensive regulatory mechanisms between miRNA and lncRNA and between miRNA and protein-coding mRNA through ceRNA effect,which can form a lncRNA-miRNA-mRNA model of regulatory network.The lncRNA-miRNA-mRNA network widely participates in the carcinogeneses and development of various types of tumors.The software predicts that DCLK1 is a target gene of miR-7-2-3p,and its mRNA has a binding site of miR-7-2-3p.ObjectivesTo investigate whether the BMPR1B-AS1/miR-7-2-3p axis affects the malignant phenotype of endometrial cancer cells by regulating the DCLK1 expression and PI3K/Akt/NF-κB pathways.MethodsBioinformatics and dual-luciferase reporter systems were used to verify miR-7-2-3p can target bind to DCLK1 mRNA.The expression levels of DCLK1 in endometrial cancer tissues and cell lines were detected by RT-qPCR and western blot,and the correlation with the expression levels of BMPR1B-AS1 and miR-7-2-3p were analyzed,respectively.DCLK1 inhibitor(DCLK1-IN-1)was used to inhibit DCLK1 activity,and the PI3K/Akt/NF-κB pathway activity was detected.ResultsThe results of the dual luciferase reporter assay showed that transfection of miR-7-2-3p mimic could reduce the luciferase activity of WT-pmirGlo-DCLK1 plasmid(P<0.01),so miR-7-2-3p can directly target DCLK1 mRNA.The mRNA and protein levels of DCLK1 in the BMPR1B-AS1-overexpression group were significantly higher than those in the control group,and cotransfection with miR-7-2-3p mimic could attenuate the up-regulation effect of BMPR1B-AS1 on DCLK1 expression.The mRNA and protein levels of DCLK1 in the BMPR1B-AS1-knockdown group were significantly lower than those in the control group,and cotransfection with miR-7-2-3p inhibitor could reverse the inhibition of DCLK1 expression caused by BMPR1B-AS1-knockdown.Spearman’s correlation analysis showed that miR-7-2-3p expression was negatively correlated with DCLK1 mRNA expression(r=-.531,P<0.01),whereas BMPR1B-AS1 expression was positively correlated with DCLK1 mRNA expression(r=.690,P<0.001).Ishikawa cells and Hec-lb cells were treated with DCLK1-IN-1 for 1,4 and 24 hours.The levels of p-PI3K p85,p-Akt and p-p65 were decreased in the DCLK1-IN-1 group,but we did not observe any significant changes in the total PI3K p85,Akt or p65 levels.ConclusionsThe expression of DCLK1 in endometrial cancer cells is regulated by the BMPR1B-AS1/miR-7-2-3p axis,which could promote the proliferation,migration and invasion of endometrial cancer cells,and inhibit apoptosis by affecting the activity of the PI3K/Akt/NF-κB pathway.Part Ⅳ Effects of BMPR1B-AS1/miR-7-2-3p/DCLKl on endometrial cancer growth in an animal model of endometrial cancerBackgroundThe dysregulated lncRNAs in tumor tissues can not only affect the biological behavior of cancer cells in vitro,but also can promote or inhibit tumor growth in vivo.In recent years,miRNA-based antitumor drug therapy has been extensively studied,which will benefit the application of the lncRNA-miRNA axis as a cancer therapeutic target.ObjectivesTo investigate the function of BMPR1B-AS1/miR-7-2-3p/DCLK axis in vivo by tumor xenografts in nude mice.MethodsThe mice were divided into 5 groups:Lv-BMPR1B-AS1 group,Lv-BMPR1BAS1+agomir-7-2-3p group,Lv-BMPR1B-AS1+agomir-7-2-3p NC group,Lv-NC group,and blank Hec-lb group.Each mouse was injected subcutaneously in the right flank area with cells.The agomir-7-2-3p 1OD or agomir NC 1OD was administrated by intra-tumoral injection after 10 days of inoculation(to allow for tumor growth).The tumor volume was calculated and recorded every 3 days.After 25 days,all the mice were sacrificed,and the tumors were removed and weighed.The tumor tissues were subjected to RT-qPCR to detect BMPR1B-AS1,miR-7-2-3p and DCLK1 mRNA expressions,and to immunohistochemistry to detect DCLK1 protein expression.ResultsBMPR1B-AS1 significantly promote tumor growth in vivo,while the tumor growth was inhibited by treatment with agomir-7-2-3p.The overexpression of BMPR1B-AS1 upregulated DCLK1 expression,while administration of agomir-7-2-3p reversed the change.In addition,spearman’s correlation analysis showed that BMPR1B-AS1 expression was negatively correlated with miR-7-2-3p expression(r=-.506,P<0.01),and miR-7-2-3p expression was negatively correlated with DCLK1 mRNA expression(r=-.452,P<0.01),whereas BMPR1B-AS1 expression was positively correlated with DCLK1 mRNA expression(r=.387,P<0.01).ConclusionsBMPR1B-AS1 promoted endometrial tumor growth in vivo through the miR-7-2-3p/DCLK1 axis. |
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