| Objective: Endometrial carcinoma(EC)is one of the three most common malignant tumors of the female reproductive system.Over recent years,the incidence of EC has increased constantly and the age of onset is a younger trend.Although the incidence of type? EC accounts for approximately 80% of EC,its etiology and pathogenesis remain unclear.Long non-coding RNA(lncRNA)is another widely concerned molecular marker after microRNA(miRNA)over recent years.LncRNA can regulate gene expression in cells at epigenetic,transcriptional and posttranscriptional levels and can participate in a range of important regulatory processes.LncRNA is closely related to the occurrence and development of tumors.Our research group previously used the Arraystar Human LncRNA V3.0 microarray to screen the lncRNA expression profile of type? EC tissue and normal endometrial tissue as a control,found that HOXA transcription antisense RNA myeloid-specific 1(HOTAIRM1)is a significantly upregulated lncRNA in type? EC tissue.Studies have demonstrated that the antisense lncRNAs often exert their biological functions by regulating its sense genes.LncRNA HOTAIRM1 is transcribed antisense to the HOXA1 gene.The expression of HOXA1 is significantly upregulated in a variety of tumors.HOXA1 is the key factor in the process of tumor occurrence and development.Therefore,based on our earlier microarray screening results and the international research progress,the present study aimed to investigate the effects of lncRNA HOTAIRM1 and its sense gene HOXA1 on the occurrence and development of type? EC,which will be the novel potential prognostic biomarker and new potential therapeutic target for type? EC.Methods: 1.To investigate the expression and significance of lncRNA HOTAIRM1 and HOXA1 gene in type? EC tissue.qRT-PCR detected the expression of HOTAIRM1 and HOXA1 mRNA in type? EC and normal endometrial tissue.Western blotting was used to detect HOXA1 protein in type? EC and normal endometrial tissue.The correlation between HOTAIRM1 and HOXA1 expression,and the associated clinical data of type? EC were analyzed.2.To study the regulation relationship between lncRNA HOTAIRM1 and HOXA1 gene,and the effects and the possible mechanisms of lncRNA HOTAIRM1 and HOXA1 gene on the biological behavior of type? EC cells.In vitro,we first established stable transfected cell lines for the knockdown and overexpression of HOTAIRM1 and knockdown of HOXA1 in Ishikawa and HEC-1A cells.qRT-PCR and Western blotting were used to verify the transfection efficiency.To determine the overexpression and silencing effects of HOTAIRM1 on EC,we divided cells into five groups: Control group(without any shRNA or virus transfection,named ‘mock control');GV367-NC group(transfection with negative control virus);GV367-HOTAIRM1 group;shRNA-NC group(transfection with negative control shRNA)and sh-HOTAIRM1 group.To determine the silencing effect of HOXA1 on EC,cells were divided into three groups: Control group;shRNA-NC group and sh-HOXA1 group.Western boltting was applied to detect the expression of HOXA1 protein in each group.The Cell Counting Kit-8 assay was used to determine the effect of HOTAIRM1 and HOXA1 on cell proliferation.Transwell assays were used to detect the ability of cell migration and invasion.The change of cell cycle proportion was detected by flow cytometry.In vivo,tumor xenograft implantation in nude mice was used to detect the ability of cell tumorigenicity and growth in each group.Western boltting was applied to determine the expression of Cyclin D1 and E-cadherin,N-cadherin which were EMT related protein,and the expression of p-p65 and p65 which were NF-?B signaling pathway related protein in each group.Results: 1.qRT-PCR showed that the expression levels of HOTAIRM1 and HOXA1 mRNA in type? EC tissues were significantly higher than those in normal endometrium tissues(P<0.01).Western blotting further revealed that the expression level of HOXA1 protein in type? EC tissues was significantly higher than that in normal endometrium tissues(P<0.01).Statistical analysis showed that there was a significant positive correlation between HOTAIRM1 and HOXA1 expression in type? EC tissues(P<0.01).The HOTAIRM1 and HOXA1 mRNA expression levels were both significantly correlated with International Federation of Gynecology and Obstetrics(FIGO)stage and lymph node metastasis(P<0.05),but were not correlated with patient age,differentiation and myometrial invasion(P>0.05).2.(1)qRT-PCR showed that the expression of HOTAIRM1 in the GV367-HOTAIRM1 group was significantly increased compared with the GV367-NC group and the Control group(P<0.05),while compared with the shRNA-NC group and the Control group,the expression of HOTAIRM1 in the sh-HOTAIRM1 group was significantly decreased.(P<0.05).The CCK8 assay showed that the proliferation rate was significantly higher in the GV367-HOTAIRM1 group than that in the GV367-NC and Control groups,and was significantly lower in the sh-HOTAIRM1 group than that in the shRNA-NC and Control groups(P<0.05).The results of flow cytometry showed that the proportion of cells in G0/G1 phase was significantly decreased and the proportion of cells in S phase was significantly increased in the GV367-HOTAIRM1 group(P<0.05),while the proportion of cells in G0/G1 phase was significantly increased and the proportion of cells in S phase was significantly decreased in the sh-HOTAIRM1 group(P<0.05).In the transwell assays,data showed that the numbers of migrating and invading transmembrane cells in the GV367-HOTAIRM1 group were both significantly increased(P<0.05),while the numbers of migrating and invading transmembrane cells in the sh-HOTAIRM1 group were significantly decreased(P<0.05).Western blotting showed the expression levels of CyclinD1,N-cadherin and p-p65 were significantly increased and the expression level of E-cadherin was significantly reduced in the GV367-HOTAIRM1 group(P<0.05),while the expression levels of CyclinD1,N-cadherin and p-p65 were significantly reduced and the expression level of E-cadherin was significantly increased in the sh-HOTAIRM1 group(P<0.05),the expression level of p65 has no obvious difference in each group(P>0.05).In vivo,transplanted tumors formed early,grew fast and the volume and weight of the transplanted tumors were significantly increased in the GV367-HOTAIRM1 group compared with the GV367-NC group and the Control group(P<0.05).In contrast,the transplanted tumors formed late,grew slowly and the volume and weight of the transplanted tumors were significantly decreased in the sh-HOTAIRM1 group compared with the shRNA-NC group and the Control group(P<0.05).(2)qRT-PCR showed that the expression level of HOXA1 mRNA in the GV367-HOTAIRM1 group was significantly increased compared with the GV367-NC group and the Control group(P<0.05),while the expression level of HOXA1 mRNA in the sh-HOTAIRM1 group was significantly decreased compared with the shRNA-NC group and the Control group(P<0.05).Western blotting showed that the expression level of HOXA1 protein in the GV367-HOTAIRM1 group was significantly increased(P<0.05),while the expression level of HOXA1 protein in the sh-HOTAIRM1 group was significantly decreased(P<0.05).(3)qRT-PCR and Western blotting showed that the expression level of HOXA1 mRNA and protein in the sh-HOXA1 group were both significantly decreased compared with the shRNA-NC group and the Control group(P < 0.05).The CCK8 assay showed that the proliferation rate was significantly lower in the sh-HOXA1 group than that in the shRNA-NC and Control groups(P<0.05).The results of flow cytometry showed that the proportion of cells in G0/G1 phase was significantly increased and the proportion of cells in S phase was significantly decreased in the sh-HOXA1 group(P<0.05).In the transwell assays,data showed that the numbers of migrating and invading transmembrane cells in the sh-HOXA1 group were both significantly decreased(P<0.05).Western blotting showed the expression levels of CyclinD1,N-cadherin and p-p65 were significantly decreased and the expression level of E-cadherin was significantly increased in the sh-HOXA1 group(P<0.05),while the expression level of p65 has no obvious difference in each group(P>0.05).In vivo,the transplanted tumors formed late,grew slowly and the volume and weight of the transplanted tumors were significantly decreased in the sh-HOXA1 group compared with the shRNA-NC group and the Control group(P<0.05).Conclusion: 1.LncRNA HOTAIRM1 and HOXA1 gene were highly expressed in type? EC.There was a significant positive correlation between the expression of HOTAIRM1 and HOXA1,and the expression levels of HOTAIRM1 and HOXA1 were both positively correlated with FIGO stage and lymph node metastasis,which showed that lncRNA HOTAIRM1 and HOXA1 gene were associated with the occurrence and development of type? EC.2.LncRNA HOTAIRM1 and HOXA1 gene could enhance the proliferation ability of Ishikawa and HEC-1A cells and enhance the ability of subcutaneous tumor formation and growth of nude mice,could regulate cell cycle distribution by increasing the expression of Cyclin D1,could enhance the ability of migration and invasion by up-regulating N-cadherin and down-regulating E-cadherin.3.LncRNA HOTAIRM1 can positively regulate the expression of HOXA1 gene.HOTAIRM1 and HOXA1 both can promote the occurrence and development of type? EC partly through the activation of NF-?B signaling pathway. |
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