| Objective: Vulvar cancer ranks fourth in malignant tumors of female reproductive organs,mainly including primary squamous cell carcinoma.In recent years,the incidence of vulvar squamous cell carcinoma(VSCC)has increased year by year,threatening the health of older women.And the five-year survival rate of advanced vulvar squamous cell carcinoma is only 20%.Long non-coding RNA(lnc RNA),more than 200 nucleotides in length,without protein encoding function,can regulate gene expression in many types of ways,including epigenetic,transcriptional or post-transcriptional regulation.In recent years,a large number of studies have found that the different expression or abnormal function of lnc RNA is closely related to the occurrence and development of many kinds of human diseases including cancer,and some lnc RNAs have been confirmed to be able to regulate each other with m RNA,micro RNA,corresponding target genes and proteins,participating in tumor progression,cell differentiation,cell cycle changes,apoptosis regulation,immunomodulatory and other processes.PCBP1-AS1(poly C binding protein-1 antisense RNA1)is an antisense lnc RNA of PCBP1.Antisense lnc RNA is a kind of endogenous lnc RNA which is complementary with other transcripts sequence and regulates the expression of target genes at the level of transcription and post-transcription,and may play a unique role in the development of the disease.Tumor necrosis factor receptor associated factor 5(TRAF5)is an important intracellular signal transduction protein.Previous studies have shown that TRAF5 mediates signal transduction of LT-?R,CD40,CD30,CD27,and activates nuclear factor kappa B(NF-?B)signal transduction pathway,thereby inhibiting apoptosis,involved in inflammatory response and immune regulation.The aim of this study was to investigate the expression of PCBP1-AS1 in VSCC,to verify that PCBP1-AS1 could regulate the biological behavior of VSCC cells through TRAF5-mediated NF-?B signaling pathway.Methods: The expression level of PCBP1-AS1 and TRAF5 m RNA in 19 pairs of vulvar squamous cell carcinoma and corresponding adjacent normal vulvar tissues were detected by q RT-PCR.The relationship between the relative expression levels of PCBP1-AS1 and TRAF5 m RNA and the clinicopathological characteristics of patients with vulvar squamous cell carcinoma were analyzed by Fisher exact test.The expression levels of TRAF5,p65,p-p65,c-Rel and I?B? in 19 pairs of vulvar squamous cell carcinoma tissues and corresponding adjacent normal vulvar tissues were detected by Western blotting(WB).After the expressions of PCBP1-AS1 and TRAF5 were successfully changed by liposome transfection in SW954 cells,the expressions of PCBP1-AS1 and TRAF5 m RNA were detected by q RT-PCR,the cell proliferation was detected by CCK-8,the cell apoptosis was detected by Annexin V-FITC & PI method,the cell migration ability was detected by Scratch Wound Healing Assay,the cell invasion ability was detected by Transwell Invasion Assay,to preliminarily verify the interacting way of PCBP1-AS1 and TRAF5 in vulvar squamous cell carcinoma SW954 cells and their biological functions.After the expressions of PCBP1-AS1 and TRAF5 were successfully changed in SW954 cells,the expressions of TRAF5,p65,p-p65,c-Rel and I?B? protein were detected by WB method,the activity of NF-?B in the cells was detected by NF-?B activation-nuclear translocation fluorescence labeling method.On the basis of the successful change in the expressions of PCBP1-AS1 and TRAF5,NF-?B signaling pathway activator and inhibitor were respectively applicated to carry out NF-?B activation-nuclear translocation fluorescence labeling method,CCK-8,Annexin V-FITC & PI,Scratch Wound Healing Assay and Transwell Invasion Assay,to investigate whether TRAF5-mediated NF-?B signaling pathway was involved in the regulation of biological behavior of SW954 cells by PCBP1-AS1.Results: Part I: 1.The expression levels of PCBP1-AS1 and TRAF5 m RNA in VSCC tissues were lower than those in the corresponding adjacent normal vulvar tissues.2.The expression level of PCBP1-AS1 in VSCC tissues was closely related to tumor size,differentiation degree,FIGO stage,lymph node metastasis,distant metastasis and squamous cell carcinoma antigen(SCC-Ag).The expression level of TRAF5 m RNA was closely related to FIGO stage and lymph node metastasis.3.The expression levels of TRAF5,p65,p-p65,c-Rel and I?B? protein in VSCC tissues were significantly lower than those in the corresponding adjacent normal vulvar tissues.Part II: 1.TRAF5 knockdown was successfully performed in SW954 cells by sh RNA technique,and TRAF5-RNAi-0/SW954 cells with higher silencing efficiency were selected for further experiments.2.After the expression of PCBP1-AS1 was up-regulated,the expression level of TRAF5 m RNA was increased accordingly,while there was no significant change in the expression level of PCBP1-AS1 after TRAF5 expression was up-regulated or down-regulated.3.Up-regulation of PCBP1-AS1 could significantly inhibit the proliferation of SW954 cells,while the down-regulation of TRAF5 markedly promote the proliferation of SW954 cells.4.The upregulation of PCBP1-AS1 could significantly promote the apoptosis of SW954 cells,while the down-regulation of TRAF5 obviously inhibit the apoptosis of SW954 cells.5.The upregulation of PCBP1-AS1 could obviously inhibit the migration of SW954 cells,while the down-regulation of TRAF5 significantly promoted the migration of SW954 cells.6.PCBP1-AS1 upregulation could markedly inhibit SW954 cell invasion,and TRAF5down-regulation could significantly promote SW954 cell invasion.Part III: 1.After exogenous increase of PCBP1-AS1 expression,TRAF5,p65,p-p65,c-Rel and I?B? protein expression levels were significantly increased,while after TRAF5 expression was exogenously decreased,p65,p-p65,c-Rel and I?B? protein expression levels were significantly decreased.2.The up-regulation of PCBP1-AS1 could significantly increase the activity of NF-?B,while the down-regulation of TRAF5 obviously reduce the activity of NF-?B.3.The activity of NF-?B was significantly increased after applying NF-?B signaling pathway activator,and was markedly decreased after NF-?B signaling pathway inhibitor was applied,verifying the construction of NF-?B signaling pathway activation and inhibition model was successfully established.4.The proliferation of SW954 cells was suppressed by the activation of NF-?B signaling pathway,and was promoted by NF-?B signaling pathway inhibition.The activation of NF-?B signaling pathway could reduce the increase of SW954 cell proliferation induced by the down-regulation of TRAF5,and the inhibition of NF-?B signaling pathway could resist the decease of SW954 cell proliferation induced by the up-regulation of PCBP1-AS1.5.The apoptosis of SW954 cells was promoted by the activation of NF-?B signaling pathway,was suppressed by NF-?B signaling pathway inhibition.The activation of NF-?B signaling pathway could resist the decrease of SW954 cell apoptosis rate induced by TRAF5 down-regulation,and the inhibition of NF-?B signaling pathway could reduce the increase of SW954 cell apoptosis rate induced by PCBP1-AS1up-regulation.6.The activation and inhibition of NF-?B signaling pathway had no significant effect on SW954 cell migration.7.The activation and inhibition of NF-?B signaling pathway had no significant effect on SW954 cell invasion.Conclusion: 1.The expression of PCBP1-AS1 and TRAF5 m RNA are both decreased in VSCC tissues.The expression of PCBP1-AS1 and TRAF5 m RNA in VSCC tissues are both closely correlated with clinicopathological characteristics.The inhibition of NF-?B/p65 and NF-?B/c-Rel signal transduction pathway existes in VSCC tissues.2.TRAF5 is the target gene of PCBP1-AS1 and plays a role in the form of PCBP1-AS1-TRAF5 m RNA as a gene pair.PCBP1-AS1 may serve as a tumor suppressor gene,inhibit tumor cell proliferation,migration and invasion,promote tumor cell apoptosis,thereby affecting the occurrence and development of VSCC.3.TRAF5-mediated NF-?B signaling pathway is involved in the regulation of proliferation and apoptosis of SW954 cells by PCBP1-AS1. |
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