| Objective: Endometrial carcinoma(EC)is a kind of gynecologic malignancy,and has a high incidence rate in many malignant tumors.It is ranked the fourth among the most common cancers in women.Its impact is only after breast,lung and colorectal cancer.Its incidence and mortality rate have been increasing in recent years,and the 5-year survival rate of patients with advanced endometrial cancer with distant metastases is less than20%.Therefore,the study of the mechanism of endometrial cancer is particularly important.Therefore,research on the mechanism of endometrial cancer is particularly important.Among many RNA molecules,long noncoding RNAs(lncRNA)have more than 200 nucleotides.Through the interaction with micro RNAs(miRNAs),the expression of m RNA is changed and plays a regulatory role.At the same time,it plays a good regulatory effect on the improvement of human cancer and inhibits the metastasis and proliferation of cancer.Long intergenic noncoding RNA 958(LINC00958)has been shown to be significantly upregulated in a variety of human tumor tissues,and downregulation of LINC00958 expression can inhibit the development of nasopharyngeal carcinoma,pancreatic cancer,lung cancer,cervical cancer,and glioma.Currently,LINC00958 has been found to be elevated in endometrial cancer patient tissues,however,its role in endometrial carcinogenesis has not been reported.YinYang 1(YY1)is a multifunctional transcription factor that is overexpressed in a variety of human cancer tissues and has regulatory effects on cell proliferation,cell viability,epithelial mesenchymal transition,metastasis,and drug/immune tolerance.YY1 was found to bind to the promoter region of lnc RNA to regulate its transcription.Upregulation of YY1 promotes proliferation and invasion of ovarian,breast,and lung cancer cells.Moreover,overexpression of YY1 promotes proliferation and migration of endometrial cancer cells both in vitro and in vivo,while inhibition of YY1 exerts the opposite effect.Currently,only bioinformatics predicts that YY1 binds to the promoter region of LINC00958,but its regulatory mechanism has not been reported.MiRNAs are composed of noncoding single stranded small RNA molecules,and the length of nucleotides reaches 18 ~ 22.They can fully express the horizontal regulation target genes even after transcription.It was found that miR-378a-3p could act as a tumor suppressor and its overexpression inhibited the proliferation of ovarian cancer and promoted apoptosis,while enhancing the sensitivity of ovarian cancer cells to cisplatin.Mi R-378a-3p also inhibited the proliferation and invasion of hepatocellular carcinoma cells and enhanced the sensitivity of hepatocellular carcinoma cells to sorafenib chemotherapy.In addition,bioinformatic predictions showed that miR 378a-3p was down-regulated in endometrial cancer patient tissues,while its role in endometrial cancer has not been reported.Currently,it has been documented that LINC00958 can interact with miR-378a-3p to regulate bladder cancer proliferation and metastasis.Facing this situation,it can be considered that LINC00958 can play a good regulatory role in mir-378a-3p in endometrial cancer.In addition,bioinformatics also predicted the presence of a binding site for miR-378a-3p in YY1,but its targeting relationship and mechanism of action have not been reported.Therefore,the aim of this study was to investigate the mechanism of LINC00958,miR-378a-3p and YY1 in regulating the malignant biological behavior of endometrial cancer,and to confirm that LINC00958 can promote the upregulation of YY1 expression by competitively inhibiting miR-378a-3p,while YY1 can target and regulate LINC00958 to play a pro-tumor role in the development of endometrial cancer.exerting a pro-tumor role.Methods:Part I:The status of LINC00958 in the uterus was detected by quantitative reverse transcription PCR(QRT PCR).The detection objects include normal endometrial tissue,endometrial cancer tissue and endometrial cancer cell line.An3 ca cell line and HEC-1-A cell line were transfected with lentivirus overexpressing LINC00958(lv-LINC00958)and lentivirus containing anti LINC00958 short hairpin RNA(lv-sh-LINC00958),respectively.The proliferation and cell cycle of endometrial cancer cells were detected by CCK-8 cell counting and flow cytometry.Cell scratch test and Transwell can detect not only the migration ability of cells,but also the invasion ability.The expression levels of cycle and migration related proteins(cyclin D1,cyclin E,MMP9,MMP2)and MEK / ERK signal transduction pathway related proteins are detected by Western blot.The activities of MMP9 and MMP2 are analyzed by gelatinase spectrum experiment,The effects of LINC00958 on tumor volume,weight,Ki67 expression,proliferation,invasion and protein expression of cyclin D1,MMP2 and p-ERK1 / 2 were detected by tumor formation experiment in nude mice.Part II:The targeted binding sites of LINC00958,mir-378a-3p and YY1 were predicted by bioinformatics analysis.In order to better understand the performance and relationship of LINC00958,mir-378a-3p and YY1 in the process of targeted binding,it needs to be verified by double luciferase experiment;At the same time,the expression of mir-378a-3p and YY1 in LINC00958 and knockdown LINC00958 endometrial cancer cell lines can be detected by QRT PCR.Through the construction of mir-378a-3p mimics and NC mimics,HEC-1-A cells were transfected.The effect of overexpression of mir-378a-3p on YY1 expression was detected by QRT PCR;The regulation of LINC00958 / mir-378a-3p / YY1 axis on the proliferation,invasion and migration of endometrial cancer cells was detected by CCK-8 cell counting method and Transwell experiment;The action axis of LINC00958/ mir-378a-3p / YY1 was detected by Western blot,and the occurrence and development of endometrial cancer was regulated by MEK / ERK signal transduction pathway.Part III:Dual luciferase assay and Chromatin immunoprecipitation(Ch IP)assay were used to further investigate the binding of YY1 as a transcription factor to the upstream promoter region of LINC00958 to regulate the expression of LINC00958.Lv-sh YY1+Lv-LINC00958 and Lv-sh NC+Lv-LINC00958 were co-transfected with AN3 CA cell lines,and the expression of LINC00958 after co-transfection with Lv-sh YY1+Lv-LINC00958 and Lv-sh NC+Lv-LINC00958 was detected by q RT-PCR.The proliferation of Lv-sh YY1+Lv-LINC00958,Lv-sh NC+Lv-LINC00958 co-transfected cells was detected by CCK-8 cell counting assay,and the proliferation of Lv-sh YY1+Lv-LINC00958,Lv-sh NC+Lv-LINC00958 co-transfected cells was detected by cell scratch assay and Transwell.In the process of detecting LINC00958 co transfected cells,cell scratch method and Transwell method are usually introduced,while the expression of proteins such as cell cycle and migration are usually detected by Western blot.Results:Part I:The performance of LINC00958 in normal tissues and endometrial cancer tissues was understood by comparative analysis.The results showed that the increase was more obvious in endometrial cancer tissues.Among human endometrial cancer cells Ishikawa,RL95-2,HEC-1-B,AN3 CA and HEC-1-A,LINC00958 expression was lowest in AN3 CA cells and highest in HEC-1-A cells,therefore,AN3 CA cell line and HEC-1-A cell line were selected for the experiment.Endometrial cancer cells develop from G1 phase to S phase due to the overexpression of LINC00958,followed by the decrease of G1 phase cells and the increase of S phase and G2 phase cells.Assuming that the expression of LINC00958 in endometrial cancer cells is reduced,the result will be opposite to overexpression.In addition,the expression of cyclin D1 and cyclin E in endometrial cancer cells will also be up-regulated with the overexpression of LINC00958,which directly speeds up the cell cycle,otherwise it plays an inhibitory role.At the same time,overexpression of LINC00958 enhanced proliferation,migration and invasion ability of endometrial cancer cells,and knockout of LINC00958 suppressed proliferation,migration and invasion ability of endometrial cancer cells.Meanwhile,overexpression of LINC00958 promoted the expression level and activity of MMP9 and MMP2 in endometrial cancer cells,and knockdown of LINC00958 inhibited the expression level and activity of MMP9 and MMP2.In endometrial cancer cells,overexpression of LINC00958 increased the expression levels of phosphorylated MEK1/2(p-MEK1/2)and phosphorylated ERK1/2(p-ERK1/2)without altering the expression levels of total MEK1/2 or total ERK1/2.p-MEK1/2 or p-ERK1/2 to total MEK1/2 or total ERK1/2 ratios increases indicate that the MEK/ERK pathway is activated by LINC00958.Conversely,knockdown of LINC00958 inhibited the activation of this pathway.The tumorigenic assay in nude mice revealed that overexpression of LINC00958 promoted the growth process of endometrial cancer tumors in vivo,promoted the proliferation and migration ability of endometrial cancer cells in vivo,and increased the protein expression levels of cyclin D1,MMP2,and p-ERK1/2,while knockdown of LINC00958 had the opposite effect.Part II:Bioinformatics analysis showed that there was a targeted binding relationship between hsa-mir-378a-3p and LINC00958,and this targeted binding relationship also existed between mir-378a-3p and YY.The detection of double luciferase reporter gene is applied to verify the targeting relationship between LINC00958 / mir-378a-3p / YY1.Lipofectamine 3000 was introduced into LINC00958 wild-type vector(wt-LINC00958)and mutant vector(mut-LINC00958)containing mir-378a-3p binding site.The results showed that miR-378a-3p could bind to LINC00958.Similarly,miR-378a-3p was also shown to bind to the 3 ’ UTR of YY1.Overexpression of LINC00958 decreased the expression level of miR-378a-3p and increased the expression level of YY1 in endometrial cancer cells,while knockdown of LINC00958 did the opposite.Transfection of miR-378a-3p mimics(miR-378a-3p mimics)in HEC-1-A cell lines with high LINC00958 expression levels.The level of YY1 m RNA in cells was negatively correlated with the expression of mir-378a-3p.The lower the former,the higher the expression of the latter.In addition,the proliferation and invasion of endometrial cancer cells will be inhibited by the overexpression of mir-378a-3p.Overexpression of miR-378a-3p inhibited the expression of cyclin D1,MMP2 and p-ERK1/2,suggesting that miR-378a-3p inhibited the proliferation and invasive ability of endometrial cancer cells and the activation of MEK/ERK signaling pathway.Part III:Dual luciferase reporter gene experiments confirmed the binding relationship between YY1 and three LINC00958 promoter regions of different lengths,suggesting that YY1,as a transcription factor,can bind directly to the LINC00958 promoter.The PCR product of the truncated LINC00958 promoter in PCR after Ch IP assay indicated that YY1 binds directly to the LINC00958 promoter.After co-infection of AN3 CA cells with Lv-sh YY1+Lv-LINC00958 or Lv-sh NC+Lv-LINC00958,the CCK-8 cell counting assay demonstrated that knockdown of YY1 reversed the promotion effect of overexpression of LINC00958 on the proliferative capacity of endometrial cancer cells,and cell scratch assays demonstrated that knockdown of YY1 reversed the The cell scratch assay demonstrated that knockdown of YY1 reversed the promotion of LINC00958 overexpression on the migration ability of endometrial cancer cells,and the Transwell assay demonstrated that knockdown of YY1 reversed the promotion of LINC00958 overexpression on the invasion ability of endometrial cancer cells.In addition,the expression levels of cyclin D1 and MMP2 were downregulated in AN3 CA cell lines transfected with Lv-sh YY1+Lv-LINC00958 compared with those transfected with Lv-sh NC+Lv-LINC00958,indicating that knockdown of YY1 could reverse the promotion of LINC00958 overexpression on cell cycle progression,migration and invasion ability.Compared with the transfected Lv-sh NC+Lv-LINC00958 group,the expression of phosphorylated ERK1/2(p-ERK1/2)was inhibited in the transfected Lv-sh YY1+Lv-LINC00958 group without altering the total protein level of ERK1/2,and the ratio of p-ERK1/2 to total ERK1/2 was reduced,suggesting that knockdown of YY1 eliminated the overexpression LINC00958 on the activation of MEK/ERK signaling pathway in endometrial cancer.Conclusion:(1)LINC00958 expression was significantly increased in endometrioid adenocarcinoma tissues compared with normal endometrial tissues.LINC00958 promotes the transition from G1 to S phase in the cell cycle and promotes the cycle progression of endometrial cancer cells.LINC00958 significantly promotes the proliferation,migration and invasive ability of endometrial cancer and is associated with the activation of the MEK/ERK signaling pathway LINC00958 promoted endometrial cancer tumor growth in nude mice,promoted endometrial cancer tumor proliferation and migration ability,and acted through activation of MEK/ERK signaling pathway.(2)In endometrial cancer,LINC00958 promoted YY1 expression by targeting and repressing miR-378a-3p expression through ce RNA mechanism,while miR-378a-3p targeted and repressed YY1 expression,while the targeting between LINC00958,miR-378a-3p and YY1 was confirmed using a dual luciferase reporter gene assay to confirm the binding relationship.miR-378a-3p inhibited the proliferation,migration and invasive ability of endometrial cancer cells,while miR-378a-3p regulated the malignant biological behavior of endometrial cancer by inhibiting the MEK/ERK signaling pathway.(3)Knockdown of YY1 reversed the promoting effects of overexpression of LINC00958 on the proliferation,migration and invasion ability of endometrial cancer cells,which was achieved with the help of regulation of MEK/ERK signaling pathway.Dual luciferase reporter gene assay and CHIP assay confirmed that YY1,as a transcription factor,could directly bind to LINC00958 promoter region and LINC00958/miR-378a-3p/YY1 regulated the malignant biological behavior of endometrial cancer by influencing MEK/ERK signaling pathway to form a positive feedback loop rather than a unidirectional axis. |
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