Long Non-coding RNA NA Promotes Malignancy In Human Endometrial Carcinoma Through Positive Regulation Of NR4A1

Endometrioid endometrial carcinoma(EEC)is one of the three major malignant tumors common in the female reproductive system.It is an epithelial malignant tumor that occurs in the endometrium.In recent years,its incidence has increased year by year.According to clinical pathological classification,endometrial cancer is divided into type I(hormone-dependent)and type II(hormone-independent),type I is mainly endometrial adenocarcinoma,accounting for 80%-90%of all cases;II Types include serous and clear cell carcinoma,so the main subject of this study is type I endometrial cancer.At present,the treatment of endometrial cancer includes surgery,radiotherapy,chemotherapy and comprehensive treatment.A thorough understanding of the pathogenesis of the disease will help to diagnose and treat the disease,so it is necessary to clarify its pathogenesis.Long non-coding RNAs(IncRNAs)are a class of non-coding RNAs that are more than 200 nucleotides in length,lack a specific open reading frame,have no protein-coding function,but have regulatory gene expression.Long-chain non-coding RNA was first sequenced in mice in 2002.With the deepening of the research,it has a regulatory role in cell proliferation,' differentiation and material metabolism,and participates in the pathological process of many cancer diseases in humans.Abnormal expression of IncRNA affects the progression of tumors,and as a diagnostic or prognostic marker,it plays an important role in the progression of diseases such as gastric cancer,colorectal cancer,breast cancer,and lung cancer.However,in endometrioid endometrial cancer,the biological functions and molecular mechanisms of long non-coding RNA have not been extensively studied,so it is important to study the role of IncRNA in endometrioid endometrial cancer.Nuclear receptor NR4A1 is a member of the nuclear receptor NR4A family and is involved in cell proliferation and apoptosis regulation.It plays an important role in important life activities such as tumorigenesis,vascular remodeling and steroid synthesis.Studies have shown that NR4A1 acts as a tumor suppressor gene and plays an important role in tumor progression.In the hematopoietic system,Nr4al and Norl double knockout mice can develop acute myeloid leukemia(AML);NR4A1 is overexpressed in certain types of lymphoma cells,and induces apoptosis and inhibits these cell-derived tumors.Growth in mice.Studies have shown that NR4A1 can also slow the growth of LNCaP prostate cancer cells.At the same time,studies have shown that NR4A1 has a certain role in tumor migration.In ZR-75-1 breast cancer cells expressing ER and PMC42 breast cancer cells with progenitor characteristics,ectopic NR4A1 expression can inhibit cell migration.Then,in endometrial cancer,whether NR4A1 has a certain correlation with the progression of tumors has not been reported in the literature.In recent years,with the study of lncRNA,lncRNA is an important regulator in transcription and translation,and plays an important role in cell function,such as chromatin remodeling,transcription and post-transcriptional regulation.When functioning,IncRNA regulates gene expression through different pathways,including cis-regulation and trans-regulation.In addition,lncRNA can play a specific role through two different approaches:protein and microRNA networks.In summary,IncRNA mainly regulates the expression of genes from epigenetic regulation,transcriptional regulation and post-transcriptional regulation.The lncRNA of this study was found to be adjacent to the nuclear receptor NR4A1 on the same chromosome.Lnc-NA is highly expressed in many reproductive related organs.NR4A1 is a gene that plays an important role in tumorigenesis and development.Previous studies have shown that IncRNA can play a role by binding adjacent genes and regulating the expression of adjacent genes.Therefore,we hypothesize whether Lnc-NA and NR4A1 may play a role in the occurrence and development of endometrial cancer.Pre-experimental results showed that the expression of Lnc-NA and NR4A1 in endometrial cancer was decreased and correlated,suggesting that Lnc-NA may play a role in the occurrence and development of endometrial cancer,and NR4A1 may also participate in it.ObjectivesTo clarify the role of Lnc-NA in endometrioid endometrial cancer,to explore the specific molecular mechanism of Lnc-NA,and provide new ideas and targets for the treatment of clinical endometrioid endometrial cancer.Methods1.Collect 30 specimens of surgically resected endometrioid endometrial cancer tissue from the provincial hospital affiliated to Shandong University.After re-diagnosis by pathologist,detect Lnc-NA and NR4A1 in endometrial cancer tissues and adjacent tissues by real-time PCR.The expression of Lnc-NA and NR4A1 in cancer and adjacent tissues and the correlation between Lnc-NA and NR4A1.2.Analyze the reliability of Lnc-NA by network software and confirm that it is the accuracy of IncRNA.3,Predict the positional relationship of Lnc-NA and NR4A1 and possible binding sites by software,and confirm the presence of binding sites by luciferase reporter gene experiments.4.Real-time PCR was used to analyze the expression of Lnc-NA in endometrioid endometrial cancer cell lines Ishikawa,KLE,RL95-2,HEC-1-b and normal endometrial cell line ESC,and the background low expression and high were screened.The Lnc-NA-expressing endometrial cancer cell line was constructed,and the Lnc-NA overexpression plasmid and shRNA plasmid were constructed.The expression of Lnc-NA in the low and high expression cell lines was up-regulated and down-regulated by plasmid transfection method.The corresponding cell lines stably transfected and down-expressed Lnc-NA were subsequently used,and then the transfection efficiency was identified by real-time PCR.5.The effects of Lnc-NA on the proliferation,apoptosis,invasion and migration of endometrioid endometrial cancer cells were detected by CCK8 proliferation,apoptosis,transwell invasion and migration assay.6.Western-blot assay was used to detect the expression of NR4A1,apoptosis-related protein(Bcl2,Bax)and migration-related protein MMP2/9 in up-regulated cells.7.Transfected sh-NR4A1 plasmid in the endometrial cancer cell line Ishikawa stably transfected with Lnc-NA,down-regulated the expression of NR4A1,and verified the proliferation,apoptosis,invasion and migration of CCK8 to verify NR4A1 in Lnc-NA-mediated role in cell proliferation,apoptosis,invasion and migration,and the expression of apoptosis-related protein(Bcl2,Bax)and migration-related protein MMP2/9 were detected by western-blot assay.8.Western blot was used to detect the expression of protein NR4A1 in endometrioid endometrial cancer cell lines Ishikawa,KLE,RL95-2 and HEC-l-b.The NR4A1 overexpression plasmid was constructed and transfected by plasmid.NR4A1 expression in cell lines expressing NR4A1;and the effects of NR4A1 on proliferation,apoptosis,invasion and migration of endometrial cancer cells by CCK8 proliferation,apoptosis,invasion and migration assays,and detection by western-blot assay Expression of apoptosis-related proteins(Bcl2,Bax)and migration-associated protein MMP2/9.9.Western-blot assay was used to detect Caspase9,Caspase-3(8G10),Caspase-7(ASP198),Caspase-7(D2Q3L),PARP and(ASP124)of caspase apoptosis signaling pathways after up-and down-regulation of Lnc-NA.Results1.Compared with normal adjacent tissues,Lnc-NA is low in human endometrioid endometrial cancer tissues;compared with normal adjacent tissues,NR4A1 is low in human endometrial cancer tissues;Lnc-NA and NR4A1 expression is positive Correlation(r=0.454,P=0.0191).2.Lnc-NA is indeed a non-coding RNA,and Lnc-NA directly binds to the upstream sequence of the promoter of NR4A1.3.In endometrioid endometrial cancer cell lines Ishikawa,KLE,RL95-2,HEC-1-b,Lnc-NA is highly expressed in KLE and HEC-1-b cell lines,whereas Lnc-NA is low expression in RL95-2 and Ishikawa cell lines.4.When the endometrioid endometrial cancer cell line Ishikawa overexpresses Lnc-NA,the cell proliferation,invasion and migration ability are decreased,and the cell apoptosis ability is significantly enhanced(P<0.05).At the same time,the anti-apoptosis related protein Bax and the expression of the migration related protein MMP2/9 was significantly decreased,while the expression of the apoptosis-related protein Bcl2 was significantly increased.When the endometrial cancer cell line HEC-1-b down-regulated Lnc-NA,the cell proliferation,invasion and migration ability were increased,while the cell apoptosis ability was significantly decreased(P<0.05).At the same time,the anti-apoptosis related protein Bax and The expression of migration-related protein MMP2/9 was significantly increased,while the expression of apoptosis-related protein Bcl2 was significantly decreased(P<0.05).5.Western blot results showed that overexpression of Lnc-NA promoted the expression of NR4A1 in the endometrial cancer cell line Ishikawa,while down-regulation of Lnc-NA inhibited the expression of NR4A1 in the endometrioid endometrial cancer cell line HEC-1-b(P<0.05).6.Compared with the control group,in the endometrioid endometrial cancer cell line that overexpressed Lnc-NA and down-regulated NR4A1,the proliferation of CCK8 showed that the proliferative capacity of the cells was significantly enhanced after the down-regulation of NR4A1,and the apoptosis test results showed cells.The apoptotic rate was significantly reduced,and the number of cells invading and migrating was significantly increased by transwell invasion and migration experiments(P<0.05).The results of western blot showed that the expression of Bcl2 and migration-related protein MMP2/9 was significantly increased and the expression of anti-apoptotic protein Bax was significantly decreased after overexpression of Lnc-NA and down-regulation of NR4A1(P<0.05).7.After overexpressing NR4A1 in endometrioid endometrial cancer cell line Ishikawa,the proliferation,invasion and migration ability of the cells were decreased,and the apoptosis ability of the cells was significantly enhanced(P<0.05).The anti-apoptotic protein Bax and migration-related protein MMP2 were also observed.The expression of/9 was significantly reduced,while the expression of the apoptosis-related protein Bcl2 was significantly increased.In the cell line up-regulated by NR4A1,the expression of Lnc-NA was down-regulated,and the proliferation,apoptosis,invasion and migration of cells did not change.The expression of apoptosis-related proteins Bcl2,Bax and migration-related protein MMP2/9 were also observed no change.8.The results of Western blot showed that the expression of Caspase9,Caspase-3(8G10),Caspase-7(ASP 198)and Caspase-7(D2Q3L)was significantly increased in the cell line upregulated by Lnc-NA(P<0.05),while the expression of protein PARP was significantly reduced,but the expression of(ASP 124)PARP did not change significantly.After down-regulation of Lnc-NA,the expression of Caspase9,Caspase-3(8G10),Caspase-7(ASP198)and Caspase-7(D2Q3L)was significantly decreased(P<0.05),the expression of PARP was significantly increased(P<0.05).Meanwhile the protein PARP(ASP124)was significantly increased(P<0.05).In the cell lines that simultaneously up-regulated Lnc-NA and down-regulated NR4A1,Caspase signaling pathway proteins Caspase9,Caspase-3(8G10),Caspase-7(ASP198),Caspase-7(D2Q3L)were decreased compared with Lnc-NA alone(P<0.05),while the expression of protein PARPwas significantly increased,but the expression of PARP(ASP124)did not change significantly.Conclusion1.Lnc-NA expression was decreased in endometrioid endometrial cancer tissues compared with normal tissues,and the expression of Lnc-NA was positively correlated with the expression of NR4A1.2.Lnc-NA inhibits the proliferation,invasion and migration of tumor cells in the endometrioid endometrial cell line and promotes the apoptosis of tumor cells.3.Lnc-NA promotes tumor apoptosis by targeting NR4A1 expression and then affecting caspase apoptosis signaling pathway.