| Endometrial carcinoma?EC?is the second most common malignant tumour of reproductive system in menopausal and perimenopausal women.It occupies the fourth place in female malignant tumors in developed countries[1].In recent years,the number of EC new cases and deaths has doubled[2-4],it threatens the health of women all over the world.Statistical analysis of the National Cancer Center of China shows that with the change of people's lifestyle,the adjustment of dietary structure and the unconscious exposure of hormone food and drugs,the incidence of endometrial carcinoma is increasing year by year and the age of onset is younger,which seriously affects the healthy life span of elderly women and the reproductive health of women of childbearing age,has a significant impact on the family and society[5].The occurrence and development of endometrial carcinoma is the result of the interaction of environmental factors and genetic variation.It involves a series of factors such as differential expression of multiple genes and transcription factors,abnormal regulation of cell signal transduction pathways and imbalance of cell microenvironment homeostasis.Different pathological and molecular characteristics determine the risk level and prognosis situation of EC patients.Long non-coding RNAs are RNA molecules that cannot encode proteins and transcribe more than 200 nucleotides.They regulate the expression of target genes through epigenetic,transcriptional and post-transcriptional pathways[6,7],which is related to the proliferation,migration and invasion of various tumors.Some abnormally expressed LncRNAs play the role of proto-oncogenes in the biological process of endometrial cancer[8-10],and promote the progress of cancer.DLEU1 is a highly conserved LncRNA which located in the 13q14.2-q14.3 region of human chromosome,also known as deleted in lymphocytic leukemia 1.It is highly expressed in many solid tumors(such as osteosarcoma[11],glioma[12],hepatocellular carcinoma[13]and bladder cancer[14]).DLEU1 also plays an important role in reproductive system tumors such as cervical cancer[15],endometrial cancer[16]and ovarian cancer[17].In ovarian epithelial malignant tumors[17],DLEU1 may act as a competitive endogenous RNA?ceRNA?sponge to adsorb miRNAs,eliminate the expression of miRNAs and regulate the expression of target genes,promote the proliferation,EMT process,migration and invasion of tumor cells,and DLEU1-miRNA-mRNA regulation axis mechanisms may exist in tumors.Many studies have shown that miR-490 can exert its anti-oncogene function by regulating downstream target gene expression,thus affecting the occurrence and development of malignant tumors such as glioma[18],prostate cancer[19],liver cancer[20],ovarian cancer[21]and breast cancer[22].SP1 is a nuclear transcription factor and a sequence-specific DNA-binding protein.SP1 participates in the proliferation,apoptosis,neovascularization,migration and invasion of cancer cells by regulating oncogenes,tumor suppressor genes,cell cycle regulators and growth-related cell signaling pathways during the growth and metastasis of tumors.It plays an important role in many malignant tumors,such as small cell lung cancer[23],cervical cancer[24],endometrial cancer[25]and breast cancer[26].The expression level of SP1 in ovarian cancer[170],endometrial cancer[225],cervical cancer[172],breast cancer[173]and hepatocellular carcinoma[171]is also regulated by a variety of upstream transcription factors including miRNAs and cell signal transduction pathways.In different tumors,different miRNAs target SP1 as upstream factors,affecting the malignant biological behavior and disease progression of tumor cells by affecting the expression of SP1.Studies have shown that DLEU1 is highly expressed in EC,but the precise expression pattern,biological function and potential molecular mechanism of DLEU1in EC are not yet clear.Therefore,this study explored the role of DLEU1 in regulating the transcription of target gene SP1 by eliminating the expression and function of miR-490 in EC,in order to elucidate the possible mechanism of DLEU1-miR-490-SP1 regulation axis in endometrial carcinoma.OBJECTIVE:To elucidate the molecular mechanism of regulating the development of endometrial carcinoma,we studied the expression level of DLEU1 in endometrial carcinoma and its correlation with clinicopathological changes and prognosis,the effect of DLEU1 expression on malignant biological behavior of endometrial carcinoma cells,the regulation of miR-490 in DLEU1 on endometrial carcinoma cells malignant biological behavior,and the mechanism of DLEU1promoting malignant biological behavior of endometrial carcinoma cells through the DLEU1-miR-490-SP1 regulatory axis,provide research basis for clinical early molecular inhibition of the development of endometrial carcinoma.Method:1.The expression of DLEU1 in EC tissues and normal endometrial tissues,HHUA,KLE,Ishikawa,ECC-1 endometrial carcinoma cell lines and normal endometrial cells were detected by qRT-PCR.2.The expression of DLEU1 in Ishikawa cells was inhibited by shRNA interference.MTT,flow cytometry,Transwell test and Western blot were used to detect the proliferation activity,apoptosis,migration and invasion of Ishikawa cells,expression of apoptosis and EMT process related proteins.3.The expression of miR-490 in EC tissues and normal endometrial tissues,HHUA,KLE,Ishikawa,ECC-1 endometrial carcinoma cell lines and normal endometrial cells were detected by qRT-PCR.4.qRT-PCR was used to detect the expression of miR-490 after sh-DLEU1 gene interference inhibited DLEU1 expression in Ishikawa cells.5.miR-490 inhibitor was used to inhibit the expression and function of miR-490in Ishikawa cells.MTT,flow cytometry,Transwell test and Western blot were used to detect the proliferation activity,apoptosis,migration and invasion of Ishikawa cells,expression of apoptosis and EMT process related proteins.6.Luciferase reporter gene technology was used to detect the target gene and its binding sites of miR-490.7.The expression of SP1 in Ishikawa cells was inhibited by siRNA interference.MTT,flow cytometry,Transwell test and Western blot were used to detect the proliferation activity,apoptosis,migration and invasion of Ishikawa cells,expression of apoptosis and EMT process related proteins.Result:1.The expression of DLEU1 in endometrial carcinoma and its correlation with clinicopathological changes and prognosis?1?The expression level of DLEU1 in EC?3.17±0.92?was significantly higher than that in normal endometrium?0.97±0.43??P<0.05?.?2?The expression level of DLEU1 in HHUA?2.74±0.22?,KLE?3.45±0.46?,Ishikawa?3.77±0.25?,ECC-1?2.52±0.24?cell lines was significantly higher than that in normal endometrial cells?0.98±0.12??P<0.05?,and the highest expression level was found in Ishikawa cell lines.?3?DLEU1 is highly expressed in EC tissues and tends to be highly expressed in advanced and high-grade endometrial carcinoma.2.Effect of DLEU1 expression on malignant biological behavior of endometrial carcinoma cells?1?The expression level of DLEU1 in Ishikawa cells transfected with sh-DLEU1?0.38±0.18?was significantly lower than that in sh-NC group?0.98±0.15??P<0.05?.?2?Compared with sh-NC group,after the decrease of DLEU1 expression the proliferative activity of Ishikawa cells decreased significantly?P<0.05?;the percentage of apoptotic cells increased significantly?P<0.05?;the expression of pro-apoptotic related proteins?Bax,cleaved-caspase-3 and cleaved-caspase-9?increased,the expression of anti-apoptotic related proteins?Bcl-2?decreased;the ability of cell migration and invasion decreased?P<0.05?;the EMT process was weakened?expression of E-cadherin protein increased,and the expression of N-cadherin,Snail and Vimentin decreased?.3.DLEU1 regulates malignant biological behavior of endometrial carcinoma cells by spone the miR-490?1?After transfection of sh-DLEU1 into Ishikawa cells,the expression level of miR-490?3.33±0.30?was significantly higher than that in sh-NC group?0.99±0.15??P<0.05?.?2?The expression level of miR-490 in EC?0.54±0.28?was significantly lower than that in normal endometrial tissue?0.92±0.41??P<0.05?;the expression level of miR-490 in HHUA?0.57±0.11?,KLE?0.35±0.11?,Ishikawa?0.24±0.08?,ECC-1?0.56±0.12?endometrial carcinoma cell lines was significantly lower than that in normal endometrium?0.97±0.14??P<0.05?.The expression level was the lowest in Ishikawa cell line.?3?Compared with sh-DLEU1 group,in the miR-490 inhibitor group the proliferation activity of Ishikawa cells increased?P<0.05?,the percentage of apoptotic cells decreased?P<0.05?,the expression of pro-apoptotic proteins?Bax,cleaved-caspase-3 and cleaved-caspase-9?decreased,and the expression of anti-apoptotic proteins?Bcl-2?increased.The ability of cell migration and invasion was enhanced?P<0.05?;the process of EMT was enhanced?the expression of E-cadherin decreased,the expression of N-cadherin,Snail and Vimentin increased?.4.The mechanism of DLEU1 regulating malignant biological behavior of endometrial carcinoma cells through DLEU1-miR-490-SP1 axis?1?The results of luciferase reporter gene test showed that the expression of luciferase in Ishikawa cells co-transfected with wild type SP1-3'UTR plasmid and miR-490 mimic plasmid?0.43±0.09?was significantly lower than that in mimic control group?0.96±0.16??P<0.05?.The expression of luciferase in Ishikawa cells co-transfected mutant SP1-Mut 3'UTR plasmid and miR-490 mimic plasmid?0.96±0.08?was similar to that in the mimic control group?0.97±0.09??P>0.05?.?2?Compared with the control group,the expression of SP1 mRNA?0.47±0.13??P<0.05?and SP1 protein in Ishikawa cells decreased after the enhancement of the expression of miR-490,and the expression of SP1 mRNA?2.95±0.21??P<0.05?and SP1 protein in Ishikawa cells increased after the inhibition of the expression of miR-490.?3?Compared with the miR-490 inhibitor group,in the si-SP1 group the proliferation activity of Ishikawa cells decreased?P<0.05?,the percentage of apoptotic cells increased?P<0.05?,the expression of pro-apoptotic related proteins?Bax,cleaved-caspase-3 and cleaved-caspase-9?increased,and the expression of anti-apoptotic related proteins?Bcl-2?decreased.The ability of cell migration and invasion was decreased?P<0.05?;the process of EMT was weakened?the expression of E-cadherin increased,the expression of N-cadherin,Snail and Vimentin decreased?.Conclusion:1.DLEU1 is highly expressed in endometrial carcinoma tissues and cells,and more higher in advanced and high-grade endometrial carcinoma tissues,it shows that DLEU1 is involved in the development of endometrial carcinoma as an oncogene.2.DLEU1 enhances the proliferation activity and inhibits the apoptotic level of endometrial carcinoma cells,enhances the migration and invasion ability of endometrial carcinoma cells and promotes the EMT process.It is demonstrated that DLEU1 plays a positive regulatory role in malignant biological behavior of endometrial carcinoma cells.3.miR-490 is negatively regulated by DLEU1 in endometrial carcinoma.The low expression of miR-490 in endometrial carcinoma tissues and cells demonstrated that miR-490 was involved in the development of endometrial carcinoma as an anti-oncogene.4.Inhibiting the expression and function of miR-490 can release the"containment state"of malignant biological behavior of endometrial carcinoma cells after inhibiting the expression of DLEU1,which proves that DLEU1 promotes malignant biological behavior of endometrial carcinoma cells by sponge and adsorb the miR-490 by and eliminating the expression and function of miR-490.5.In endometrial carcinoma cells,miR-490 regulates SP1 targeting at the binding site of 3'UTR,and miR-490 plays a negative regulatory role on SP1.6.DLEU1 promotes the malignant biological behavior of endometrial carcinoma cells and the progression of endometrial carcinoma lesions through the DLEU1-miR-490-SP1 regulatory axis. |
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