| Endometrial carcinoma(EC)is one of the three major malignant tumors of the female reproductive system,ranking No.4 in women with systemic malignancies.In recent years,its mortality rate has been on the rise with the change of people and development,the treatment is still mainly surgery,supplemented by radiotherapy and chemotherapy,for those with advanced or recrudescent endometrial cancer,due to the loss of timing of surgery,can only accept hormone and other drug therapy.At present,the clinical researches on endometrial cancer is relatively abundant in the world,while its etiology is relative few.With the further studies on molecular mechanism,more and more molecular targeted drugs,such as epidermal growth factor receptor(EGFR2)inhibitors,m TOR signaling pathway inhibitors,have been preliminary clinical application and achieved certain effect.However,the search for more effective treatment is still the main direction nowadays.LINC01016 is an estrogen and estrogen receptor-related Lnc RNA found in breast cancer,which is rarely reported around the world.Phillip et al.detected the genes differential expression in breast cancer cell lines MCF7 and T47 D by single-molecule sequencing,and found the expression of ER?-related LINC01016 was highly expressed by the methods of subsequent chromatin immunoprecipitation,PCR and survival analysis,its expression was directly related to the clinical prognosis and survival rate of breast cancer.Endometrial cancer is an estrogen-related tumor.Our previous research confirmed that LINC01016 was highly expressed and played a role in endometrial cancer.In recent years,many studies focused on the regulation of mi RNA by Lnc RNA thereby affecting the biological behavior of tumor.In our previous work,mi R-302a-3p and mi R-3130-3p were screened out by mi Randa,PITA,RNAhybrid and dual luciferase reporter system in a strong interaction with LINC01016 and significantly were down-regulated in endometrial carcinoma.The mi R-302/367 gene cluster is encoded by the human chromosome 4p25 and encodes nine RNAs in the form of polycistrons:mi R-302 a,mi R-302a*?mi R-302b?mi R-302b*?mi R-302c?mi R-302c*?mi R-302d?mi R-367 and mi R-367*(*indicated low expression levels).mi R-302 can improve the sensitivity of hepatoma cells and testicular neuroblastoma cell line to chemotherapy and the radiotherapy sensitivity of breast cancer cells,inhibits the proliferation of prostate cancer cells and cervical cancer cells.In endometrial cancer,mi R-302 inhibits the tumorigenicity of cancer cells.However,there is no correlation between mi R-302 a and endometrial cancer in the establishment of other signaling pathways,transcriptional level and changes of downstream protein epigenetics.While there is no relevant researches of mi R-3130-3p.Using Target Scan,we initially demonstrated the binding sites of has-mi R-302a-3p/mi R-3130-3p to the 3'UTR of NFYA.Through literature retrieved,we found that nuclear transcription factor(NF-Y)is a transcription factor that binds specifically to the CCAAT consensus site.Clinical researches has identified that up-regulation of NF-Y target genes in breast and lung cancer is associated with poor prognosis.Shear variants of NFYA bind to the promoter region of the ALDHA1 gene,which determines the ALDH activity of the aldehyde dehydrogenase by the CCAAT binding region,and the expression of the short hairpin RNA(NF-YA)in the endometrial cancer tissues expressing high level of ALDH is preferentially increased poor prognosis with endometrial adenocarcinoma.Database search revealed a 1000 bp upstream of the transcription initiation site of Special AT-rich sequence-binding protein1(SATB1),suggesting the potential binding site for NFYA(ccaat).At the same time,we found that SATB1 was the first cell-specific nuclear matrix-attached protein that was found to play an important role in chromosomal recombination in T cells.SATB1 can promote tumor growth and metastasis,expressing in many tumors and indicating poor prognosis,including endometrial cancer,which is considered the major gene for tumor metastasis and an independent prognostic factor.The aim of this study was to determine the expression level of LINC01016,mi R-302a-3p,mi R-3130-3p,NFYA and SATB1 in endometrial carcinoma through our previous analysis and hybridization in situ and chromatin immunoprecipitation,elucidating the interaction between LINC01016 and mi R-302a-3p/mi R3130-3p,mi R-302a-3p/mi R3130-3p and NFYA,NFYA and SATB1 by using endometrial carcinoma cell lines Ishikawa and RL-95-2.Endometrial cancer cells proliferation,apoptosis,migration,invasion and cell cycle after up-regulation or down-regulation of LINC01016,mi R-302a-3p/mi R-3130-3p or NFYA.To clarify the specific mechanism of LINC01016-mi R-302a-3p/mi R-3130-3p-NFYA-SATB1 regulatory axis affecting the occurrence and development of endometrial cancer,and to provide new ideas and new strategies for the diagnosis and treatment of endometrial cancer.Methods: 1.The expression of LINC01016 in endometrial carcinoma and normal endometrium was detected by Real-time PCR,and the relationship between the expression of LINC01016 and the clinical pathological parameters of endometrial carcinoma was analyzed.The overexpression and knockdown of LINC01016 transfection in Ishikawa and RL-95-2 cell lines was performed,and the transfection efficiency was verified by Real-time PCR.CCK-8 assay was used to detect the proliferation ability of LINC01016 in both cell lines.The changes of proliferation of Ishikawa and RL-95-2cells were also detected by Ed U assay.The changes of cell cycle and apoptosis were detected by flow cytometry after the intervention of LINC01016 expression;Wound healing assay was used to detected cell migration ability;Transwell assay was used to detected cell invasion ability.2.The mi RNAs interacting with LINC01016 were screened by bioinformatics software mi Randa,PITA and RNAhybrid.The expression levels of mi RNAs in endometrial carcinoma tissues and normal tissues were detected by Real-time PCR and mi RNAs that meet the two requirements at the same time were mi R-302a-3p and mi R-3130-3p.The relationship between the expression level and clinical parameters was analyzed.mi R-302a-3p and mi R-3130-3p were overexpressed and knocked down and they were validated by Real-time PCR.The proliferation ability of mi R-302a-3p and mi R-3130-3p transfected cells were detected by CCK-8 and Ed U respectively.Flow cytometry were used to detect the cell cycle and apoptosis in the same method.Wound healing assay was used to detect the cell migration abilities.The Transwell assay was used to detect the cell invasion ability.After cells interfered with LINC01016,the expression of mi R-302a-3p and mi R-3130-3p was detected by Real-time PCR.The expression of LINC01016 was detected by Real-time PCR after the intervention of mi R-302a-3p and mi R-3130-3p.The luciferase vector ligated with LINC01016 was co-transfected with mi R-302a-3 p and mi R-3130-3p.The binding sites of LINC01016 with mi R-302a-3p and mi R-3130-3p were detected.3.The bioinformatics software Target Scan screened the transcription factor NFYA potentially binding to mi R-302a-3p and mi R-3130-3p,and the database searched for the potential binding sequence of NFYA in the promoter region of SATB1.Real-time PCR and Western blot were used to detect the transcriptional and translational levels of NFYA and SATB1 in endometrial carcinoma.NFYA wild-type and mutant-type dual-luciferase reporter vectors were constructed and was co-transfected with mi R-302a-3p/mi R-3130-3p.The expression of NFYA and SATB1 were detected in LINC01016 transfected cells and mi R-302a-3p/mi R-3130-3p transfected cells by real-time PCR.After NFYA were overexpressed and knocked down in cells,CCK-8,Ed U,cell cycle,apoptosis,wound healing and Transwell assay were used to detect the changes of cell biological behavior.The wild-type and predicted mutant luciferase reporter vector of SATB1 promoter region were co-transfected with NFYA and potential binding sites were detected;CHIP experiment was used to furtherly detect interaction between NFYA and SATB1 promoter region;Real-time PCR and western blot were used to detect SATB1 expression level in NFYA transfected cells.The effects of LINC01016,mi R-302a-3p and mi R-3130-3p on the size of xenografts in nude mice were analyzed by real-time PCR and Western blot.Expression levels of NFYA and SATB1 in the tumor tissues at transcription and translation levels were analyzed.Results: 1.The expression of LINC01016 in endometrial carcinoma tissues was significantly higher than that in normal endometrial tissues(p <0.05).The CCK-8 and Ed U showed that the cell proliferation was increased after high-LINC01016 transfection and the cells in G2-M phase were significantly increased.The overall apoptosis rate(early apoptosis + late apoptosis)decreased.Wound healing experiments showed that the healing ability of LINC01016 increased after transfection.The results of Transwell showed that the cell invasion ability also increased after high transfection of LINC01016(all p <0.05).2.202 mi RNAs interacting with LINC10106 were screened by bioinformatics software mi Randa,PITA and RNAhybrid.Through dual-luciferase assay and Real-time PCR,we chose mi R-302a-3p and mi R-3130-3p since they are significantly down expressed in endometrial cancer tissues and can interact with LINC01016.The proliferation of mi R-302a-3p and mi R-3130-3p transfection cells detected by CCK-8 and Ed U respectively was decreased.The cell cycle and apoptosis activity were detected by flow cytometry.Wound healing assay showed cell migration abilities decreased.The Transwell assay showed cell invasion ability decreased.After LINC01016 was increased,Real-time PCR showed expression of mi R-302a-3p and mi R-3130-3p was decreased.In contrary,the expression of LINC01016 was decreased after the overexpression of mi R-302a-3p and mi R-3130-3p.The dual luciferase reporter assay of LINC01016 showed 2 binding sites with mi R-302a-3p and 1 with mi R-3130-3p.3.In endometrial cancer tissues,NFYA and SATB1 were highly expressed in both RNA and protein levels.Dual luciferase reporter assay indicated that mi R-302a-3p /mi R-3130-3p interacted with 3'UTR of NFYA;NFYA and SATB1 were highly expressed in endometrial cancer cells with high transfection of LINC01016,but low expression of NFYA and SATB1 in cells transfected with mi R-302a-3p / mi R-3130-3p;CCK-8,Ed U,flow cytometry,wound healing assay and Transwell assay were used to detect the changes of cell biological behavior after intervention with NFYA.Namely,the proliferation of cells was enhanced detected by CCK-8 and Ed U;the cells in G2-M phase were significantly increased,and the overall apoptosis rate(early apoptosis + late apoptosis)were decreased;wound healing experiments showed that the healing ability was enhanced,and the cell invasion ability was also enhanced detected by Transwell(all p <0.05);Both luciferase reporter gene and CHIP assay showed that there was binding site between NFYA and SATB1 promoter region.The change of NFYA expression could regulate the expression level of SATB1,which was positively correlated with the increase of LINC01016 and the decrease of mi R-302a-3p / mi R-3130-3p.With the tumor volume increased,NFYA and SATB1 expression levels were increased.Conclusions: 1.LINC01016 is highly expressed in endometrial cancer tissues and exogenous increased LINC01016 expression can promote the development of malignant biological behavior of cells.2.LINC01016 and mi R-302a-3p/mi R-3130-3p form a negative feedback regulatory loop,as one of the extrinsic increase can inhibit the other expression.mi R-302a-3p/mi R-3130-3p were low expressed in endometrial carcinoma and the extrinsic increase of either can inhibit the malignant behavior of endometrial cancer cells.The effect of LINC01016 on cell biology can be mediated by mi R-302a-3p/mi R-3130-3p.3.NFYA and SATB1 were highly expressed in endometrial cancer tissues,and the exogenous increase of NFYA could promote the malignant biological behavior of cancer cells.mi R-302a-3p/mi R-3130-3p has binding sites with NFYA 3'UTR,and NFYA can regulate SATB1 expression as a transcription factor by the promoter region of SATB1.The exogenous increase of LINC01016 and the exogenous decrease of mi R-302a-3p/mi R3130-3p can enhance the transcriptional and translational expression of NFYA and SATB1 in both cellular level and nude mice.The biological effects of LINC01016 regulating mi R-302a-3p/mi R3130-3p on endometrial cancer can be mediated by regulating NFYA and SATB1. |
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