| Objective: Endometrial cancer is one of the most important malignant tumors of female reproductive system.Most patients with endometrial cancer can be diagnosed early and have a good prognosis,but there are still some patients with tumor recurrence and metastasis after treatment.Rna-binding proteins are involved in the regulation of RNA stability,variable splicing,transport and translation,and the formation of RNAprotein complexes by binding RNA to regulate the occurrence and development of many diseases,including tumors.Upframeshiftmutant 1(UPF1)plays the role of an important controller of transcripts.It is known to be one of the key elements involved in m RNA decay pathways,and is involved in several different aspects of transcript and protein quality control.Previous studies of our research group have shown that UPF1 is elevated in endometrial cancer tissues and can act as an RNA binding protein to promote a variety of malignant biological behaviors of endometrial cancer cells.Long non-coding RNAs(lncRNAs)are a class of Rnas with a length of more than 200 nucleotides and no protein coding ability.Lnc RNAs participate in the occurrence and development of tumors through epigenetic modification,transcription and posttranscriptional regulation,and play an important regulatory role in cell proliferation,invasion,metastasis and other processes.lncRNA can act as a molecular sponge to competitively bind miRNA,and antagonize the inhibitory effect of miRNA on its target genes,thus affecting the occurrence and development of tumors.The purpose of this study was to investigate the possible important role of the UPF1/MIR100HG/SNHG20/miR-2467-3p/RASA2 axis in the genesis and development of endometrial cancer cells.And further study the regulatory relationship and action mechanism between its molecules.That is,by reducing the stability of MIR100 HG and SNHG20,UPF1 regulates miR-2467-3p and down-regulates the expression of RASA2,promoting the malignant biological behavior of endometrial cancer cells,and providing a new theoretical and experimental basis for the treatment of endometrial cancer.Methods:Part Ⅰ: Firstly,the RNA expression levels of MIR100 HG,SNHG20,miR-2467-3p and RASA2 in human endometrial cancer tissues and normal endometrial tissues were detected by q RT-PCR method,and the relationship between mir100 Hg,SNHG20,Mir-2467-3p and RASA2 in patients with endometrial cancer was analyzed.The expression of RASA2 protein in endometrial carcinoma was verified by immunohistochemical method,and the relationship between RASA2 expression and prognosis was analyzed.Part Ⅱ: Culture of human endometrial carcinoma Ishikawa and HEC-1A cell lines.The expression localization of UPF1 and MIR100HG/SNHG20 in cells was detected by fluorescence in situ hybridization combined with immunofluorescence.RNA coimmunoprecipitation was used to verify the binding effect of UPF1 and MIR100HG/SNHG20.Endometrial carcinoma Ishikawa and HEC-1A cell lines with UPF1,MIR100HG/SNHG20 overexpression and silence were constructed by cell transfection.In endometrial cancer cells overexpressed or silenced with UPF1 or cotransfected with MIR100HG/SNHG20,the half-life of MIR100HG/SNHG20 was detected by stability assay.The effects of UPF1 and MIR100HG/SNHG20 on the biological function of endometrial cancer cells were verified in vitro by EDU,the scar test,Transwell chamber test and Annexin V-FITC/PI apoptosis assay.A subcutaneous transplanted tumor model was established in nude mice to verify the effects of MIR100HG/SNHG20 overexpression and UPF1 knockdown on the growth rate of the transplanted tumor and RASA2 protein.Part Ⅲ: Culture of HEK-293 T cell line and verify the targeted binding of miR-2467-3p to MIR100 HG,SNHG20 and RASA2 by using dual luciferase gene reporter system.miR-2467-3p,overexpression and silence of intrauterine carcinoma Ishikawa and HEC-1A cell lines as well as MIR100HG/SNHG20 and miR-2467-3p overexpression co-transfected cell lines were constructed.Overexpression or silencing of miR-2467-3p and co-transfected with MIR100HG/SNHG20 overexpression in endometrial cancer cells were verified in vitro by Western blot,EDU,scar assay,Transwell chamber assay and Annexin V-FITC/PI apoptosis assay The expression of RASA2,and the effect of cell proliferation,migration and invasion,apoptosis.Results:Part Ⅰ: The expression of MIR100 HG and SNHG20 was decreased in endometrial cancer tissues,and the low expression of MIR100 HG was associated with FIGO stage III-IV.Low expression of SNHG20 was associated with muscle infiltration depth ≥1/2 and FIGO stage III-IV.miR-2467-3p expression was increased in endometrial cancer tissues,and its high expression was associated with FIGO stage III-IV and lymphatic vasculature metastasis.Expression of RASA2 m RNA was decreased in endometrial carcinoma tissues,and its low expression was associated with muscle infiltration depth ≥1/2 and FIGO stage III-IV(all P < 0.05).Immunohistochemistry showed that RASA2 protein was underexpressed in endometrial carcinoma tissues,and the absence of RASA2 expression showed a trend of worse prognosis(P=0.063).Part Two: EDU experiment showed that overexpression of MIR100 HG and SNHG20 could inhibit the proliferative activity of Ishikawa and HEC-1A cell line,Transwell experiment showed that overexpression of MIR100 HG and SNHG20 could inhibit the invasion ability of Ishikawa and HEC-1A cell line.Scratch healing experiment verified that overexpression of MIR100 HG and SNHG20 could inhibit the metastasis ability of Ishikawa and HEC-1A cell lines,and flow test showed that the overall apoptosis rate(early apoptosis + late apoptosis)of Ishikawa and HEC-1A cell lines increased after overexpression of MIR100 HG and SNHG20.RIP experiments confirmed the existence of targeted binding of UPF1 and MIR100 HG with SNHG20.UPF1 downregulates the expression of MIR100 HG and SNHG20 by reducing the stability of mir100 Hg and Sn HG20 in vitro through EDU,scar assay,Transwell Chamber assay and Annexin V-FITC/PI apoptosis assay.Promote the proliferation,migration and invasion ability of Ishikawa and HEC-1A cell lines,and inhibit the overall apoptosis rate.The results of subcutaneous transplantation tumor model in nude mice verified the inhibitory effect of overexpression of MIR100HG/SNHG20 on the growth of the transplanted tumor,and the co-transfection of knockdown UPF1 and overexpression of MIR100HG/SNHG20 produced stronger inhibitory effect on the tumor.(P < 0.05 for all the above).Part Ⅲ: Bioinformatics software and double luciferase gene reporting experiments confirmed that miR-2467-3p and MIR100 HG and SNHG20 target binding through predicted sites.It was verified in vitro that overexpression of miR-2467-3p could significantly promote cell proliferation,migration and invasion of Ishikawa and HEC-1A cell lines,and reduce the overall apoptosis rate by EDU,Transwell chamber assay and Annexin V-FITC/PI apoptosis assay.Bioinformatics software and dual luciferase gene reporting system confirmed the existence of binding sites in the 3’UTR region of miR-2467-3p and RASA2.It was verified in vitro that overexpressed MIR100HG/SNHG20 could reduce the content of miR-2467-3p and up-regulate the expression of RASA2 by Edu,scar assay,Transwell chamber assay and Annexin VFITC/PI apoptosis assay.Thus,the proliferation,migration and invasion of endometrial cancer cells were inhibited,and the overall apoptosis rate was increased.(P < 0.05 for all the above).Conclusions:1.MIR100 HG,SNHG20 and RASA2 were low expressed in endometrial cancer tissues,while miR-2467-3p was high expressed in endometrial cancer tissues,which were correlated with muscle infiltration,FIGO staging and other clinicopathologic parameters.2.Overexpression of MIR100 HG and SNHG20 can inhibit the proliferation,migration and invasion of endometrial cancer cells,and promote cell apoptosis.UPF1 can affect the biological behavior of endometrial cancer cells by binding to MIR100 HG and SNHG20 and reducing their stability.3.As ce RNA,MIR100 HG and SNHG20 competitively bind miR-2467-3p with RASA2.Binding inhibition of miR-2467-3p was produced,and RASA2 level was upregulated,thus inhibiting the proliferation,migration and invasion of endometrial cancer cells and promoting cell apoptosis.The UPF1/ MIR100HG/SNHG20/miR-2467-3p/RASA2 axis provides a new idea and a new target for further exploration of the molecular regulatory mechanism of endometrial cancer cells. |
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