| Objective:Endometrial carcinoma is one of the most common malignancies in gynecology.It mainly originates from endometrial glands.Among all gynecological tumors,endometrial cancer ranks second and third in terms of morbidity and mortality,respectively.Currently,endometrial cancer is treated with surgery,chemotherapy and radiation therapy,but the efficacy of these methods is still limited to advanced endometrial cancer.The mortality of endometrial cancer doubled from 2008 to 2018.Therefore,it is extremely important to understand the pathogenesis of endometrial cancer and find potential therapeutic targets for this fatal disease.RNA-binding proteins are very important proteins in cells.They can interact with both coding and noncoding RNAs to participate in various posttranscriptional regulatory processes,such as RNA transport,splicing,localization and translation.An increasing number of studies have shown that posttranscriptional regulation mediated by RNA binding proteins is crucial for tumor development.Insulin growth factor m RNA binding protein 3(IGF2BP3)is an RNA-binding protein that is a member of the insulin-like growth factor m RNA binding protein family and can maintain the integrity of embryonic epithelial tissue.Although it has been reported that IGF2BP3 expression is increased in endometrial cancer tissues by immunohistochemical staining,its function and mechanism in endometrial cancer cells have not yet been studied.Long noncoding RNAs are nonprotein-coding RNA transcripts containing at least 200 bases.They can regulate gene expression at the transcriptional,posttranscriptional and translational levels and perform many functions in cells.At present,many lnc RNAs have been found to be involved in the proliferation,invasion,metastasis and drug resistance of tumors.Although it has been reported that some lnc RNAs can combine with IGF2BP3 to directly or indirectly increase or decrease the stability of downstream m RNA,thus affecting the progression of tumors,the understanding of how lnc RNAs regulate the function of IGF2BP3 in the development and progression of endometrial cancer is still limited.The aim of this study was to identify lnc RNA molecules that interact with IGF2BP3 in endometrial cancer,observe whether the interaction between them can change the malignant biological behavior of endometrial carcinoma and provide new ideas and new targets for endometrial cancer risk assessment,prognosis prediction,diagnosis and treatment through a series of molecular mechanism studies.Method:1.RT–q PCR and immunohistochemical staining were used to detect the expression levels of IGF2BP3 in endometrial carcinoma.The median expression levels were used to divide the patients with endometrial carcinoma into a high expression group and a low expression group.Then,the relationship between IGF2BP3 expression and clinicopathological parameters and the overall survival rate of endometrial cancer was analysed.2.Lentiviruses overexpressing IGF2BP3 and hairpin–derived small RNAs targeting IGF2BP3 were employed to construct IGF2BP3-overexpressing or IGF2BP3-knockdown Ishikawa and HEC–1–A cells.CCK-8 and Ed U assays were applied to detect the proliferation of IGF2BP3-overexpressing or IGF2BP3-knockdown Ishikawa and HEC–1–A cells.Transwell assays were employed to detect the migration and invasion of IGF2BP3-overexpressing or IGF2BP3-knockdown Ishikawa and HEC–1–A cells.3.RNA immunoprecipitation sequencing and The Cancer Genome Atlas(TCGA)database were used to identify lnc RNAs that interacted with IGF2BP3,and the results were verified in both Ishikawa and HEC–1–A cells by RNA immunoprecipitation–q PCR and RNA pulldown assays.4.RT–q PCR was used to detect the expression of LINC00958 in endometrial carcinoma tissue.Then,the relationship between LINC00958 and clinicopathologic features was analysed.The Pearson correlation coefficient method was used to analyse whether the m RNA expression of LINC00958 and IGF2BP3 in endometrial carcinoma tissues was correlated.5.Lentiviruses overexpressing LINC00958 and hairpin–derived small RNAs targeting LINC00958 were employed to construct LINC00958-overexpressing or LINC00958-knockdown Ishikawa and HEC–1–A cells.In LINC00958-overexpressing or LINC00958-knockdown Ishikawa and HEC–1–A cells,the m RNA and protein expression levels of IGF2BP3 were detected by RT–q PCR and Western blot.Similarly,in IGF2BP3-overexpressing or IGF2BP3-knockdown Ishikawa and HEC–1–A cells,the expression levels of LINC00958 were detected by RT–q PCR.Finally,RNA fluorescence in situ hybridization and fluorescence immunocytochemical staining were used to detect the subcellular localization of LINC00958 and IGF2BP3.6.Cotransfected cell lines were constructed.CCK-8 and Ed U assays were performed to detect the proliferation of Ishikawa cells and HEC-1-A cells upon ectopic expression of IGF2BP3 combined with LINC00958-silenced expression.Transwell assays were carried out to observe the migration and invasion of Ishikawa cells and HEC-1-A cells upon ectopic expression of IGF2BP3 combined with LINC00958-silenced expression.7.Transcriptome sequencing and TCGA database analysis were used to identify possible downstream genes coregulated by LINC00958 and IGF2BP3 in Ishikawa cells.Overexpression or RNAi lentivirus was used to overexpress or knock out LINC00958 or IGF2BP3 in Ishikawa and HEC–1–A cells.RT-q PCR and Western blot were used to verify the regulatory effects of LINC00958 and IGF2BP3 on E2F3.Rescue assays were used to determine whether IGF2BP3 regulates E2F3 protein expression through LINC00958.8.E2F3 si RNA was transfected into Ishikawa cells and HEC–1–A cells.CCK-8,Ed U and Transwell assays were employed to detect the proliferation,migration and invasion of E2F3 knockout Ishikawa and HEC–1–A cells.9.Cotransfected cell lines were constructed and CCK-8,Ed U and Transwell assays were performed to observe the proliferation,migration and invasion of cotransfected cells in each group.10.The subcutaneous xenotransplanted tumor model of nude mice was used to observe the growth rates and volumes of xenotransplanted tumors when IGF2BP3 knocked out or overexpressed HEC–1–A cells were injected subcutaneously into nude mice.At the same time,immunohistochemistry was used to detect the expression of E2F3 and Ki67.11.RNA decay assays were performed to detect the half–life of E2F3 m RNA in LINC00958-overexpressing endometrial carcinoma cell lines and IGF2BP3 knockout cell lines.Rescue assays were also conducted to analyse whether IGF2BP3 affects the stability of E2F3 via LINC00958,thus affecting m RNA output.Results:1.The expression of IGF2BP3 was increased in endometrial carcinoma tissues,and high expression of IGF2BP3 was associated with stage(P=0.01),grade(P=0.03),vascular invasion(P=0.03),distant metastasis(P=0.047),and poor prognosis(P=0.0334).2.Knockdown of IGF2BP3 inhibited the proliferation,migration and invasion of endometrial carcinoma cells.Overexpression of IGF2BP3 promoted the progression of endometrial carcinoma cells.3.The intersection of the RNA immunoprecipitation sequencing results and the list of differentially expressed genes obtained from the TCGA database found a total of 10 potential lnc RNA molecules that bound to IGF2BP3 in endometrial carcinoma.RNA immunoprecipitation–q PCR and RNA pulldown results showed that,in fact,only LINC00958 could bind to IGF2BP3.4.LINC00958 is highly expressed in endometrial carcinoma tissues,and high expression of LINC00958 is associated with stage(P=0.01),lymphnode metastasis(P=0.02),and distant metastasis(P=0.03).The results of Pearson correlation coefficient method showed that the expression of LINC00958 was not related to the expression of IGF2BP3(R~2=0.035).5.After LINC00958 was overexpressed or knocked down in Ishikawa cells and HEC-1–A cells,IGF2BP3 had no significant changes at either the m RNA or protein level.The expression level of LINC00958 did not change significantly after IGF2BP3 was overexpressed or knocked down.The results of RNA fluorescence in situ hybridization and fluorescence immunocytochemical staining showed that LINC00958 interacted with IGF2BP3 mainly in the cytoplasm.6.The results of rescue assays indicated that in Ishikawa and HEC-1-A cells,knockdown of LINC00958 partially reversed the proliferation,migration and invasion of IGF2BP3-overexpressing cells,suggesting that LINC00958 might exert its tumor-promoting effects by interacting with IGF2BP3.7.Transcriptome sequencing results of LINC00958-silenced Ishikawa cells intersected with the list of genes coexpressed with IGF2BP3 obtained from TCGA database,and nine potential downstream effector genes were obtained.RT–q PCR and Western blot results showed that only the m RNA and protein expression levels of E2F3 were changed when LINC00958 and IGF2BP3 were knocked out or overexpressed in Ishikawa and HEC-1–A cells,suggesting that E2F3 was regulated by both LINC00958 and IGF2BP3.Moreover,rescue assays demonstrated that IGF2BP3 could regulate E2F3 protein expression through LINC00958.8.Knockdown of E2F3 by si RNAs in the two endometrial cancer cell lines inhibited the proliferation,migration and invasion of endometrial cancer cells,indicating that E2F3 may promote the progression of endometrial carcinoma.9.Overexpression of IGF2BP3 in Ishikawa and HEC–1–A cells accompanied by knockdown of E2F3 partially restored the carcinogenic effect of overexpression of IGF2BP3.10.In vivo experiments demonstrated that knockdown of IGF2BP3 can inhibit the growth of xenotransplanted tumors,whereas overexpression of IGF2BP3 can promote the growth of xenotransplanted tumors.Immunohistochemical results showed that the protein expression levels of E2F3 and Ki67 were also decreased or increased after IGF2BP3 silencing or overexpression.Rescue assays showed that IGF2BP3affected the growth of xenotransplanted tumors and the expression of E2F3 and Ki67 via LINC00958.11.RNA decay assays showed that overexpression of LINC00958prolonged the half-life of E2F3 in Ishikawa and HEC–1–A cells,while knockdown of IGF2BP3 shortened the half-life of E2F3.Moreover,the elevated m RNA stability of E2F3 in IGF2BP3-overexpressing endometrial carcinoma cells was restored by LINC00958 knockdown and consequently affected the E2F3 m RNA expression output.Conclusion:1.IGF2BP3 is highly expressed in endometrial carcinoma,and its increased expression is associated with many poor clinicopathological types and poor prognosis.IGF2BP3 can promote the proliferation,migration and invasion of endometrial cancer cells.2.LINC00958 combines with IGF2BP3 in the cytoplasm and assists IGF2BP3 in influencing the progression of endometrial cancer by changing the stability of E2F3m RNA and regulating the expression of E2F3 m RNA. |
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