| Endometrial carcinoma is one of the three malignant tumors of female genital tract,which originates from endometrial epithelium and often occurs in perimenopausal and postmenopausal women.In recent years,the incidence of endometrial carcinoma has been increasing year by year,and endometrial adenocarcinoma from endometrial epithelium accounts for about 85-90%of it.So far,the common treatment methods of endometrial carcinoma mainly include surgical treatment,radiotherapy and chemotherapy,hormone therapy,targeted therapy and so on.Most endometrial carcinoma can appear symptoms early,early diagnosis,surgery-based comprehensive treatment prognosis is better.invasive metastasis occurs in advanced or poorly differentiated endometrial carcinoma,the recurrence rate is high after comprehensive treatment,and the prognosis is poor.Therefore,it is of great significance to find biomarkers of endometrial carcinoma and to study the molecular mechanism of its occurrence and development.Numb normal physiological conditions can regulate cell differentiation,its role in malignant tumors has also been reported,but its role in different types of cancer is different.Numb has been shown to inhibit the growth of prostate tumor xenografts and the development of castration-resistant prostate cancer,which involves various functions related to signal transduction(such as regulating Notch activation and TP53 activation pathways).Low Numb levels in breast cancer patients are associated with poor prognosis.nevertheless,Numb overexpression can not inhibit tumor cell proliferation in glioma in vitro experiments,which may be associated with variable shear(alternative splicing,AS)of Numb.Alternative splicing(AS)is a pre-mRNA that can produce multiple mRNA isoforms or proteins with different functions that may be under different post-transcriptional regulation.Recent massively parallel RNA sequencing(RNA-seq)transcriptome analysis shows that more than 90%of human genes have undergone alternative splicing.In human cells,alternative cleavage is accomplished by cleavage bodies,which are dynamic enzyme complexes composed of small ribonucleoprotein particles.The composition and process of the alternative splicing system are strictly regulated at different developmental stages,and their disorders are closely related to various human diseases including cancer.Normally,the alternative splicing mode is unique to tissues and developmental stages,and is precisely regulated by the synergy between trans-RNA binding proteins(RBPs)and cis-regulatory elements in the nascent transcript.Numb gene alternative splicing mainly produces two subtypes of transcripts,namely Numb-L and Numb-S,corresponding to the translation into two Numb protein subtypes.The main difference is the length of the proline-rich region(PRR).These two protein subtypes play opposite roles in regulating cell function.Numb-L can promote the proliferation of mouse embryonic cancer cells,but does not promote differentiation.Numb-S can promote differentiation,but does not promote proliferation.It is reported that the expression of Numb-L is increased in lung cancer,bladder cancer,breast cancer and colon cancer.Studies have reported that Numb is highly expressed in endometrial cancer.However,there is no relevant research on the expression of Numb-L and Numb-S in endometrial cancer.Mutations in splicing regulators and abnormal splicing of RNA targets are associated with many human diseases.However,up to now,the exact molecular mechanism of controlling alternative splic process under physiological and pathological conditions is not very clear.RBM10 encodes a protein of 930 amino acids,including two RNA recognition motifs(RRM),two zinc fingers and a G patch motif.These motifs are usually present in RNA-binding proteins involved in pre-mRNA splicing,such as the protein components of heterogeneous ribonucleoprotein(HnRNPs)and small ribonucleoprotein(SnRNPs).Through mass spectrometry analysis,it has been reported that RBM10 binds to the purified splicing complex and has been further identified as a component of U2 snRNPs,spliceosome A(or pre-spliceosome)and B complex.In addition,based on the yeast two-hybrid method,a study on the interaction between more than 200 known proteins in the splice can prove the physical interaction between RBM10 and a variety of splice components.Although all these observations suggest the potential role of RBM10 in the regulation of pre-mRNA splicing,it is still unclear whether RBM10 is involved in splicing and how to regulate splicing.Moreover,the regulatory mechanisms of alternative splicing are histone modification and microRNA(mirna),microRNA are a class of non-coding small molecules involved in post-transcriptional regulation of genes RNA,which inhibit their translation by post-transcriptional binding to the 3'-non-translational region(UTR)of target genes,regulating different biological processes.The purpose of this study is to detect the expression of Numb alternative splicing in endometrial carcinoma RBM to study its effect on the proliferation function of endometrial carcinoma cells.And further explore the possible mechanism of regulating Numb alternative splicing.Part I Expression and function of Numb alternative splicing in endometrial carcinomaObjectives:1.Verify the difference of expression between Numb-L and Numb-S in endometrial carcinoma;2.To explore the correlation between the expression level of Numb-L and the clinical features of endometrial carcinoma;3.Screen relevant proteins that can target Numb alternative splicing in endometrial carcinoma and verify the expression of alternative splicing regulator RBM in endometrial carcinoma;4.Explore the correlation between the level RBM10 alternative spliceosome regulators and clinical characteristics and Numb-L,and verify that alternative splicing is regulated by RBM10 in endometrial cancer cells;5.Study the Numb-L and Numb-S of two alternative splicing body expression levels and endometrial cancer prognosis.Methods:1.Study included endometrial cancer patients in obstetrics and gynecology of the first affiliated Hospital of Zhejiang University of traditional Chinese Medic'ine.From January 2016 to May 2019,the patients did not receive radiotherapy and chemotherapy before operation.The tumor tissues of endometrial carcinoma and paired adjacent normal endometrial tissues were collected.Finally,29 matched specimens were collected.2.mRNA and protein levels of Numb-L?Numb-S subtypes in paired endometrial carcinoma and adjacent non-tumor control tissues were detected by semi-quantitative polymerase chain reaction(PCR)and Western blotting.3.Collected clinical characteristic data to analyze the difference of Numb-L level in endometrial carcinoma patients with different clinical characteristics.4.Western blotting was used to determine the expression levels of the three alternative-cut related proteins RBM5,RBM6 and RBM 10 and the downstream gene Notchl of Numb.5.Collect patient clinical characteristics data to analyze the influence of different clinical characteristics on the expression level of alternative splicing-related protein RBM 10 in patients with endometrial cancer,and analyze the correlation between Numb-L and RBM 10.6.Effects of RBM 10 on Numb-L and Numb-S expression were determined by immunoblotting by constructing HEC-1A and stable cell lines that knock down or overexpression.7.By constructing HEC-1A and RL95-2 stable cell lines that express Numb-L and Numb-S,MTT analysis and plate cloning were used to determine the difference of cell proliferation activity,semiquantitative polymerase chain reaction(PCR)to detect the mRNA levels of target molecular HES1 and HEY2 of the Notch pathway.Results:1.Numb-L mRNA and protein expression levels were significantly higher in endometrial carcinoma than in adjacent normal endometrial tissues,but there was no significant difference in Numb-S mRNA and protein expression levels.2.Higher the Numb-L level,the more lymph node metastasis and the higher the FIGO stage in patients with endometrial carcinoma.3.Expression level of only RBM10 proteins in the three related proteins with alternative splicing in endometrial carcinoma decreased,and there was no significant difference between RBM5 and RBM6 protein expression levels.4.Lower the RBM10 level,the more lymph node metastasis and the higher the FIGO stage,and there was a negative correlation with the Numb-L level.5.The protein expression of 5.overexpression RBM10 decreased in HEC-1A and RL95-2 cells,and the expression of Numb-S protein increased.6.The HEC-1A and RL95-2 cell proliferation activity of overexpression Numb-L increased,and the HEC-1A and RL95-2 cell proliferation activity decreased.7.The expression of Notch 1 protein in endometrial carcinoma tissue was significantly higher than that in adjacent normal endometrial tissue.8.Target gene HES1 and HEY2mRNA expression were increased in HEC-1 A and RL95-2 cells with overexpression,while HES1 and HEY2mRNA expression decreased in HEC-1 A and RL95-2 cells with overexpression.Conclusions:1.The abnormal expression of Numb-L in endometrial carcinoma tissue is related to the malignant degree of tumor.The proliferative activity of overexpression Numb-L endometrial carcinoma cells is increased,and its possible mechanism is to activate Notch signaling pathway.2.The expression of RBM10 in endometrial carcinoma was abnormal and correlated with the degree of malignancy.The expression of RBM10 in endometrial carcinoma cells was negatively correlated with the expression of Numb-L.3.Numb-L expression may be regulated by variable shear-related protein RBM10,and the relationship between RBM10 and Numb variable shear may provide new biological markers for the diagnosis and treatment of endometrial carcinoma.Part ? miR-335 alternative splicing of Numb by RBM10 regulationObjectives:1.Verify the relationship between RBM10 of alternative splicing-related proteins and post-transcriptional regulation of microRNA;2.Screening miRNA targeting RBM10 action;3.Verify the correlation between miR-335 and RBM10 expression levels in endometrial carcinoma and their clinical characteristics;4.Study the effect of nude mouse model miR-335 on proliferation of endometrial carcinoma cells;5.explore the related mechanisms miR-335 the effect of alternative splicing on endometrial cancer cell proliferation through RBM10 regulation.Methods:1.As in the first part,a total of 29 specimens of endometrial cancer tissues and matched patients with normal adjacent endometrial tissues were collected.2.Detect the mRNA and protein levels of RBM10 in matched endometrial cancer and adjacent non-tumor control tissues by semi-quantitative polymerase chain reaction(PCR)and western blotting.3.Using bioinformatics tools to predict miRNAs targeting RBM10,and confirm their interaction with RBM10 through dual-luciferase reporter gene analysis and western blotting,and further screen miRNAs by analyzing the correlation between clinical miRNAs levels and RBM10.4.Screening miRNA,regulating alternative splic protein RBM10 to collect clinical characteristic data of patients with endometrial carcinoma to analyze the effect of different clinical features on the expression of miRNA in patients with endometrial carcinoma,and to analyze the effect of RBM10 and screened miRNA on the prognosis of patients with endometrial carcinoma by Kaplan-Meier Plotter online tool.5.Furthermore,the endometrial cancer cell line HEC-1-A overexpressing this miRNA was planted in nude mice,the tumor growth was observed and the tumor volume was calculated,and the expression level of the miRNA was detected by the PCR method.The immunoblotting method was used to detect the expression of the alternative-spliced proteins of RBM10 and Numb in the implanted mice.Results:1.Compared with the normal endometrial tissue adjacent to the cancer,the RBM10 protein level in the endometrial tumor tissue was significantly reduced,and the prognosis of the patient was poor,but the mRNA level did not change significantly.2.The miRNAs screened by bioinformatics tools include miR-22,miR-29a,miR-29b,miR-122,miR-133a,miR-133b,miR-179,miR-335,miR-382.3.The luciferase reporter gene analysis showed that the luciferase activity was significantly inhibited after co-transfection with RBM10 was miR-133a,miR-133b and miR-335.4.The expression of RBM10 was reduced in HEC-1A cells overexpressing miR-133a/b or miR-335.In endometrial cancer,the level of miR335 was significantly up-regulated,which was negatively correlated with the level of RBM10 protein,but the level of miR-133a/b was not significantly different,and there was no significant correlation between the level of miR-133a/b and the level of RBM10 protein.5.After overexpressing miRNA-335 in a xenograft mouse model,the tumor volume increased significantly faster and was higher than that of mice that did not overexpress miR-335.PCR confirmed that miR-335 was overexpressed in mice,and the expression level of RBM10 protein decreased.The expression level of Numb-L protein increased.Conclusions:1.The expression level of Numb-L is related to RBM10,which is further regulated by the expression of miRNA-335.2.miRNA-335 may regulate the alternative splicing of Numb through RBM10 to promote the proliferation of endometrial cancer cells,which also provides new ideas for the study of the mechanism of endometrial cancer. |
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