| Ovarian cancer is one of the three major malignant tumors of female reproductive system.Because the ovary is located in the deep pelvic cavity of women,it is not easy to palpate,and there is a lack of early screening means,most patients with ovarian cancer are treated as advanced stage.According to the American Cancer Society in 2020,there were 21,750 new cases of ovarian cancer,accounting for 2.38%of new malignant tumors,and 13,940 deaths of ovarian cancer,accounting for 4.89%of malignant cancer deaths,ranking fifth after bronchial/lung cancer,breast cancer,colorectal cancer,and pancreatic cancer.In recent years,with the development of second-generation sequencing technology and The Cancer Genome Atlas(TCGA)project,it has been found that there are many gene expression disorders in ovarian cancer,such as TP53 mutation,MYC amplification,homologous recombination defects,activation of FOXM1 and NORCH pathways.However,The pathogenesis of ovarian cancer remains unclear.In recent years,with the use of poly ADP-ribose polymerase(PARP)inhibitors,the clinical prognosis of ovarian cancer has been greatly improved,but these drugs are only suitable for patients with platinum-sensitive and homologous recombination defects.It is still of vital importance to study the pathogenesis of ovarian cancer and to screen molecular targets for targeted therapy.RNA splicing is the process of removing introns,joining exons,and producing mature mRNA.In recent years,with the widespread application of second-generation sequencing,high-throughput sequencing studies have shown that 100%of human genes produce at least two alternative mRNA isoforms.Alternative splicing enables a kind of DNA sequence to produce multiple messenger RNAs,and then produce multiple proteins,which is one of the important reasons for protein diversity.Alternative splicing is involved in cell proliferation,differentiation and other biological processes,and is closely related to tumor genesis and development.Alternative splicing is regulated by splicing enhancers,splicing silencers,splicing factors,etc.USP39 was originally identified as a deubiquitinase,but later analysis showed that it lacked three key amino acids for deubiquitinase activity.In addition,in vitro enzyme activity assay also found that USP39 lacked deubiquitinase activity,but bound to the U5/U6-SnRNP complex.Makarova et al.found that USP39 was involved in the assembly of U5/U6-snRNP splicing complex.Yesenia Rios et al.found that the deletion of USP39 in zebrafish resulted in the splicing defect of RB1 mRNA.Ding K et al.found that USP39 played an important role in the maturation of TAZ mRNA in gliomas.We analyzed the expression of splicing factors in ovarian cancer from CPTAC database,and found that most splicing factors were overexpressed in ovarian cancer,and USP39 was one of the overexpressed splicing factors in ovarian cancer.This study mainly focused on the role and mechanism of USP39 in promoting ovarian cancer,and searched for the upstream regulatory genes of USP39.This study is mainly divided into the following four parts:Part Ⅰ:The expression and clinical significance of USP39 in ovarian cancer Research purposeTo explore the expression of USP39 in ovarian cancer,to clarify the relationship between USP39 expression and clinical prognosis of ovarian cancer patients.Research methodsThe expression of ovarian cancer protein and pathway analysis in the CPTAC database were analyzed,and the splicing factor USP39 was found to be highly expressed in ovarian cancer.The RNA expression of USP39 in ovarian cancer patients was explored through the analysis of the TCGA database.The expression of USP39 protein in ovarian cancer tissue samples collected from the department of obstetrics and gynecology of Qilu Hospital was detected by tissue microarray,and the relationship between USP39 expression and the clinical characteristics of ovarian cancer patients such as age,FIGO stage,CA125 level,platinum sensitivity,lymph node metastasis,omental metastasis and prognosis was analyzed.Research results1.USP39 is highly expressed in ovarian cancerThe CPTAC analysis found that proteins associated with the splicing pathway were significantly activated in ovarian cancer compared with normal ovarian tissue,and 140 splicing-related proteins were highly expressed in ovarian cancer.USP39 was one of the splicing-related proteins that were highly expressed in ovarian cancer.Analysis of TCGA data showed that USP39 RNA was highly expressed in a variety of tumors,including ovarian cancer.2.High expression of USP39 was associated with higher FIGO stage and platinum resistance in ovarian cancer patientsImmunohistochemical results showed that in ovarian cancer tissue samples collected from the Department of Gynaecology and Obstetrics of Qilu Hospital,the expression of USP39 in ovarian cancer was significantly higher than that in normal tubal umbrella tissue.The expression of USP39 was not correlated with age,CA125 level,lymph node metastasis,or omental metastasis of ovarian cancer patients.The high expression of USP39 was correlated with advanced FIGO stage and platinum resistance.3.High expression of USP39 is associated with poor prognosis in ovarian cancer patientsSurvival analysis of ovarian cancer tissue samples collected from the Department of Gynaecology and Obstetrics of Qilu Hospital showed that the Overall Survival(OS)and Progression-Free Survival(PFS)of ovarian cancer patients with high USP39 expression were shorter.Kaplan-Meier Plotter analysis also showed that ovarian cancer patients with high USP39 expression had shorter overall survival and progression-free survival.Part Ⅱ:the effect of USP39 on malignant biological behavior of ovarian cancer Research purposeTo investigate the effects of USP39 on the proliferation,invasion,tumor formation and other malignant biological behaviors of ovarian cancer cells.Research methodsUSP39 overexpression and knockdown cell lines were constructed to investigate the effect of USP39 on malignant biological behavior of ovarian cancer cells.CCK8 assay was used to detect cell proliferation,EDU assay was used to detect cell division,and transwell assay was used to detect the invasion ability of ovarian cancer cells.Meanwhile,USP39 overexpression and knockdown cell lines were injected into immunodeficient nude mice to observe the effect of USP39 on tumorigenicity of ovarian cancer cells in vivo.Research results1.Construction of USP39 overexpression and knockdown cell linesOvarian cancer cell lines with overexpression of USP39 were constructed by retrovirus infection,and that with knockdown of USP39 were constructed by lentivirus infection.The efficiency of overexpression and knockdown were detected by Western Blot,which proved that the overexpression and knockdown cell lines were successfully constructed.2.USP39 promotes the proliferation of ovarian cancer cells in vitroCCK8 and EDU assay were used to detect proliferation and cell division ability of ovarian cancer cells after overexpression or knockdown of USP39.The results showed that overexpression of USP39 promoted the proliferation of ovarian cancer cells,while knockdown of USP39 decreased the proliferation of ovarian cancer cells.3.USP39 promotes in vitro invasion of ovarian cancer cellsTranswell assay was used to detect the effect of USP39 on the invasion ability of ovarian cancer cells.The results showed that overexpression of USP39 promoted the invasion ability of ovarian cancer cells and knocked down of USP39 decreased the invasion ability of ovarian cancer cells.Meanwhile,Western Blot results showed that USP39 promoted the EMT process.4.USP39 promotes tumor formation of ovarian cancer cells in nude miceThe effect of USP39 on the tumorigenesis ability of ovarian cancer cells in nude mice was detected by subcutaneous and abdominal tumorigenesis experiments.The results showed that the subcutaneous and abdominal tumorigenesis ability of ovarian cancer cells in nude mice were decreased when USP39 was knocked down,while the subcutaneous and abdominal tumorigenesis ability of ovarian cancer cells in nude mice were increased when USP39 was overexpressed.Part Ⅲ:The mechanism of USP39 promoting malignancy of ovarian cancer cells Research purposeTo search the upstream regulatory factor of USP39 and explore the regulatory mechanism of upstream factor on USP39.To investigate the role of USP39 in ovarian cancer cells and whether it is involved in splicing regulation.To explore the downstream target genes of USP39 and the regulatory mechanisms.Research methodsThe promoter sequence of USP39 was used to search for transcription factors binding to it.The binding sites of upstream transcription factors to the USP39 promoter were predicted using an online prediction website.Western Blot and RT-qPCR were used to verify the regulation of upstream transcription factors on USP39 at the protein and RNA levels,respectively.The dual luciferase reporter assay examined the regulation of upstream transcription factors on the promoter of USP39.ChIP assay verified the direct binding of upstream transcription factors to the USP39 promoter region.Proteins interacting with USP39 were pulled down by immunoprecipitation and detected by mass spectrometry to analyze the types and functions of USP39 binding proteins.Phosphorylated SF3B1 levels were used to detect spliceosome activity.Splicing reporter plasmid was used to verify the effect of USP39 on splicing efficiency.RNA-Seq was used to detect the effect of USP39 on alternative splicing and splicing efficiency,and RIP-Seq was used to screen USP39-bound RNA.The effect of USP39 on HMGA2 transcripts expression was detected by RNA-seq.The change of 5 ’and 3’ splicing efficiency of HMGA2 after USP39 knockdown was analyzed.The ratio of pre-mRNA to mRNA indicates splicing efficiency.Minigene assay was used to detect the changes of HMGA2 splicing efficiency in cells after USP39 knockdown.The binding of USP39 to HMGA2 RNA was verified by RIP-PCR and RNA-pulldown assay.The regulation effect of USP39 on HMGA2 protein and RNA was detected by Western Blot and qPCR.The rescue effect of HMGA2 on malignant biological behavior of ovarian cancer was verified by colony formation assay,transwell assay and nude mice xenografts assay.Research results1.c-MYC transcriptionally activates USP39 expression in ovarian cancer cellsOvarian cancer cell lines with c-MYC overexpression and knockdown were constructed,and the RNA and protein expressions of USP39 were detected by qPCR and Western Blot.The results showed that the overexpression of c-MYC promoted the expression of USP39,and knockdown of c-MYC reduced the expression of USP39.The binding sites of c-MYC and USP39 promoter were predicted,and the wild type and mutant vectors of USP39 promoter were constructed.The dual luciferase reporting experiment showed that c-MYC activated the activity of USP39 wild type promoter,but had no regulatory effect on the USP39 promoter mutated at the c-MYC binding site.The results of ChIP assay showed that c-MYC could directly bind to the promoter of USP39.2.USP39 is involved in the regulation of splicing in ovarian cancer cellsProteins interacting with USP39 were pulled down by immunoprecipitation and detected by mass spectrometry.The results showed that most of the proteins binding to USP39 were RNA-binding proteins and splicing related proteins.The localization of USP39 in cells was detected by immunofluorescence,and the results showed that USP39 was located in nuclear speckle.Nuclear speckle is a site where splicing factors gather and assemble,suggesting that USP39 may be involved in splicing.Phosphorylated SF3B1 is a marker of catalytically active splicosome.Western Blot results showed that overexpression of USP39 increased the level of phosphorylated SF3B1,whereas knockdown of USP39 reduced the level of phosphorylated SF3B1.Splicing report plasmid experiments showed that the splicing efficiency decreased when USP39 was knocked down.The RNA-seq results showed that USP39 regulates several alternative splicing events,of which the most common is cassette exons.After USP39 knock down,percentage of introns increased.Analysis of splicing efficiency at 5’ and 3’ splicing sites showed that both 5’ and 3’ splicing efficiency were reduced after USP39 knockdown.RIP-Seq identified USP39 binding RNA.Reads analysis and peak analysis showed that USP39 mainly bind to intron region,expecially bind to intron-exon and exon-intron junction.The 161 genes identified by RIP-seq were performed GO analysis and the results showed that transcriptional coactivators accounted for the largest proportion.Genes of which the expression and splicing efficiency were both reduced after USP39 knockdown and genes to which RIP experiment proved USP39 bound were intersected,and a total of 11 candidate target genes of USP39 were screened out.The regulatory effect of USP39 on 4 of 11 candidate target genes was verified by semi-quantitative PCR and HMGA2 was selected as the target gene for further study.3.USP39 regulates HMGA2 splicing efficiencyRNA-seq results showed that the expression of 11 HMGA2 transcripts was decreased after USP39 knockdown.The splicing efficiency analysis showed that the 5 and 3’ splicing efficiency of HMGA2 decreased when USP39 was knocked down.The ratio of pre-mRNA to mRNA detected by qPCR indicated splicing efficiency.The results showed that the over-expression of USP39 resulted in a decrease in the ratio and an increase in splicing efficiency.When USP39 is knocked down,the ratio increases and the splicing efficiency decreases.Minigene assay showed that knocking down USP39 increased HMGA2 pre-mRNA and decreased mature mRNA.RIP-Seq and RIP-PCR results showed that USP39 could bind to HMGA2 RNA.RNA-pulldown test further verified the combination of the two.4.Overexpression of HMGA2 can rescue malignant biological behavior of ovarian cancerThe regulation of USP39 on HMGA2 was detected by qPCR and Western Blot.Overexpression of USP39 increased the expression of HMGA2,while knockdown of USP39 decreased the expression of HMGA2.The TCGA database analysis showed a linear correlation between USP39 and HMGA2 RNA expression.The protective effect of HMGA2 on malignant biological behavior of ovarian cancer was verified by plate cloning assay,Transwell assay and tumor formation assay in nude mice.The results showed that overexpression of HMGA2 could save the reduction of malignant biological behavior of ovarian cancer caused by USP39 knockdown.Conclusions1.USP39 is highly expressed in ovarian cancer and is associated with poor prognosis.2.Splicing factor USP39 promotes proliferation,invasive behavior and epithelial-mesenchymal transformation of ovarian cancer.3.c-MYC directly binds to the promoter region of USP39 and transcriptionally activates the expression of USP39.4.Splicing factor USP39 regulates a number of selective splicing events,including cassette exon.5.Splicing factor USP39 binds to HMGA2 pre-mRNA directly and promotes effective splicing of HMGA2,upregulateing its expression level.6.HMGA2 is a key target gene of USP39,and overexpression of HMGA2 can rescue the biological effects caused by USP39 knockdown.The innovation of this study1.In this study,it was found that splicing pathway is abnormally activated and USP39 was highly expressed in ovarian cancer.And expression of USP39 was associated with prognosis of ovarian cancer.2.In this study,RIP-Seq combined with RNA-Seq clarified the global regulation of splicing by USP39 and its downstream molecular network.3.In this study,it was found that the mechanism of USP39 promoting the development of ovarian cancer is realized by promoting the effective splicing of HMGA2,which is the direct target gene of USP39.Shortcomings of this study1.The study did not explore the effects of USP39 on drug resistance,angiogenesis and stemness of ovarian cancer cells.2.The motif of USP39 binding RNA has not been determined,and the USP39 binding motif can be identified by clip-seq and other methods in the future.3.The splicing process is often coupled with transcription.The effect of USP39 on splicing-coupled transcription has not been investigated.We can next study the influence of USP39 on transcription by means of ChIP-seq,newborn RNA detection and other methods. |
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