Yang Zhao

SNORA73B promotes endometrial cancer progression through targeting MIB1 and regulating host gene RCC1 alternative splicing

AbstractEndometrial cancer (EC) is a common gynaecological malignant tumour with unclear pathogenesis. Small nucleolar RNA (snoRNA) is involved in many biological processes, including those of cancers. Using the Cancer Genome Atlas (TCGA) database, the expression pattern of a snoRNA, SNORA73B, was analysed. The biological functions of SNORA73B were assessed by in vitro proliferation, apoptosis, migration, and invasion assays and in vivo by the xenograft model. RNA sequencing (RNA‐seq) and RNA immunoprecipitation assays were performed to determine the relationship between SNORA73B and its target genes. High‐performance liquid chromatography (HPLC) was performed to detect the pseudouridine content of the mindbomb E3 ubiquitin protein ligase 1 gene (MIB1). The stability of MIB1 mRNA was evaluated using a transcription inhibitor, actinomycin D. By performing co‐immunoprecipitation assays, the change in the ubiquitin levels of the Jagged canonical Notch ligand 1 (Jag 1), caused by SNORA73B and MIB1, was identified. RNA‐seq and qRT‐PCR were performed to detect the alternative splicing of the regulator of the chromosome condensation 1 gene (RCC1). The TCGA database analysis showed that SNORA73B was highly expressed in EC. SNORA73B promoted cell proliferation, migration, and invasion and inhibited apoptosis. SNORA73B modified the pseudouridine content in MIB1 and increased the stability of MIB1 mRNA and protein; thus, it affected Jag 1 ubiquitination and further activated the Notch pathway. SNORA73B also affected the alternative splicing of RCC1, increasing the number of transcripts, RCC1‐T2 and RCC1‐T3, which promoted cell proliferation, migration, and invasion. SNORA73B can be a potential target for EC.

C/D box small nucleolar RNA SNORD104 promotes endometrial cancer by regulating the 2ʹ-O-methylation of PARP1

Abstract Background Small nucleolar RNAs (snoRNAs) are dysregulated in many cancers, although their exact role in tumor genesis and progression remains unclear. Methods The expression profiles of snoRNAs in endometrial cancer (EC) tissues were analyzed using data from The Cancer Genome Atlas, and SNORD104 was identified as an upregulated snoRNA in EC. The tumorigenic role of SNORD104 in EC was established in CCK8, colony formation, EdU, apoptosis, Transwell, and in vivo xenograft experiments. The molecular mechanisms of SNORD104 were analyzed by RNA immunoprecipitation (RIP), Nm-seq, RTL-P assay, RNA stability assay, qRT-PCR, and western blotting. Results Antisense oligonucleotide (ASO)-mediated knockdown of SNORD104 in Ishikawa cells significantly inhibited their proliferation, colony formation ability, migration, and invasion in vitro and increased apoptosis. On the other hand, overexpression of SNORD104 promoted EC growth in vivo and in vitro. RIP assay showed that SNORD104 binds to the 2ʹ-O-methyltransferase fibrillarin (FBL), and according to the results of Nm-seq and RTL-P assay, SNORD104 upregulated PARP1 (encoding poly (ADP-ribose) polymerase 1) 2ʹ-O-methylation. The binding of FBL to PARP1 mRNA was also verified by RIP assay. Furthermore, SNORD104 expression was positively correlated with PARP1 expression in EC tissues. In the presence of actinomycin D, SNORD104 increased the stability of PARP1 mRNA and promoted its nuclear localization. Finally, silencing FBL or PARP1 in the HEC1B cells overexpressing SNORD104 inhibited their proliferative and clonal capacities and increased apoptosis rates. Conclusions SNORD104 enhances PARP1 mRNA stability and translation in the EC cells by upregulating 2ʹ-O-methylation and promotes tumor growth.

SNORD60 promotes the tumorigenesis and progression of endometrial cancer through binding PIK3CA and regulating PI3K/AKT/mTOR signaling pathway

AbstractEndometrial carcinoma is a common gynecological malignant tumor, small nucleolar RNAs (snoRNAs) are involved in cancer development. However, researches on the roles of snoRNAs in endometrial carcinoma are limited. The expression levels of snoRNAs in endometrial cancer tissues were analyzed using The Cancer Genome Atlas (TCGA) database. Antisense oligonucleotides (ASOs) and plasmids were used for transfection. Moreover, CCK‐8, EdU, wound‐healing assay, transwell, cell apoptosis, western blotting, and xenograft model were employed to examine the biological functions of related molecules. real‐time reverse transcription polymerase chain reaction and western blotting were performed to detect messenger RNA (mRNA) and protein levels. Including bioinformatics, fluorescence in situ hybridization, RNA pulldown, actinomycin D and RTL‐P assays were also carried out to explore the molecular mechanism. Analysis of data from TCGA showed that the expression level of small nucleolar RNA, C/D box 60 (SNORD60) in endometrial cancer tissues is observably higher than that in normal endometrial tissues. Further research suggested that SNORD60 played a carcinogenic role both in vitro and in vivo, and significantly upregulated the expression of PIK3CA. However, the carcinogenic effects can be reversed by knocking down fibrillarin (FBL) or PIK3CA. SNORD60 forms complexes by binding with 2′‐O‐methyltransferase fibrillarin, thus catalyzes the 2′‐O‐methylation (Nm) modification of PIK3CA mRNA and modulates the PI3K/AKT/mTOR signaling pathway, so as to promote the development of endometrial cancer. In short, SNORD60 might become a new biomarker for the therapy of endometrial cancer in the future and provide new strategies for diagnosis and treatment.

CircRNA WHSC1 targets the miR‐646/NPM1 pathway to promote the development of endometrial cancer

AbstractCircular RNAs (circRNAs) play important roles in human cancer progression. Their high stability and tissue specificity make circRNAs important molecular targets for clinical diagnosis, treatment and prognosis. However, the functions and molecular mechanisms of circRNA WHSC1 in endometrial cancer are unknown. CircWHSC1 expression in normal endometrial and endometrial cancer tissues was detected using PCR. Overexpression or knockdown of circWHSC1 in endometrial cancer cell lines HEC‐1B or Ishikawa, respectively, cell function experiments were used to detect the impact of circWHSC1 on endometrial cancer cells. A nude mouse xenograft model was used to detect changes in tumorigenesis of HEC‐1B cells after circWHSC1 overexpression. Bioinformatics and dual luciferase reporter gene technology were used to predict and validate the sponging ability of circWHSC1 on microRNAs. Gene expression changes were detected by using Western blotting. CircWHSC1 expression was increased in endometrial cancer tissues. CircWHSC1 overexpression promoted the proliferation, migration and invasion of endometrial cancer cells and decreased apoptosis. CircWHSC1 knockdown had the opposite effect. CircWHSC1 overexpressed nude mice showed increased tumorigenicity. Bioinformatics predicted that circWHSC1 binds to miR‐646, which was confirmed using luciferase reporter gene assays. High expression of miR‐646 could reverse the effect of circWHSC1 on endometrial cancer cells. Western blotting showed increased or decreased levels of nucleophosmin 1 (NPM1), an miR‐646 downstream target, after circWHSC1 overexpression or knockdown, respectively. CircWHSC1 promotes endometrial cancer development through sponging miR‐646 and targeting NPM1.

Circ_PUM1 promotes the development of endometrial cancer by targeting the miR‐136/NOTCH3 pathway

Abstract Endometrial cancer is one of the most common gynaecological malignancies and the sixth most common cause of cancer‐related death among women. Here, we define the role and molecular mechanism of circ_0000043 (hereafter referred to as circ_PUM1) in the development and progression of endometrial carcinoma. QRT‐PCR was used to detect the expression of circ_PUM1 in normal endometrial tissue and endometrial carcinoma tissues. Changes in cell function and tumorigenicity in nude mice were examined after circ_PUM1 overexpression or knockdown. Bioinformatic analysis and dual‐luciferase reporter assay were used to predict and analyse the miRNAs that circ_PUM1 binds. Gene expression changes were analysed using Western blot. Circ_PUM1 was expressed at significantly higher levels in endometrial cancer tissues than in normal tissues. Up‐regulation of circ_PUM1 promoted the proliferation, migration and invasion of endometrial carcinoma cells. Opposite results were observed with circ_PUM1 knockdown, and the tumorigenic ability of endometrial cancer cells after circ_PUM1 knockdown was reduced compared to control cells. Circ_PUM1 is capable of binding to miR‐136, and up‐regulating its target gene NOTCH3, which can be reversed by overexpression of miR‐136. Circ_PUM1 can compete with miR‐136, leading to up‐regulation of NOTCH3, and thereby promote the development of endometrial cancer.

piR-26441 inhibits mitochondrial oxidative phosphorylation and tumorigenesis in ovarian cancer through m6A modification by interacting with YTHDC1

Abstract Ovarian cancer (OC) is a heterogeneous cancer. In contrast to other tumor cells, which rely primarily on aerobic glycolysis (Warburg effect) as their energy source, oxidative phosphorylation (OXPHOS) is also one of its major metabolic modes. Piwi-interacting RNAs (piRNAs) play a regulatory function in various biological processes in tumor cells. However, the role and mechanisms of piRNAs in OC and mitochondrial OXPHOS remain to be elucidated. Here, we found that piR-26441 was aberrantly downregulated in OC, and its overexpression suppressed the malignant features of OC cells and tumor growth in a xenograft model. Moreover, overexpression of piR-26441 significantly reduced mitochondrial OXPHOS levels in OC cells. Furthermore, piR-26441 directly binds to and upregulates the expression of YTHDC1 in OC cells. piR-26441 also increased m6A levels, thereby interacting with YTHDC1 to destabilize the mRNA of TSFM. The resultant TSFM loss reduced mitochondrial complex I activity and mitochondrial OXPHOS, leading to mitochondrial dysfunction in OC cells, increased reactive oxygen species levels, and thus, DNA damage and apoptosis in OC cells, thereby inhibiting OC progression. Additionally, ago-piR-26441 suppressed tumor growth and mitochondrial metabolism in the patient-derived organoid model. Altogether, piR-26441 could inhibit OC cell growth via the YTHDC1/TSFM signaling axis, underscoring its significant importance in the context of OC, as well as offering potential as a therapeutic target.

Role of HPV E7/miR-143-3p/SH2D3A pathway in regulating the occurrence and development of cervical cancer

This study aims to explore the role of SH2D3A in cervical cancer, as well as its potential interaction with human papillomavirus (HPV) E7 and microRNA (miRNA). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to compare the expressions of SH2D3A in tissues. To assess the effects of SH2D3A on cervical cancer cell phenotypes, SH2D3A was knocked down in SiHa and HeLa cells, followed by cell proliferation (Cell Counting Kit-8 assay), apoptosis (flow cytometry), and invasion (Transwell assay) analyses. A transplantation tumor model was established to compare the tumorigenic ability of cervical cancer cells before and after SH2D3A silencing. Bioinformatics analysis predicted and dual-luciferase reporter assays verified the sponge adsorption effect of SH2D3A on miRNA. Western blot and qRT-PCR analyses were conducted to examine the impact on target genes following the downregulation of HPV E7 and SH2D3A. SH2D3A expression was significantly elevated in cervical cancer tissues. SH2D3A silencing inhibited cell proliferation and invasion, induced apoptosis, and reduced tumorigenesis in nude mice. Bioinformatics tools identified a binding relationship between SH2D3A and miR-143-3p, confirmed by the luciferase reporter assays. Western blot analysis revealed that SH2D3A knockdown led to decreased levels of Janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) proteins. Additionally, qRT-PCR showed that SH2D3A mRNA levels decreased after HPV E7 silencing, whereas miR-143-3p levels significantly increased. HPV E7 influences SH2D3A expression through miR-143-3p, thereby regulating the JAK1/STAT3 pathway. This mechanism promotes the occurrence and development of cervical cancer.

CircCRIM1 promotes ovarian cancer progression by working as ceRNAs of CRIM1 and targeting miR-383-5p/ZEB2 axis

Abstract Background Ovarian cancer is the leading cause of death in patients with gynecologic cancer, and circular RNAs (circRNAs) are involved in cancer progression. However, there are limited studies on the roles of circRNAs in ovarian cancer. Methods We designed divergent and convergent primers, used sanger sequencing and RNase R digestion to verify the source of circCRIM1. We detected the expression of circCRIM1 and its parental gene cysteine rich transmembrane BMP regulator 1 (CRIM1) in ovarian cancer and normal ovarian samples via qRT-PCR. MTT viability assay, apoptosis assay, wound healing assay and invasion assay were used to investigate the function of circCRIM1 and CRIM1 in ovarian cancer cell lines OVCAR3 and CAOV3. Mice xenografts experiment was performed. Bioinformatics predicted the microRNAs that bond with circCRIM1 and CRIM1, and dual luciferase reporter system confirmed it. Rescue experiments of microRNAs mimics transfection on the basis of circCRIM1 over-expression were carried out to uncover the mechanism by which circCRIM1 played cancer-promoting roles in ovarian cancer. Results CircCRIM1 was derived from CRIM1 by back-splicing. CircCRIM1 and CRIM1 had higher expression in ovarian cancer than in normal ovarian tissues, and both of them promoted ovarian cancer progression in vitro. In vivo circCRIM1 promoted the growth of tumors. CircCRIM1 and CRIM1 had a positive correlation relationship in the same cohort of ovarian cancer tissues. Bioinformatics predicted and dual luciferase assay confirmed circCRIM1 and CRIM1 bond with miR-145-5p, and circCRIM1 bond with miR-383-5p additionally. CircCRIM1 positively affected the expression of CRIM1. After circCRIM1 was over-expressed, miR-145-5p mimics transfection reversed the expression of CRIM1. Western blot discovered circCRIM1 positively affected the expression of zinc finger E-box binding homeobox 2 (ZEB2). Rescue experiments found miR-383-5p mimics reversed ZEB2 expression and the cancer-promoting effects of circCRIM1. Conclusions CircCRIM1 bond with miR-145-5p to work as competing endogenous RNA (ceRNA) of CRIM1, and circCRIM1 bond with miR-383-5p to improve the expression of ZEB2 in ovarian cancer. CircCRIM1 and CRIM1 promoted the ovarian cancer progression and supplied a novel insight into the researches of ovarian cancer.

SNORA70E promotes the occurrence and development of ovarian cancer through pseudouridylation modification of RAP1B and alternative splicing of PARPBP

AbstractThe present study demonstrated for the first time that SNORA70E, which belongs to box H/ACA small nucleolar noncoding RNAs (snoRNAs) who could bind and induce pseudouridylation of RNAs, was significantly elevated in ovarian cancer tissues and was an unfavourable prognostic factor of ovarian cancer. The over‐expression of SNORA70E showed increased cell proliferation, invasion and migration in vitro and induced tumour growth in vivo. Further research found that SNORA70E regulates RAS‐Related Protein 1B (RAP1B) mRNA through pseudouracil modification by combing with the pyrimidine synthase Dyskerin Pseudouridine Synthase 1 (DKC1) and increase RAP1B protein level. What's more, the silencing of DKC1/RAP1B in SNORA70E overexpression cells both inhibited cell proliferation, migration and invasion through reducing β‐catenin, PI3K, AKT1, mTOR, and MMP9 protein levels. Besides, RNA‐Seq results revealed that SNORA70E regulates the alternative splicing of PARP‐1 binding protein (PARPBP), leading to the 4th exon‐skipping in PARPBP‐88, forming a new transcript PARPBP‐15, which promoted cell invasion, migration and proliferation. Finally, ASO‐mediated silencing of SNORA70E could inhibit ovarian cancer cell proliferation, invasion, migration ability in vitro and inhibit tumorigenicity in vivo. In conclusion, SNORA70E promotes the occurrence and development of ovarian cancer through pseudouridylation modification of RAP1B and alternative splicing of PARPBP. Our results demonstrated that SNORA70E may be a new diagnostic and therapeutic target for ovarian cancer.

LncRNA linc01194 promotes the progress of endometrial carcinoma by up-regulating SOX2 through binding to IGF2BP1

Endometrial carcinoma (EC) is one of the most common gynecological malignant tumors. Our study showed that long non-coding RNA (lncRNA) linc01194 plays an important role in EC. We explored the mechanism of lncRNA linc01194 in EC. The expression of lncRNA linc01194 was detected in The Cancer Genome Atlas database and starBase database. The potential targeted protein of linc01194 was predicted through the starBase database. To determine the role of linc01194 in EC, we downregulated or upregulated the level of linc01194 in EC cell lines and analyzed the cell behaviors and the changes of its potential target proteins. The expression of linc01194 in EC tissues is higher than that in normal endometrial tissues. The knockdown of linc01194 inhibited the cell proliferation, invasion and migration and promoted the apoptosis of EC cells, while overexpression of linc01194 promoted cell proliferation, invasion and migration and inhibited the apoptosis of EC cells. The starBase database revealed that linc01194 could bind to insulin-like growth factor 2 binding protein 1 (IGF2BP1). Previous results showed that in EC, IGF2BP1 could promote the expression of sex-determining region Y-box 2 (SOX2) by promoting the stability of SOX2 mRNA. Our results showed that linc01194 regulate the expression of IGF2BP1 and SOX2. Linc01194 can promote the expression of downstream protein SOX2 through binding to IGF2BP1, thus promoting the occurrence and development of EC.