Cell Biology International

OTUB2 promotes the progression of endometrial cancer by regulating the PKM2‐mediated PI3K/AKT signaling pathway

AbstractEndometrial carcinoma (EC) morbidity and mortality have been increasing in recent years. Otubain 2 (OTUB2) was shown to be upregulated in EC patients, so the aim of this study was to explore the role of OTUB2 in EC. Cell Counting Kit‐8 (CCK‐8), colony formation, enzyme‐linked immunosorbent assay, the wound healing assay, and Transwell invasion assays were used to investigate the specific role of OTUB2 in EC tumorigenesis. Real‐time polymerase chain reaction and western blot analysis were used to detect the expression of OTUB2 in EC tissues and cells. OTUB2 is upregulated in EC patients and cell lines and is associated with a poor prognosis. The overexpression of OTUB2 promoted glycolysis and induced the proliferation, migration, and invasion of endometrial cancer cells. The silencing of OTUB2 had the opposite effect. In addition, the silencing of OTUB2 significantly suppressed the expression levels of PKM2. Importantly, inhibition of the PKM2/PI3K/AKT signaling pathway significantly reversed the promoting effect of OTUB2 overexpression on EC. OTUB2 regulated the proliferation and invasion of EC cells by regulating the PKM2/PI3K/AKT signaling pathway. OTUB2 may serve as a potential prognostic and therapeutic target in EC.

FLOT1 Is a Novel Serum Biomarker of Ovarian Cancer Targeted by N6‐methyladenosine Modification Inhibition

ABSTRACTOvarian cancer (OC) is a deadly disease and lacks a precise marker for diagnosis worldwide. Our previous work has shown the overexpression of flotillin‐1 (FLOT1) in OC tissue. To improve diagnostic sensitivity and accuracy, we evaluated the serum level of FLOT1 in OC patients and found that the serum concentration of FLOT1 as well as CA125 was significantly increased in patients with OC compared with healthy control (p < 0.01) and those with benign tumors (p < 0.05). The detection rate (above the upper limit of a cut‐off value) of FLOT1 and CA125 was 77.78% and 72.22%, respectively, in patients with OC, which was increased to 88.89% in combination. The elevation of FLOT1 was confirmed in the serum of nude mice after the implantation of human OC cells. A high level of FLOT1 protein in the serum was positively correlated with the overexpression of FLOT1 protein in OC tissues. Furthermore, the level of m6A modification of FLOT1 mRNA was significantly high in OC cells compared with normal ovarian epithelial cells, leading to an increase in FLOT1 mRNA expression. Application of a methylation inhibitor, 3‐deazaadenosine, decreased FLOT1 mRNA expression in OC cells and suppressed tumor formation in a xenograft mouse model. In conclusion, the current study demonstrated that FLOT1 is a novel serum biomarker of OC and can be targeted by m6A modification inhibition. These data highlight the potential application of FLOT1 as a diagnostic marker and a therapeutic target for patients with OC.

In silico analysis of DYNLL1 expression in ovarian cancer chemoresistance

AbstractOvarian cancer (OC) is the most lethal gynecological cancer and chemoresistance is responsible for the treatment failure and unfavorable clinical outcome in this disease. The deletion of DYNLL1 was reported to result in increased chemoresistance in BRCA1‐mutant high‐grade serous ovarian carcinoma cells. Considering its role in chemoresistance, a better understanding of DYNLL1 expression is needed to develop novel strategies in the treatment of OC. In the current study, we aimed to investigate the differential expression of DYNLL1 in OC with respect to cell types, chemosensitivity profiles, certain drug treatments, and cancer progression. DYNLL1 levels were analyzed using expression profiling data sets from Gene Expression Omnibus and quantitative reverse‐transcription polymerase chain reaction in R. We found that the level of DYNLL1 was higher in OC histotypes compared with normal ovarian cells. DYNLL1 expression is decreased in OC cells of epithelial type; but, it is increased in OC cells of stromal type, compared with matched control cells. Chemoresistant OC cells were shown to have lower DYNLL1 expression than chemosensitive OC cells. Carboplatin and NSC319726 treatments resulted in slightly decreased DYNLL1 expression and DYNLL1 levels were decreased in the course of cancer progression in OC epithelial cells. The results suggest that changes in DYNLL1 expression in OC might be cell‐type dependent and lower DYNLL1 levels may be associated with increased chemoresistance in OC. Although further studies are needed, certain drugs and cancer progression may lead to lower DYNLL1 levels, possibly resulting in increased chemoresistance. Therefore, it can be stated that DYNLL1 might be an important player in OC progression and chemoresistance.

Reversing microtubule‐directed chemotherapeutic drug resistance by co‐delivering α2β1 inhibitor and paclitaxel with nanoparticles in ovarian cancer

AbstractPrevious reports indicated that integrins associated signals are tightly related to tumor progression. Here, we observed elevated expression of integrin α2β1 in tumor tissues from microtubule‐directed chemotherapeutic drugs (MDCDs) resistant patients compared with the samples from chemosensitive patients. More importantly, we sorted the integrin α2β1+ tumor cells and found those cells revealed high MDCDs resistance, whereas MDCDs shows effective cytotoxicity to those integrin α2β1− tumor cells in vitro and in vivo. Mechanistically, we demonstrated that integrin α2β1 could induce MDCDs resistance through the activation of the PI3K/AKT pathway. Applying MPEG‐PLA to co‐encapsulate the integrin α2β1 inhibitor E7820 and MDCDs could effectively reverse MDCDs resistance, resulting in enhanced anticancer effects while avoiding potential systemic toxicity in vitro and in vivo. In conclusion, the expression of integrin α2β1 contributes to MDCDs resistance, while applying E7820 combination treatment by MPEG‐PLA nanoparticles could reverse the resistance.

Overexpression of Fut 2, 4, and 8, and nuclear localization of Fut 4 in ovarian cancer cell lines induced by ascitic fluids from epithelial ovarian cancer patients

AbstractFucosyltransferases (Fut) regulate the fucosylation process associated with tumorogenesis in different cancer types. Ascitic fluid (AF) from patients diagnosed with advanced stage of epithelial ovarian cancer (EOC) is considered as a dynamic tumor microenvironment associated with poor prognosis. Previous studies from our laboratory showed increased fucosylation in SKOV‐3 and OVCAR‐3, cancer‐derived cell lines, when these cells were incubated with AFs derived from patients diagnosed with EOC. In the present work we studied three fucosyltransferases (Fut 2, Fut 4, and Fut 8) in SKOV‐3, OVCAR‐3 and CAOV‐3 cell lines in combination with five different AFs from patients diagnosed with this disease, confirming that all tested AFs increased fucosylation. Then, we demonstrate that mRNAs of these three enzymes were overexpressed in the three cell lines under treatment with AFs. SKOV‐3 showed the higher overexpression of Fut 2, Fut 4, and Fut 8 in comparison with the control condition. We further confirmed, in the SKOV‐3 cell line, by endpoint PCR, WB, and confocal microscopy, that the three enzymes were overexpressed, being Fut 4 the most overexpressed enzyme compared to Fut 2 and Fut 8. These enzymes were concentrated in vesicular structures with a homogeneous distribution pattern throughout the cytoplasm. Moreover, we found that among the three enzymes, only Fut 4 was located inside the nuclei. The nuclear location of Fut 4 was confirmed for the three cell lines. These results allow to propose Fut 2, Fut 4, and Fut 8 as potential targets for EOC treatment or as diagnostic tools for this disease.

Exosomal miR‐21‐5p derived from cisplatin‐resistant SKOV3 ovarian cancer cells promotes glycolysis and inhibits chemosensitivity of its progenitor SKOV3 cells by targeting PDHA1

AbstractOvarian cancer (OC) is a common reason for gynecologic cancer death. Standard treatments of OC consist of surgery and chemotherapy. However, chemoresistance should be considered. Exosomal miR‐21‐5p has been shown to regulate the chemosensitivity of cancer cells through regulating pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1). However, the role of miR‐21‐5p/PDHA1 in OC is unclear. The levels of miR‐21‐5p and PDHA1 in clinical samples and cells were investigated. Exosomes derived from SKOV3/cisplatin (SKOV3/DDP) cells (DDP‐Exos) were isolated and used to treat SKOV3 cells to test DDP‐Exos effects on SKOV3 cells. Extracellular acidification rate and oxygen consumption rate were tested with a Seahorse analyzer. Cell apoptosis was analyzed by a flow cytometer. PDHA1 was overexpressed and miR‐21‐5p was silenced in SKOV3 cells to study the underlying mechanism of miR‐21‐5p in OC. Quantitative real‐time PCR and immunoblots were applied to measure gene expression at mRNA and protein levels. The levels of PDHA1 in DDP‐resistant SKOV3 or tumor tissues were significantly decreased while the levels of miR‐21‐5p were remarkably upregulated. miR‐21‐5p in DDP‐Exos was sharply increased compared to that of Exos. Data also indicated that DDP‐Exos treatment suppressed the sensitivity of SKOV3 cells to DDP and promoted cell viability and glycolysis of SKOV3 cells through inhibiting PDHA1 by exosomal miR‐21‐5p. miR‐21‐5p derived from DDP‐resistant SKOV3 OC cells promotes glycolysis and inhibits chemosensitivity of its progenitor SKOV3 cells by targeting PDHA1. Our data highlights the important role of miR‐21‐5p/PDHA1 axis in OC and sheds light on new therapeutic development.

LncRNA HOTTIP inhibits cell pyroptosis by targeting miR‐148a‐3p/AKT2 axis in ovarian cancer

AbstractLong noncoding RNA HOTTIP is a crucial regulator in multiple types of cancer, including ovarian cancer (OC). However, the biological roles and underlying mechanisms of HOTTIP in OC have rarely been studied. Hence, this study aimed to investigate the functional correlation between HOTTIP and pyroptosis in OC progression. The expression of HOTTIP in OC tissues and cell lines was characterized by quantitative real‐time PCR. Cell proliferation was evaluated using Cell Counting Kit‐8 and clone formation assays. Western blot was performed to quantify protein levels. A dual‐luciferase reporter assay was used to analyze the molecular interaction among HOTTIP, miR‐148a‐3p, and AKT2. The expression of HOTTIP was significantly upregulated in OC tissue samples and cell lines. The silencing of HOTTIP led to the inhibition of cell proliferation and NLRP1 inflammasome‐mediated pyroptosis. In addition, HOTTIP increased AKT2 expression by negatively regulating miR‐148a‐3p and then inhibited ASK1/JNK signaling. Further rescue experiments revealed that downregulation of miR‐148a‐3p and overexpression of AKT2 obviously diminished the effects of HOTTIP downregulation in OC cells. Thus, our study elucidated a novel pyroptosis‐related mechanism by which HOTTIP participated in OC progression, which might provide a theoretical reference for clinical treatment.

Roles of metformin‐mediated girdin expression in metastasis of epithelial ovarian cancer

AbstractAntimetastatic effect of Metformin has been documented in epithelial ovarian cancer (EOC). Presently, we investigated the regulatory mechanism of Metformin in EOC metastasis. First, Girdin was significantly enhanced in EOC tumorous tissues and cell lines. Seconded, knockdown of Girdin significantly suppressed EOC cell viability, migration, and invasion, while upregulation of Girdin produced the opposite effects in vitro and facilitated lung metastasis in EOC cell xenograft in vivo. In addition, we confirmed that the inhibitory effect of Metformin on Girdin expression. Mechanistically, the oncogenic effects of Girdin could be reversed by LY294002 (an AKT pathway inhibitor) and Metformin. These results suggested that Metformin attenuated EOC metastasis through Girdin and targeting Girdin may be a promising therapeutic strategy for EOC in the future.

HPV16 E6/E7 stabilize PGK1 protein by reducing its poly‐ubiquitination in cervical cancer

AbstractThis study aimed to explore the expression profile, prognostic value, regulatory effect, and the underlying mechanism of dysregulation of phosphoglycerate kinase 1 (PGK1) in high‐risk human papillomavirus (HPV)‐positive cervical epithelial squamous cell carcinoma (CESC). Bioinformatic analysis was performed using the CESC subset of The Cancer Genome Atlas (TCGA)‐Cervical Cancer (CESC) and normal cervix in The Genotype‐Tissue Expression (GTEx) project. HPV‐16 positive CaSki and SiHa cells were used as in vitro cell models. Results showed that compared to the normal cervix, CESC tissues had significantly higher expression of PGK1. CESC patients with the higher 50% expression of PGK1 had substantially shorter disease‐specific survival (DSS), and progression‐free survival (PFS) compared to the cases with the lower 50% expression of PGK1. PGK1 knockdown impaired, but PGK1 overexpression enhanced the proliferation, colony formation, aerobic glycolytic activities (lactate production, intracellular ATP levels, glucose uptake, and extracellular acidification rate), migration, and invasion of CaSki and SiHa cells. HPV‐16 E6/E7 knockdown in CaSki and SiHa cells had limited influence on PGK1 transcription but significantly decreased the half‐life of PGK1 protein. E6/E7 knockdown mediated PGK1 downregulation could be blocked by adding MG‐132. PGK1 poly‐ubiquitination was significantly enhanced after E6/E7 knockdown. In conclusion, this study showed that PGK1 expression might serve as a prognostic biomarker in cervical cancer. Its upregulation contributes to enhanced aerobic glycolysis, migration, and invasion of CESC cells. HPV16 E6/E7 stabilizes PGK1 protein by reducing its poly‐ubiquitination.

HPV16 E6 promotes cell proliferation, migration, and invasion of human cervical cancer cells by elevating both EMT and stemness characteristics

AbstractIn most cases of cervical cancer, the high risk of the disease is caused by the human papilloma virus (HPV). Surgery or radiation usually benefits patients with early cervical cancer, while the metastatic one is uncurable and new therapeutic strategies and approaches are required. In this study, HPV16 E6 silence or overexpression were carried out to evaluate the possible mechanisms of HPV16 E6 function in cervical cancer cells with different HPV16 E6 expression background. HPV16 E6‐positive cervical cancer cell Siha exerts significantly stronger cell invasion and migration potentials than the HPV16 E6‐negative C33A cells. HPV16 E6 silence significantly weakened the potentials of cell invasion and migration, cell proliferation and stemness characteristic in Siha cells. Meanwhile, the overexpression of HPV16 E6 effectively promoted the cell proliferation and stemness characteristic in C33A cells. Our data also indicated a positive association between HPV16 E6 and the levels of epithelial to mesenchymal transition (EMT), and cell stemness. The ectopic expression of OCT4 could effectively reverse the inhibitory roles of HPV16 E6 silence on cell migration, invasion, and stemness in Siha cells. More interestingly, we found that HPV16 E6 might promote the OCT4 expression by impairing the direct binding of p53 on the promoter and activate its transcription. Taken together, our results indicated that HPV16 E6 could promoted the potential cell proliferation, migration, and invasion of human cervical cancer cells by modulating EMT and cell stemness. Our data provide a novel mechanism for how HPV16 E6 acts as a key risk factor for cervical cancer development and progression.

OTUD6B promotes cholangiocarcinoma growth by regulating STAT3 phosphorylation through deubiquitination of PTK2

AbstractCholangiocarcinoma (CCA) is a hepatobiliary carcinoma with uncontrolled cell proliferation, poor prognosis, and high mortality. The ovarian tumor structural domain (OTU) containing protein 6B (OTUD6B) belongs to the OTU deubiquitin family and is vital in tumor development. However, its expression and biological function in CCA remain unknown. The expression of OTUD6B in CCA was analyzed using TIMER2.0, UALCAN, and GEO databases. MTT, clonal formation assay, immunofluorescence staining, immunohistochemistry staining, and flow cytometry examined the regulation of OTUD6B on cell proliferation, cycle, and apoptosis. The effects of OTUD6B on tumor volume and weight were assessed using the xenograft tumor model. The activities of PTK2 and STAT3 were detected by western blot and CO‐IP. The biological database identified that OTUD6B was upregulated in CCA. In CCA cells, OTUD6B knockdown reduced CCA cell proliferation and promoted apoptosis. Cell cycle analysis indicated that the cycle stopped at the G0/G1 phase after OTU6B downregulation. Furthermore, OTUD6B knockdown resulted in a decrease in tumor volume and weight in xenograft tumor models. Mechanistically, OTUD6B is involved in the deubiquitination of PTK2. PTK2 further affected the phosphorylation of STAT3 thereby regulating the CCA process. Our study demonstrates that OTUD6B knockdown participates in the ubiquitination of PTK2 and phosphorylation of STAT3 to alleviate the process of CCA. These results suggest that OTUD6B may be a potential new strategy for CCA treatment.

Development of a method to diagnose endometrial cancer based on the AuNRs‐AntiVimentin optical probe

AbstractEndometrial cancer is one of the common gynecological malignancies, and its incidence has been increasing year by year in recent years, raising higher requirements for its rapid diagnosis. In this article, gold nanorods (AuNRs) with localized surface plasmon resonance properties (LSPR) has used to prepare AuNRs‐antibody to waveform protein (AuNRs‐AntiVimentin) optical probes, and a new method has been constructed that could rapidly detect and identify endometrial cancer tissue sections by polarized light microscopy. AuNRs were prepared by seed growth method using gold chloride as raw material, and the morphology of AuNRs and the optical characteristics of AuNRs‐AntiVimentin has characterized by transmission electron microscopy (TEM), ultraviolet–visible spectroscopy (UV–Vis), and zeta potential; immunohistochemistry (IHC) and AuNRs‐AntiVimentin optical probes have used to detect clinical endometrial cancer, respectively. The AuNRs‐AntiVimentin optical probe has been used to detect endometrial cancer tissue sections and found to have good bio‐specificity, with no significant difference in the detection of AuNRs‐AntiVimentin compared with conventional IHC techniques (p > .05). An optical probe generated by coupling AuNRs with Vimentin antibodies has been obtained to detect and identify endometrial cancer with simple operation and comparable effect to conventional IHC, providing a new method and idea for the rapid detection of endometrial cancer.

High MutS homolog 2 expression predicts poor prognosis and is related to immune infiltration in endometrial carcinoma

AbstractSeveral studies have shown that MutS homolog 2 (MSH2) is highly expressed in many cancer tissues. Transcriptome expression data were collected from the Cancer Genome Atlas (TCGA) database. We analyzed the expression of MSH2 in normal and tumor tissues, the relationship between MSH2 expression and various prognostic factors, and the relationship between MSH2 expression and overall survival, disease specific survival, and progression free interval. We also examined MSH2 promoter methylation between endometrial cancer and normal endometrial tissues, and identified the prognostic value of MSH2 methylation in endometrial cancer. MSH2 was highly expressed in endometrial cancer tumor tissues compared with normal tissues. High MSH2 expression might be an independent prognostic factor for OS, DSS, and PFI. Further, high MSH2 expression was correlated with age and histological type, but not with BMI, clinical stage, tumor invasion, or other clinical features. MSH2 promoter methylation in endometrial cancer was significantly lower than in normal tissues. Additionally, MSH2 levels, OS, DSS, and PFI were associated with BMI, age, tumor invasion, and histological type. ssGSEA showed that MSH2 expression was positively correlated with the infiltration of Th2 cells, Tcm cells, T helper cells, and Tgd cells, whereas it was negatively correlated with NK CD56 bright cells, pDC cells, iDC cells, cytotoxic cells, and neutrophils. Increased MSH2 expression and reduced MSH2 methylation in endometrial cancer predicts poor prognosis. MSH2 may be used as a biomarker for the diagnosis and prognosis of endometrial cancer and as an immunotherapy target.

Sirtuin 1 promotes autophagy and proliferation of endometrial cancer cells by reducing acetylation level of LC3

AbstractEndometrial cancer (EC) constitutes a common female genital tract tumor with a rising incidence rate. Sirtuin 1 (SIRT1) is a member of histone deacetylase, which extensively participates in the progression of aging, cell death, and tumorigenesis. This study explored the effect of SIRT1‐mediated LC3 acetylation on autophagy and proliferation of EC cells. SIRT1 expression in EC tissues and adjacent tissues, EC cell lines and normal human epithelial cells was detected. SIRT1 expression was elevated in EC cell lines and tissues. Knockdown of SIRT1 inhibited proliferation, migration, and invasion of EC cells. Then, EC cells were starved in serum‐free medium, and levels of autophagy‐related proteins were detected. Starvation induced autophagy of EC cells. The starvation‐treated EC cells showed an increased SIRT1 expression, a decreased LC3 acetylation level and an increased autophagy level. The proliferation and autophagy of EC cells under different treatments were evaluated. In EC cells transfected with overexpressing SIRT1, LC3 acetylation was inhibited and cell proliferation was promoted. Moreover, overexpressing SIRT1 facilitated growth and autophagy of transplanted tumors in nude mice. In conclusion, SIRT1 promoted autophagy and proliferation of EC cells by reducing acetylation level of LC3.

Knockdown of YKL‐40 inhibits angiogenesis through regulation of VEGF/VEGFR2 and ERK1/2 signaling in endometrial cancer

AbstractStudies have demonstrated that small interfering RNA (siRNA) targeting YKL‐40 (siYKL‐40) inhibits the proliferation, migration, invasion, and induces antiapoptotic abilities of endometrial cancer (EC) HEC‐1A cells. However, its effect on angiogenesis is unclear. The present study aimed to investigate the role of YKL‐40 in endometrial cancer and the related molecular mechanisms. YKL‐40 was knocked down by transfection with siYKL‐40 and the effects on angiogenesis, cell viability, and signaling pathways were investigated. The results showed that siYKL‐40 inhibited VEGFA levels and tube formation in endothelial cells. Additionally, inhibition of YKL‐40 decreased the expression levels of vascular endothelial growth factor (VEGF), phosphorylated vascular endothelial growth factor receptor 2 (pVEGFR2), and phosphorylated extracellular signal‐regulated kinases 1 and 2 (pERK1/2). Furthermore, a nude mice xenograft model of EC showed that siYKL‐40 inhibited tumor growth. Inhibition of YKL‐40 led to suppression of angiogenesis and reduction of microvessel density through VEGF/VEGFR2 and ERK1/2 signaling in endometrial cancer cells. Taken together, this study demonstrated novel molecular mechanisms for role of YKL‐40 in EC.

WTAP facilitates progression of endometrial cancer via CAV‐1/NF‐κB axis

AbstractThe N6‐methyladenosine (m6A) modification is one of the most prevalent methylations in eukaryotic messenger RNA (mRNA), and it is essential for the development of many important biological processes such as multiple types of tumors. One of the most important enzymes catalyzing generation of m6A on mRNA is Wilms' tumor 1‐associating protein (WTAP); however, the potential role of WTAP in endometrial cancer (EC) still remains unknown. Here, we investigated WTAP expression level in cancer tissue and paracancerous tissue from an EC patient. Subsequently, WTAP was knocked down by small interfering RNA in EC cell line of Ishikawa and HEC‐1A, respectively. Cell proliferation, migration, and invasion were studied. The expression of caveolin‐1 (CAV‐1) was detected by quantitative polymerase chain reaction (qPCR). The enrichments of m6A and METTL3 on CAV‐1 were detected using RNA immunoprecipitation‐qPCR. The activity of nuclear factor‐κB (NF‐κB) was studied using Western blot. We observed that WTAP was dramatically upregulated in the cancer tissue, and there was an enhancement in cell proliferation, migration, and invasion and a decrease in EC apoptosis in vivo and in vitro, which indicated higher tumor malignancy and worse survival outcome. After WTAP was knocked down in EC cells, CAV‐1 was significantly upregulated and the enrichments of m6A and METTL3 at 3′‐untranslated region (UTR) region of CAV‐1 were decreased. Moreover, the activity of NF‐κB signaling pathway was inhibited by its regulator CAV‐1. Taken together, we concluded that WTAP could methylate 3′‐UTR of CAV‐1 and downregulate CAV‐1 expression to activate NF‐κB signaling pathway in EC, which promoted EC progression.

METTL3 regulates m6A in endometrioid epithelial ovarian cancer independently of METTl14 and WTAP

AbstractN6‐methyladenosine (m6A) RNA methylation, one of the common RNA modifications, has been determined to execute crucial functions in tumorigenesis and cancer development. The m6A “writers” including methyltransferase like 3 (METTL3), METTL14, and Wilms tumor 1‐associated protein (WTAP) contribute to the m6A modification process initiation. However, the coordination of m6A methyltransferase complex is not fully understood in endometrioid epithelial ovarian cancer (EEOC). In this study, mRNA and protein levels of METTL3, METTL14, and WTAP were detected in 33 EEOC cases using quantitative polymerase chain reaction (qPCR), immunohistochemistry, and western blot analysis. The overall m6A methylation was detected by dot plot. The METTL3 expression and overall m6A level were elevated in EEOC tissues, while the expressions of METTL14 and WTAP have no significant difference in EEOC compared to the adjacent tissues. The expression of METTL3 was an independent factor that correlated with poor malignancy and survival of EEOC patients. Moreover, METTL3 knockdown in TOV‐112D and CRL‐11731D cells weakened the capability of cell proliferation and migration, and promoted cell apoptosis compared to negative control and cells with WTAP or METTL14 knockdown using CCK‐8 assay, transwell assay, wound healing assay, and TUNEL assay. Furthermore, METTL3 knockdown also reduced m6A enrichment of the genes associated with ovarian cancer including EIF3C, AXL, CSF‐1, FZD10 in TOV‐112D, and CRL‐11731D cells by RIP‐qPCR assay. Taken together, the high expressed METTL3 indicated poor malignancy and survival of EEOC via modulating the aberrant m6A RNA methylation. METTL3‐mediated m6A modification, independent of WTAP and METTL14, was considered as a novel mechanism underlying m6A modulation and a potential therapeutic target of EEOC.

COMMD1 Inhibits Epithelial Mesenchymal Transition (EMT) and Liver Metastasis in Cervical Cancer Through Modulation of the Twist1/E‐Cadherin Pathway

ABSTRACTOur investigation was aimed at deciphering the potential role of copper metabolism MURR1 domain containing 1 (COMMD1) in cervical cancer tumorigenesis and metastasis, along with its underlying molecular mechanism, both in vitro and in vivo. To validate the research objectives, cervical cancer cell lines with stably overexpressed and knockdown COMMD1 were generated. In addition, an orthotopic murine model of cervical cancer with liver metastasis was constructed to elucidate the metastatic impact of COMMD1. Functional assays including CCK‐8 assay, colony formation assay, scratch assay, and transwell invasion assay were conducted to evaluate the proliferation, migration, and invasion capabilities of cervical cancer cells. Western blot analysis and immunofluorescence double staining were performed to detect protein expression profiles and visualize actin cytoskeleton remodeling. Hematoxylin‐eosin (H&E) staining and immunohistochemistry were utilized to characterize tumor histopathology and protein expression. Key findings revealed that COMMD1 was markedly downregulated in cervical cancer cell lines. Ectopic expression of COMMD1 potently impeded cell proliferation, colony formation, migration, and invasion of cervical cancer cells. Mechanistically, COMMD1, suppressed epithelial–mesenchymal transformation (EMT) by antagonizing the twist family bHLH transcription factor 1 (Twist1)/epithelial (E)‐cadherin pathway, as evidenced by reduced expression of EMT‐associated markers and restored E‐cadherin membrane localization. In the orthotopic mouse model, COMMD1 overexpression significantly attenuated liver metastatic foci formation and blunted EMT progression. In conclusion, COMMD1 acts as a tumor suppressor in cervical cancer, with its antitumor effects primarily mediated by inhibiting Twist1‐driven EMT and metastatic cascade.

LRP8 promotes tumorigenesis in ovarian cancer through inhibiting p53 signaling

AbstractOvarian cancer (OC) is the most lethal gynecological malignancy with a high mortality rate. Low‐density lipoprotein (LDL) receptor‐related protein 8 (LRP8) is a cell membrane receptor belonging LDL receptor family and is involved in several tumor progressions. However, there is limited understanding of how LRP8 mediates OC development. LRP8 expression level was identified in human OC tissues and cells using immunohistochemical staining and quantitative polymerase chain reaction assays, respectively. Functions of LRP8 in OC progression were evaluated by Celigo cell counting, wound healing, transwell and flow cytometry assays, and the xenograft models. The human phospho‐kinase array analysis was used for screening potential signaling involved in OC development. We observed that LRP8 was overexpressed in OC tissues, and high expression of LRP8 was associated with poor prognosis of OC patients. Functionally, LRP8 knockdown remarkably reduced proliferation and migration of OC cells, and induced apoptosis and S phase cycle arrest. LRP8 deficiency attenuated in vivo tumor growth of OC cells. Moreover, the addition of p53 inhibitor partially reversed the effects of LRP8 knockdown on OC cell proliferation and apoptosis, indicating the involvement of p53 signaling in LRP8‐mediated OC progression. This study confirmed that LRP8/p53 axis contributed to OC progression, which might serve as a novel potential therapeutic target for OC patients.

Anti‐oncogenic PTEN induces ovarian cancer cell senescence by targeting P21

AbstractDeletion and mutation of phosphatase and tensin homolog deleted on chromosome10 (PTEN) are closely associated with the occurrence of tumors. Tumor suppressor gene PTEN mutation plays an important role in the pathogenesis of ovarian cancer. However, it has been unclear whether it can regulate the senescence of ovarian cancer cells. We speculated that PTEN might inhibit the occurrence and development of ovarian cancer by promoting the expression of P21. We found that the expression of TRIM39 in human ovarian cancer was significantly diminished. In SKOV3 cells treated with naringin, the expression of TRIM39, which binds P21 and inhibits P21 degradation, was significantly elevated. Real‐time polymerase chain reaction (PCR), Western blot, and immunofluorescence were used to detected the expression of PTEN, p21, and TRIM39, β‐galactosidase Staining was used to detect cell senescence, Ki67 staining was used to observe cell proliferation, Trim39 interference or overexpression assay was used to detect its function. We speculated that PTEN might promote SKOV3 cell senescence by increasing TRIM39 expression and decreasing P21 degradation. Furthermore, by interfering with TRIM39 in SKOV3 cells, we found that the expression of P21 was downregulated, and the number of senescent SKOV3 cells decreased. With overexpression of TRIM39 in SKOV3 cells, the expression of P21 was upregulated, and the number of senescent SKOV3 cells increased. When naringin, a PTEN agonist, was added to SKOV3 cells in which TRIM39 protein was interfered with, the expression of P21 was significantly lower than that in the control group, and the number of senescent ovarian cancer cells was significantly diminished. Our results indicated that PTEN maintained the stability of P21 and decreased the degradation of P21 by increasing TRIM39 expression, thus promoting the senescence of SKOV3 cells, and PTEN maintained the stability of p21 and promoted the aging of SKOV3 cells might be a novel therapeutic target for ovarian cancer.

Astragaloside II enhanced sensitivity of ovarian cancer cells to cisplatin via triggering apoptosis and autophagy

AbstractCisplatin (DDP) based chemotherapy occurs a reduced therapeutic effect on the later treatment of ovarian cancer (OC) due to DDP resistance. Astragaloside II (ASII), a natural product extracted from Radix Astragali, has shown promising anticancer effects. However, the effects of ASII on OC have not been clarified. In this study, we found that ASII inhibited cell growth and promoted cell apoptosis of DDP‐resistant OC cells in vitro and in vivo. Further study showed that ASII downregulated multidrug resistance‐related protein MDR1 and cell cycle‐related protein Cyclin D1 and PCNA, and also upregulated apoptosis‐related protein leaved PRAP and cleaved caspase‐3. In addition, ASII induced autophagy, characterized by upregulation of LC3II expression, downregulation of p62 expression, and elevation of LC3 punctuation, may be associated with inhibition of the AKT/mTOR signaling pathway. Moreover, the messenger RNA‐sequencing was used to identify potential molecules regulated by ASII. In conclusion, these findings indicated that ASII increased sensitivity of DDP in the treatment of OC.

UBP43 promotes epithelial ovarian carcinogenesis via activation of β‐catenin signaling pathway

AbstractDysregulation of the deubiquitinating protease, UBP43, has been implicated in many human diseases, including cancer. Here, we evaluated the functional significance and mechanism of action of UBP43 in epithelial ovarian cancer. We found that UBP43 was significantly upregulated in the tumor tissues of patients with epithelial ovarian cancer. Similar results were observed in OVCAR‐3, Caov‐3, TOV‐112D, A2780, and SK‐OV‐3 cells. Furthermore, in vitro functional assays of A2780 and TOV‐112D cells demonstrated that UBP43 overexpression promoted cell proliferation, migration, and invasion. Upregulation of UBP43 might result in epithelial–mesenchymal transition by inducing the nuclear transport of β‐catenin, which was accompanied by enhanced N‐cadherin but decreased E‐cadherin expression. These malignant phenotypes were reversed by UBP43 silencing. Further investigation revealed that the knockdown of UBP43 inhibited cell proliferation by inducing a cell cycle arrest at the G2/M phase. The oncogenic characteristics of UBP43 were validated in a subcutaneous xenograft mouse model. In vivo, tumor growth was delayed in the UBP43‐silenced group but accelerated after UBP43 overexpression. Finally, we demonstrated that β‐catenin is a key protein in the UBP43‐mediated malignant development of epithelial ovarian cancer. Specifically, overexpression of UBP43 decreased the ubiquitination degradation of β‐catenin and enhanced its protein stability. Also, we observed that the downstream genes of beta‐catenin such as cyclin D1, MMP2, and MMP9 were upregulated due to UBP43 overexpression. Thus, we concluded that UBP43 promoted epithelial ovarian cancer tumorigenesis and metastasis through activation of the β‐catenin pathway, suggesting that UBP43 may be a potential therapeutic target for this intractable disease.

Matrix metalloproteinase‐2 delivery to extracellular vesicles produced by HPV‐positive cervical cancer cells enhances metastasis via the Akt pathway

AbstractHere, we aimed to analyze the effects of matrix metalloproteinase‐2 (MMP‐2) delivery to extracellular vesicles (EVs) secreted by human papillomavirus (HPV)‐associated cervical cancer cells on human umbilical vein endothelial cell (HUVEC) angiogenesis. First, MMP‐2 expression was compared among SiHa (HPV16), HeLa (HPV18), and C‐33A (negative) cells. Then, EVs were isolated from these cells, and MMP‐2 expression in the EVs was compared. SiHa and HeLa cells were transfected with MMP‐2 or control siRNA. HUVECs were treated with EVs isolating from transfected cells. Migration and angiogenesis of HUVECs were measured, and p‐Akt protein expression in HUVECs was detected. An Akt inhibitor or activator was used to analyze the effect of MMP‐2 delivery to EVs on the migration of HUVECs. The SiHa‐induced xenograft tumors were treated with 2 µg of EVs every 3 d for a total of 27 d. Tumor growth, and the expression levels of p‐Akt, MMP‐2, and vascular endothelial growth factor (VEGF) were observed in the tumors. The results showed that MMP‐2 expression was higher in SiHa‐ and HeLa‐derived EVs than that in the C‐33A‐derived EVs. Interference with MMP‐2 suppressed the invasion of SiHa and HeLa cells. The migration and angiogenesis of HUVECs were enhanced by MMP‐2 delivery to EVs secreted by SiHa and HeLa cells through regulation of the Akt pathway. The growth of xenograft tumors was accelerated by EVs secreted by SiHa cell with differential MMP‐2 expression. Our results indicate the delivered MMP‐2 in EVs acts as a messenger between HPV‐associated cancer cells and HUVECs.

HPV E6 promotes cell proliferation of cervical cancer cell by accelerating accumulation of RBM15 dependently of autophagy inhibition

AbstractThe mechanism of m6A modification in HPV‐related cervical cancer remains unclear. This study explored the role of methyltransferase components in HPV‐related cervical cancer and the mechanism. The levels of methyltransferase components and autophagy, ubiquitylation of RBM15 protein and the co‐localization of lysosomal markers LAMP2A and RBM15 were measured. CCK‐8 assay, flow cytometry, clone formation experiment and immunofluorescence assay were conducted to measure cell proliferation. The mouse tumor model was developed to study the cell growth in vivo. The binding of RBM15 to c‐myc mRNA and m6A modifcation of c‐myc mRNA were analyzed. The expressions of METTL3, RBM15 and WTAP were higher in HPV‐positive cervical cancer cell lines than those in HPV‐negative cells, especially RBM15. HPV‐E6 knock‐down inhibited the expression of RBM15 protein and promoted its degradation, but couldn't change its mRNA level. Autophagy inhibitor and proteasome inhibitor could reverse those effects. HPV‐E6 siRNA could not enhance ubiquitylation modification of RBM15, but could enhance autophagy and the co‐localization of RBM15 and LAMP2A. RBM15 overexpression could enhance cell proliferation, block the inhibitory effects of HPV‐E6 siRNA on cell growth, and these effects could be reserved by cycloeucine. RBM15 could bind to c‐myc mRNA, resulting in an increase to m6A level and protein expression of c‐myc, which could be blocked by cycloeucine. HPV‐E6 can downregulate autophagy, inhibit the degradation of RBM15 protein, induce the accumulation of intracellular RBM15, and increase the m6A modification on c‐myc mRNA, resulting in an increase of c‐myc protein and a growth promotion for cervical cancer cells.

The negative feedback between miR‐143 and DNMT3A regulates cisplatin resistance in ovarian cancer

AbstractEmerging evidence suggests that miR‐143 plays an important role in the regulation of tumor sensitivity to chemotherapeutic agents. The study explores the underlying mechanism of miR‐143 in reversing cisplatin resistance in ovarian cancer. The cisplatin‐resistant ovarian cancer cell line A2780/CDDP was induced and established via treating A2780 cells by gradually increasing cisplatin concentrations. The IC50 values of A2780/CDDP and A2780 to cisplatin were 218.10 ± 1.12 and 21.99 ± 1.12 μM, respectively. Quantitative real‐time polymerase chain reaction (qRT‐PCR) results showed that miR‐143 was significantly decreased in A2780/CDDP cells compared with A2780 cells. miR‐143 overexpression decreased cisplatin resistance in A2780/CDDP, and miR‐143 inhibition decreased A2780 sensitivity to cisplatin. Results of qRT‐PCR, Western blot analysis, and luciferase reporter assay indicated that the direct target of miR‐143 was DNMT3A, which, in turn, was upregulated in A2780/CDDP. DNMT3A overexpression antagonized the sensitizing effect of miR‐143 on A2780/CDDP to cisplatin. Knocking down of DNMT3A reduced cisplatin resistance in A2780/CDDP, while overexpression of DNMT3A increased cisplatin resistance in A2780. Methylation‐specific polymerase chain reaction results showed that the methylation level in the promoter region of the miR‐143 precursor gene was higher in A2780/CDDP cells than in A2780 cells. DNMT3A mediated the hypermethylation of the miR‐143 precursor gene, resulting in miR‐143 downregulation in A2780/CDDP. miR‐143 inhibited cell growth of A2780/CDDP cell in nude mice. Our findings indicated the negative feedback between miR‐143 and DNMT3A as a crucial epigenetic modifier of cisplatin resistance in ovarian cancer.

DAP1 negatively regulates autophagy induced by cardamonin in SKOV3 cells

AbstractAutophagy is closely related to the formation and development of multiple human tumors including ovarian cancer. As a major regulator of this process, the role of mTOR (mammalian target of rapamycin) has been well proven. Cardamonin, a kind of flavonoid from plants, has effects on induction of autophagy and thus antiproliferation of cancer cells. However, the detailed mechanism remains unclear. DAP1 (death‐associated protein 1) is a proline‐rich protein, which is involved in the regulation of cellular growth and programmed cell death including autophagy and apoptosis. The aim of this study was to investigate whether DAP1 is involved in proliferation inhibition and autophagy induced by cardamonin in tumor cells. Using online bioinformatics tools, we found that DAP1 expression is closely related to the survival of patients with ovarian cancer. Our study showed that autophagy induced by cardamonin was associated with mTOR inhibition, and DAP1 was involved in this process. Silence of DAP1 decreased cell proliferation but enhanced the antiproliferative effect of cardamonin in SKOV3 cells. The level of autophagy was elevated by DAP1 silencing in SKOV3 cells. Notably, cardamonin showed higher autophagy flux in the DAP1 small interfering RNA group. Taken together, our results implied that DAP1 negatively regulates autophagy induced by cardamonin, and it may be a potential target for ovarian cancer therapy.

Antineoplastic drug‐loaded polymer‐modified magnetite nanoparticles: Comparative analysis of EPR‐mediated drug delivery

AbstractThis study aimed to design and evaluate enhanced permeation and retention (EPR)‐mediated anticancer effect of polymer‐modified and drug‐loaded magnetite nanocomposites. The preformulated bare (10 nm), chitosan‐superparamagnetic iron oxide (SPIO; 69 nm), heparin‐SPIO (42 nm), and (3‐aminopropyl)triethoxysilane‐polyethylene glycol‐SPIO (17 nm) nanocomposites were utilized to evaluate the EPR‐mediated localized cancer targeting and retention of doxorubicin (DOX) and paclitaxel (PTX) in human ovarian cancer cell lines, A2780 and OVCAR‐3 in vitro and in the tumor‐baring Balb/c mice in vivo. Fluorescence microscopy showed that DOX‐ and PTX‐loaded SPIO nanoparticles caused long‐term accumulation and cytoplasmic retention in A2780 and OVCAR‐3 cells, as compared to free drugs in vitro. In vivo antiproliferative effect of present formulations on immunodeficient female Balb/c mice showed a tremendous amount of ovarian tumor shrinkage within 6 weeks. The present nanocomposite systems of targeted drug delivery proved to be efficient drug carrier with sustained drug release and long‐term retention with enhanced cytotoxic properties in vitro and in vivo.

UNC5B‐AS1 promoted ovarian cancer progression by regulating the H3K27me on NDRG2 via EZH2

AbstractThe role of long non‐coding RNAs (lncRNAs) in tumorigenesis and development of ovarian cancer (OC) has caught the attention of scientists. UNC5B antisense RNA 1 (UNC5B‐AS1) is a newly identified carcinogenic lncRNA in thyroid papillary carcinoma, but its role in OC remains unclear. This study is proposed to investigate the function and mechanism of UNC5B‐AS1 in OC. UNC5B‐AS1 expression in OC samples was obtained from gene expression profiling interactive analysis (GEPIA) based on The Cancer Genome Atlas data. Gene expressions were detected by quantitative real‐time polymerase chain reaction (RT‐qPCR) and western blot. Biological functions of UNC5B‐AS1 were assessed by cell counting kit‐8, colony formation, and caspase‐3 analysis. GEPIA revealed the UNC5B‐AS1 upregulation in OC samples. RT‐qPCR assay confirmed the upregulation of UNC5B‐AS1 in OC cells. Functionally, depletion of UCN5B‐AS1 hindered proliferation and prompted apoptosis in OC cells. Mechanistically, we found that UNC5B‐AS1 interacted with zeste 2 polycomb repressive complex 2 subunit (EZH2) to trigger trimethylation of histone H3 at lysine 27 (H3K27me3) on N‐myc downstream regulated gene‐2 (NDRG2) promoter and epigenetically repressed NDRG2. Rescue assay indicated the participation of NDRG2 in the regulation of UNC5B‐AS1 on OC progression. Together, we first illustrated that UNC5B‐AS1 promoted OC progression by regulating the H3K27me on NDRG2 via EZH2, indicating UNC5B‐AS1 as a potential molecular target for OC treatment.

Long noncoding RNA SNHG16 promotes the tumorigenicity of cervical cancer cells by recruiting transcriptional factor SPI1 to upregulate PARP9

AbstractLong noncoding RNA (lncRNA) small nucleolar RNA host gene 16 (SNHG16) has been linked to multiple cancers including breast, ovarian, bladder, and colorectal cancer. However, the role of SNHG16 in cervical cancer is unclear. Here, quantitative analysis of SNHG16 and PARP9 expression levels in cervical cancer tissues and cell lines indicated that both SNHG16 and PARP9 were highly expressed compared with controls. Using the dual‐luciferase reporter gene assay, RNA immunoprecipitation, chromatin immunoprecipitation, we were able to determine that SNHG16 recruited SPI1 protein to promote transcription of PARP9 to upregulate its transcription in cervical cancer cells. After ectopic expression and knockdown experiments were conducted, it was observed that silencing SNHG16 inhibited PARP9 expression, proliferation, and invasion of cervical cancer cells, which was rescued by co‐transfection of SNHG16 silencing and PARP9 overexpression. Moreover, in vivo experimental results showed that silencing SNHG16 reduced the expression of PARP9 and suppressed tumor growth. These data indicate that SNHG16 recruits SPI1 to upregulate PARP9, which promotes the tumorigenicity of cervical cancer cells. The regulation of their expression might provide a new direction for treating cervical cancer.