Target-Triggered Aggregation of Modified E. coli for Diagnosis of Ovarian Cancer.

Bacteria inherently possess the capability of quorum sensing in response to the environment. In this work, we have proposed a strategy to confer bacteria with the ability to recognize targets with quorum-sensing behavior. Meanwhile, we have successfully achieved artificial control over the target-triggered aggregation of Escherichia coli (E. coli) by modifying the bacteria surface in a new way. Furthermore, by making use of green fluorescent protein (GFP) expressed by E. coli as the output signal, the aggregation of modified E. coli can be observed with the naked eye. Therefore, via the detection of the target, MUC1, an ovarian cancer biomarker, a simple and conveniently operated method to diagnose ovarian cancer is developed in this work. Experimental results show that the developed low-background and enzyme-free amplification method enables the highly sensitive detection of MUC1, achieving a remarkable limit of detection (LOD) of 5.47 fM and a linear detection range spanning from 1 pM to 50 nM and 50 nM to 100 nM, respectively. Clinical samples from healthy donors and patients can give distant assay results, showing great potential for clinical applications of this method.

Asparagine: A key metabolic junction in targeted tumor therapy.

Nutrient bioavailability in the tumor microenvironment plays a pivotal role in tumor proliferation and metastasis. Among these nutrients, glutamine is a key substance that promotes tumor growth and proliferation, and its downstream metabolite asparagine is also crucial in tumors. Studies have shown that when glutamine is exhausted, tumor cells can rely on asparagine to sustain their growth. Given the reliance of tumor cell proliferation on asparagine, restricting its bioavailability has emerged as promising strategy in cancer treatment. For instance, the use of asparaginase, an enzyme that depletes asparagine, has been one of the key chemotherapies for acute lymphoblastic leukemia (ALL). However, tumor cells can adapt to asparagine restriction, leading to reduced chemotherapy efficacy, and the mechanisms by which different genetically altered tumors are sensitized or adapted to asparagine restriction vary. We review the sources of asparagine and explore how limiting its bioavailability impacts the progression of specific genetically altered tumors. It is hoped that by targeting the signaling pathways involved in tumor adaptation to asparagine restriction and certain factors within these pathways, the issue of drug resistance can be addressed. Importantly, these strategies offer precise therapeutic approaches for genetically altered cancers.

Tripartite-motif 3 represses ovarian cancer progression by downregulating lactate dehydrogenase A and inhibiting AKT signaling.

The E3 ubiquitin ligase Tripartite-motif 3 (TRIM3) is known to play a crucial role in tumor suppression in various tumors through different mechanisms. However, its function and mechanism in ovarian cancer have yet to be elucidated. Our study aims to investigate the expression of TRIM3 in ovarian cancer and evaluate its role in the development of the disease. Our findings revealed a significant decrease in TRIM3 mRNA and protein levels in ovarian cancer tissues and cells when compared to normal ovarian epithelial tissues and cells. Furthermore, we observed a negative correlation between the protein level of TRIM3 and the FIGO stage, as well as a positive correlation with the survival of ovarian cancer patients. Using gain and loss of function experiments, we demonstrated that TRIM3 can inhibit cell proliferation, migration and invasion of the ovarian cancer cells in vitro, as well as suppress tumor growth in vivo. Mechanistic studies showed that TRIM3 interacts with lactate dehydrogenase A, a key enzyme in the glycolytic pathway, through its B-box and coiled-coil domains and induces its ubiquitination and proteasomal degradation, leading to the inhibition of glycolytic ability in ovarian cancer cells. RNA-sequencing analysis revealed significant alterations in the phosphatidylinositol signaling pathways upon TRIM3 overexpression. Additionally, overexpression of TRIM3 inhibited the phosphorylation of AKT. In conclusion, our study demonstrated that TRIM3 exerts a tumor-suppressive effect in ovarian cancer, at least partially, by downregulating LDHA and inhibiting the AKT signaling pathway, and thus leading to the inhibition of glycolysis and limiting the growth of ovarian cancer cells.

An electrochemical biosensor designed with entropy-driven autocatalytic DNA circuits for sensitive detection of ovarian cancer-derived exosomes.

Intelligent artificial DNA circuits have emerged as a promising approach for modulating signaling pathways and signal transduction through rational design, which may contribute to comprehensively realizing biomolecular sensing of organisms. In this work, we have fabricated an electrochemical biosensor for the sensitive and accurate detection of ovarian cancer-derived exosomes by constructing an entropy-driven autocatalytic DNA circuit (EADC). Specifically, the robust EADC is prepared by the self-assembly of well-designed DNA probes, and upon stimulation of the presence of ovarian cancer cells-derived exosomes, numerous inputs can be produced to feedback and accelerate the reaction. The catalytic abilities of the generated input sequences play a pivotal role in EADC and dramatically enhance the signal amplification capability. Through the combination of the autocatalytic circuit and circular cleavage reactions, significantly changed electrochemical signals can be recorded for sensitive analysis of the exosomes with a remarkably low detection limit of 30 particles/µL. Moreover, the proposed enzyme-free biosensor shows exceptional performance in distinguishing patient samples from healthy samples, which exhibits promising prospects for the clinical diagnosis of ovarian cancer.

Engineering extracellular vesicles mimetics for targeted chemotherapy of drug-resistant ovary cancer.

Aim: To develop nanocarriers for targeting the delivery of chemotherapeutics to overcome multidrug-resistant ovarian cancer. Materials & methods: Doxorubicin-loaded nanovesicles were obtained through serial extrusion, followed by loading of P-glycoprotein siRNA and folic acid. The targeting ability and anticancer efficacy of the nanovesicles were evaluated. Results: The doxorubicin-loaded nanovesicles showed a high production yield. The presence of P-glycoprotein siRNA and folic acid resulted in reversed drug resistance and tumor targeting. This nanoplatform tremendously inhibited the viability of multidrug-resistant ovarian cancer cells, which was able to target tumor tissue and suppress tumor growth without adverse effects. Conclusion: These bioengineered nanovesicles could serve as novel extracellular vesicles mimetics for chemotherapeutics delivery to overcome multidrug resistance.

When treating cancer affecting the ovaries, which is an organ in the female reproductive system, two challenges that arise are the inefficient delivery of chemotherapeutic drugs and the development of drug resistance inside the tumor. In this study, very small nano-scale particles called nanovesicles, which contain a chemotherapeutic drug called doxorubicin, were developed in an attempt to overcome both of these concerns. These nanovesicles were secreted by a healthy cell from an ovary, isolated and loaded with doxorubicin. These nanovesicles were also loaded with siRNA, which, in this case, prevents the synthesis of a protein in ovarian tumor cells called P-glycoprotein. This protein is responsible for pumping chemotherapy drugs back out of tumor cells, so preventing its synthesis was intended to counter chemotherapeutic resistance. The targeting ability of the nanovesicle was also enhanced with folic acid, as folic acid receptors are present on the surface of these tumor cells in higher numbers. These nanovesicles were readily and specifically taken up by ovarian tumor cells in mice with induced ovarian cancer. This reversed drug resistance and enhanced the toxic effects of doxorubicin on the tumor cells, which, in turn, increased tumor cell death and prevented tumor cell migration. No obvious adverse effect was found in mice treated with the nanovesicle system compared with the free chemotherapy drug with critical systematic toxicity. This research provides new avenues for ovarian cancer treatment, with combined therapies of siRNAs and chemotherapeutic drugs, targeted to tumor cells specifically, within nanovesicles.

LncRNA IDH1-AS1 sponges miR-518c-5p to suppress proliferation of epithelial ovarian cancer cell by targeting RMB47.

Long noncoding RNA (lncRNA) IDH1 antisense RNA 1 ( IDH1-AS1) is involved in the progression of multiple cancers, but its role in epithelial ovarian cancer (EOC) is unknown. Therefore, we investigated the expression levels of IDH1-AS1 in EOC cells and normal ovarian epithelial cells by quantitative real-time PCR (qPCR). We first evaluated the effects of IDH1-AS1 on the proliferation, migration, and invasion of EOC cells through cell counting kit-8, colony formation, EdU, transwell, wound-healing, and xenograft assays. We then explored the downstream targets of IDH1-AS1 and verified the results by a dual-luciferase reporter, qPCR, rescue experiments, and Western blotting. We found that the expression levels of IDH1-AS1 were lower in EOC cells than in normal ovarian epithelial cells. High IDH1-AS1 expression of EOC patients from the Gene Expression Profiling Interactive Analysis database indicated a favorable prognosis, because IDH1-AS1 inhibited cell proliferation and xenograft tumor growth of EOC. IDH1-AS1 sponged miR-518c-5p whose overexpression promoted EOC cell proliferation. The miR-518c-5p mimic also reversed the proliferation-inhibiting effect induced by IDH1-AS1 overexpression. Furthermore, we found that RNA binding motif protein 47 (RBM47) was the downstream target of miR-518c-5p, that upregulation of RBM47 inhibited EOC cell proliferation, and that RBM47 overexpressing plasmid counteracted the proliferation-promoting effect caused by the IDH1-AS1 knockdown. Taken together, IDH1-AS1 may suppress EOC cell proliferation and tumor growth via the miR-518c-5p/RBM47 axis.

NAT10-dependent N4-acetylcytidine modification mediates PAN RNA stability, KSHV reactivation, and IFI16-related inflammasome activation.

N-acetyltransferase 10 (NAT10) is an N4-acetylcytidine (ac4C) writer that catalyzes RNA acetylation at cytidine N4 position on tRNAs, rRNAs and mRNAs. Recently, NAT10 and the associated ac4C have been reported to increase the stability of HIV-1 transcripts. Here, we show that NAT10 catalyzes ac4C addition to the polyadenylated nuclear RNA (PAN), a long non-coding RNA encoded by the oncogenic DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV), triggering viral lytic reactivation from latency. Mutagenesis of ac4C sites in PAN RNA in the context of KSHV infection abolishes PAN ac4C modifications, downregulates the expression of viral lytic genes and reduces virion production. NAT10 knockdown or mutagenesis erases ac4C modifications of PAN RNA and increases its instability, and prevents KSHV reactivation. Furthermore, PAN ac4C modification promotes NAT10 recruitment of IFN-γ-inducible protein-16 (IFI16) mRNA, resulting in its ac4C acetylation, mRNA stability and translation, and eventual inflammasome activation. These results reveal a novel mechanism of viral and host ac4C modifications and the associated complexes as a critical switch of KSHV replication and antiviral immunity.

IGF2BP2 promotes ovarian cancer growth and metastasis by upregulating CKAP2L protein expression in an m6 A-dependent manner.

Ovarian cancer (OC) is the second leading cause of gynecological cancer-related death in women worldwide. N6-methyladenosine (m6 A) is the most abundant internal modification in eukaryotic RNA. Human insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), an m6 A reader, can enhance mRNA stability and promote translation by recognizing m6 A modifications. Its tumor-promoting effects have been demonstrated in several cancers. However, the roles of m6 A modification and IGF2BP2 in OC remain unclear. Here, by using methylated RNA immunoprecipitation sequencing, we demonstrated that there is widespread dysregulation of m6 A modification in OC tissues. The m6 A modification and the mRNA and protein levels of IGF2BP2 were significantly elevated in OC. Overexpression of IGF2BP2 facilitated OC cell proliferation, migration, and invasion in vitro and accelerated tumor growth and metastasis in vivo. While IGF2BP2-knockdown showed the opposite effect. Mechanistically, we identified cytoskeleton-associated protein 2-like (CKAP2L) as a target of IGF2BP2. IGF2BP2 promoted CKAP2L translation dependent on m6 A modification, rather than affecting mRNA and protein stability. Overexpression of CKAP2L rescued the tumor-suppressive effect of IGF2BP2 knockdown in OC cells. In conclusion, this study revealed the potential role of IGF2BP2 in tumor progression, at least partially via promoting the translation of CKAP2L in an m6 A-dependent manner.

High Hepcidin Levels Promote Abnormal Iron Metabolism and Ferroptosis in Chronic Atrophic Gastritis.

BACKGROUND: Chronic atrophic gastritis (CAG) is a chronic inflammatory disease and premalignant lesion of gastric cancer. As an antimicrobial peptide, hepcidin can maintain iron metabolic balance and is susceptible to inflammation. OBJECTIVES: The objective of this study was to clarify whether hepcidin is involved in abnormal iron metabolism and ferroptosis during CAG pathogenesis. METHODS: Non-atrophic gastritis (NAG) and chronic atrophic gastritis (CAG) patient pathology slides were collected, and related protein expression was detected by immunohistochemical staining. The CAG rat model was established using MNNG combined with an irregular diet. RESULTS: CAG patients and rats exhibited iron deposition in gastric tissue. CAG-induced ferroptosis in the stomach was characterized by decreased GPX4 and FTH levels and increased 4-HNE levels. Hepcidin, which is mainly located in parietal cells, was elevated in CAG gastric tissue. The high gastric level of hepcidin inhibited iron absorption in the duodenum by decreasing the protein expression of DMT1 and FPN1. In addition, the IL-6/STAT3 signaling pathway induced hepcidin production in gastric tissue. CONCLUSION: Our results showed that the high level of gastric hepcidin induced ferroptosis in the stomach but also inhibited iron absorption in the intestines. Inhibiting hepcidin might be a new strategy for the prevention of CAG in the future.

Luteolin alleviates ulcerative colitis in rats via regulating immune response, oxidative stress, and metabolic profiling.

Ulcerative colitis (UC) is an inflammatory bowel disease and associated with metabolic imbalance. Luteolin (LUT) reportedly exhibits anti-inflammatory activity. However, its regulatory effects on metabolites remain indistinct. Here, the effects of LUT on immune response and oxidative stress in UC were determined. Serum metabolomics profiles of UC rats treated with LUT were obtained utilizing liquid chromatography-mass spectrometry. The results revealed that LUT treatment alleviated colon tissue injury, colon shortening, weight loss, and inflammatory response in UC rats. Additionally, the levels of superoxide dismutase and total antioxidant capacity were elevated, but malondialdehyde content was reduced in serum of UC rats, while these changes were abrogated by LUT. Metabolomics analysis unveiled that l-malic acid, creatinine, l-glutamine, and l-lactic acid levels were remarkably decreased, while dimethyl sulfone, 5-methylcytosine, cysteine-S-sulfate, and jasmonic acid levels were notably increased after LUT treatment. Furthermore, differential metabolites primarily participated in d-glutamine and d-glutamate metabolism, glutathione metabolism, and citrate cycle pathways. In summary, these results demonstrated that LUT improved immune response, alleviated oxidative stress, and altered metabolites in UC rats. This study lays the root for further exploring the mechanism of LUT in the treatment of UC.

Study on the function of Huazhuo Jiedu Decoction in promoting the homing of bone marrow mesenchymal stem cells and contributing to the treatment of ulcerative colitis.

Objective: To study the function of Huazhuo Jiedu Decoction (HZJD) in promoting the homing of bone marrow mesenchymal stem cells (BMSCs) and contributing to the reconstruction of the intestinal mucosal barrier in ulcerative colitis. Methods: Bone mesenchymal stem cells derived from mice were isolated and cultured, osteogenic and adipogenic assays to study the differentiation ability of BMSCs, and flow cytometry was used to detect the surface marker of the third generation cells. 30 mice were selected and divided into blank group, model group, HZJD group, BMSCs group, and HZJD combined with BMSCs group. Mouse colon length, body weight, and DAI score were used to assess efficacy. The levels of IL-6, IL-1ß, TNF-α, and IFN-γ in serum were measured by ELISA. BMSCs transfected with GFP were used to mark the homing of BMSCs in mice. The BMSCs tagging protein CD90+/CD29+ was detected by immunofluorescence. H&E staining detects damage to the colon and the inflammatory response. The expression levels of claudin-2, claudin-4, occludin, and ZO-1 in colon tissues were detected by Western blot. Results: After subculture, the cell grew with adherence. Flow cytometry showed that the cells were CD73+/CD90+/CD29+/CD45-/CD34-, which belonged to bone mesenchymal stem cells. ELISA showed that the treatment with HZJD and BMSCs suppressed the DSS-induced inflammatory response. BMSCs carrying GFP can be detected in intestinal tissues. Immunofluorescence showed that the HZJD combined with the BMSCs group had more BMSCs homing to the colonic tissue. The results of H&E and Western blot showed that DSS-induced intestinal mucosal damage in UC mice was repaired by HZJD and BMSCs, and the abnormal tight junction proteins claudin-2, claudin-4, occludin, and ZO-1 were normalized. Conclusion: HZJD has a therapeutic effect on ulcerative colitis by promoting the migration of BMSCs to ulcers of the colon and contributing to the reconstruction of the intestinal mucosal barrier in ulcerative colitis.

A DNA-based hydrogel for exosome separation and biomedical applications.

Exosomes (EXOs) have been proven as biomarkers for disease diagnosis and agents for therapeutics. Great challenge remains in the separation of EXOs with high-purity and low-damage from complex biological media, which is critical for the downstream applications. Herein, we report a DNA-based hydrogel to realize the specific and nondestructive separation of EXOs from complex biological media. The separated EXOs were directly utilized in the detection of human breast cancer in clinical samples, as well as applied in the therapeutics of myocardial infarction in rat models. The materials chemistry basis of this strategy involved the synthesis of ultralong DNA chains via an enzymatic amplification, and the formation of DNA hydrogels through complementary base-pairing. These ultralong DNA chains that contained polyvalent aptamers were able to recognize and bind with the receptors on EXOs, and the specific and efficient binding ensured the selective separation of EXOs from media into the further formed networked DNA hydrogel. Based on this DNA hydrogel, rationally designed optical modules were introduced for the detection of exosomal pathogenic microRNA, which achieved the classification of breast cancer patients versus healthy donors with 100% precision. Furthermore, the DNA hydrogel that contained mesenchymal stem cell-derived EXOs was proved with significant therapeutic efficacy in repairing infarcted myocardium of rat models. We envision that this DNA hydrogel-based bioseparation system is promising as a powerful biotechnology, which will promote the development of extracellular vesicles in nanobiomedicine.

Analysis of the Secreted Peptidome from Omental Adipose Tissue in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is a preferential omental metastasis malignancy. Since omental adipose tissue is an endocrine organ, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to compare the peptides secreted from omental adipose tissues of HGSOC and benign serous ovarian cysts (BSOC). Among the differentially secreted peptides, we detected 58 upregulated peptides, 197 downregulated peptides, 24 peptides that were only in the HGSOC group and 20 peptides that were only in the BSOC group (absolute fold change ≥ 2 and P < 0.05). Then, the basic characteristics of the differential peptides were analyzed, such as lengths, molecular weights, isoelectric points, and cleavage sites. Furthermore, we summarized the possible functions according to the precursor protein functions of the differentially expressed peptides by Gene Ontology (GO) analysis with the Annotation, Visualization, and Integrated Discovery (DAVID) database and canonical pathway analysis with IPA. For the GO analysis, the differentially secreted peptides were mainly associated with binding in molecular function and cellular processes in biology process. For the canonical pathways, the differentially secreted peptides were related to calcium signaling, protein kinase A signaling, and integrin-linked kinase (ILK) signaling. We also identified 67 differentially secreted peptides that located in the functional domains of the precursor proteins. These functional domains were mainly related to energy metabolism and immunoregulation. Our study might provide drugs that could potentially treat HGSOC or omental metastases of HGSOC cells.

Immune evasion strategy involving propionylation by the KSHV interferon regulatory factor 1 (vIRF1).

Post-translational modifications (PTMs) are essential for host antiviral immune response and viral immune evasion. Among a set of novel acylations, lysine propionylation (Kpr) has been detected in both histone and non-histone proteins. However, whether protein propionylation occurs in any viral proteins and whether such modifications regulate viral immune evasion remain elusive. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral interferon regulatory factor 1 (vIRF1) can be propionylated in lysine residues, which is required for effective inhibition of IFN-ß production and antiviral signaling. Mechanistically, vIRF1 promotes its own propionylation by blocking SIRT6's interaction with ubiquitin-specific peptidase 10 (USP10) leading to its degradation via a ubiquitin-proteasome pathway. Furthermore, vIRF1 propionylation is required for its function to block IRF3-CBP/p300 recruitment and repress the STING DNA sensing pathway. A SIRT6-specific activator, UBCS039, rescues propionylated vIRF1-mediated repression of IFN-ß signaling. These results reveal a novel mechanism of viral evasion of innate immunity through propionylation of a viral protein. The findings suggest that enzymes involved in viral propionylation could be potential targets for preventing viral infections.

Circular RNA hsa_circ_0007444 inhibits ovarian cancer progression through miR-23a-3p/DICER1 axis.

Ovarian cancer is the second leading cause of death in women with gynecological malignancy in China. Circular RNAs are a class of noncoding regulatory RNAs reported to be involved in cancer development and progression. Previous studies, including our own, have indicated that hsa_circ_0007444 is downregulated in ovarian cancer tissues. This study aims to elucidate the function and mechanism of hsa_circ_0007444 in ovarian cancer progression. The expression of hsa_circ_0007444 is determined by quantitative real-time PCR (qRT-PCR). Cell proliferation, invasion, migration and apoptosis are examined by cell counting-kit 8 (CCK-8), transwell and flow cytometry assays. Tumor growth and metastasis are assessed in vivo using Balb/c nude mouse xenograft model and tail vein injection model. And the mechanism of action of hsa_circ_0007444 is analysed by RNA-binding protein immunoprecipitation (RIP), luciferase reporter and rescue assays. hsa_circ_0007444 is downregulated in ovarian cancer tissues and cell lines compared with that in normal ovarian tissues and normal epithelial cell line. Gain- and loss-of-function results indicate that hsa_circ_0007444 inhibits cell proliferation, invasion, migration and increases cell apoptosis of ovarian cancer cells in vitro, and inhibits tumor growth and lung metastasis in vivo. Mechanistically, hsa_circ_0007444 can interact with AGO2 and sponge miR-23a-3p, thereby upregulating DICER1 expression, which is an important tumor suppressor in ovarian cancer. And miR-23a-3p mimics can rescue the inhibitory effect of hsa_circ_0007444 on ovarian cancer cell proliferation, invasion and migration. Therefore, hsa_circ_0007444 can inhibit ovarian cancer progression through the hsa_circ_0007444/miR-23a-3p/DICER1 axis.

LncRNA SLC25A21-AS1 increases the chemosensitivity and inhibits the progression of ovarian cancer by upregulating the expression of KCNK4.

Owing to high mortality rate, ovarian cancer seriously threatens women's health. Extensive abdominal metastasis and chemoresistance are the leading causes of ovarian cancer deaths. Through lncRNA sequencing, our previous study identified lncRNA SLC25A21-AS1, which was significantly downregulated in chemoresistant ovarian cancer cells. In this study, we aimed to evaluate the role and mechanism of SLC25A21-AS1 in ovarian cancer. The expression of SLC25A21-AS1 was analyzed by qRT-PCR and online database GEPIA. The biological functions of SLC25A21-AS1 and KCNK4 were analyzed by CCK-8, transwell, and flow cytometry. The specific mechanism was analyzed by RNA-sequencing, RNA binding protein immunoprecipitation, rescue experiments, and bioinformatic analysis. SLC25A21-AS1 was decreased in ovarian cancer tissues and cell lines. Overexpression of SLC25A21-AS1 enhanced the sensitivity of ovarian cancer cells to paclitaxel and cisplatin, and inhibited cell proliferation, invasion, and migration, while SLC25A21-AS1-silencing showed the opposite effect. Potassium channel subfamily K member 4 (KCNK4) was significantly up-regulated upon enforced expression of SLC25A21-AS1. Overexpression of KCNK4 inhibited cell proliferation, invasion, migration ability, and enhanced the sensitivity of ovarian cancer cells to paclitaxel and cisplatin. Meanwhile, KNCK4-overexpression rescued the promotive effect of SLC25A21-AS1-silencing on cell proliferation, invasion and migration. In addition, SLC25A21-AS1 could interact with the transcription factor Enhancer of Zeste Homolog 2 (EZH2), while EZH2 knockdown increased the expression of KCNK4 in some of the ovarian cancer cell lines. SLC25A21-AS1 enhanced the chemosensitivity and inhibited the proliferation, migration, and invasion ability of ovarian cancer cells at least partially by blocking EZH2-mediated silencing of KCNK4.

Circulating CD4+ Treg, CD8+ Treg, and CD3+ γδ T Cell Subpopulations in Ovarian Cancer.

Background and Objectives: Regulatory T cells (Tregs) are usually enriched in ovarian cancer (OC), and their immunosuppressive function plays a key role in tumorigenesis and progression. We mainly explored the phenotypical characterization of Treg-related markers on αß and γδ T cell subsets in patients with OC. Materials and Methods: Thirty-six untreated patients with OC at the Women's Hospital of Nanjing Medical University from September 2019 to August 2021 were enrolled. Phenotypical characterization of Tregs-related markers were detected by flow cytometry (FCM). Enzyme-linked immunosorbent assay was used to detect the levels of carbohydrate antigen (CA125) and transforming growth factor ß (TGF-ß). The level of human epididymis protein 4 (HE4) was detected by electrochemiluminescence immunoassay. Results: Circulating CD4+ Tregs, CD8+ Tregs, and CD3+γδ T cell subpopulations from OC patients have elevated Foxp3, CD25, CD122, Vδ1, and reduced CD28 expression compared to benign ovarian tumor (BOT) patients and healthy controls (HC). The upregulation of Foxp3 and Vδ1 and the downregulation of CD28 were highly specific for maintaining the immunosuppression function of CD4+ Tregs, CD3+γδ T cells, and CD8+ Tregs in OC patients. These Treg subpopulations were able to discriminate OC from BOT and HC. The levels of CA125, HE4, and TGF-ß were increased in OC patients. A significant positive correlation between Treg subpopulations and CA125, HE4, and TGF-ß was revealed. Conclusions: Proportions of CD4+ Tregs, CD8+ Tregs, and CD3+γδ T cell subsets were significantly increased in OC patients and were positively correlated with FIGO stage/metastasis status, CA125, HE4, and TGF-ß. These indicators have the potential to be used as immunosurveillance biomarkers for OC.

Differential effects of the LncRNA RNF157-AS1 on epithelial ovarian cancer cells through suppression of DIRAS3- and ULK1-mediated autophagy.

Analyses of several databases showed that the lncRNA RNF157 Antisense RNA 1 (RNF157-AS1) is overexpressed in epithelial ovarian cancer (EOC) tissues. In our study, suppressing RNF157-AS1 strikingly reduced the proliferation, invasion, and migration of EOC cells compared with control cells, while overexpressing RNF157-AS1 greatly increased these effects. By RNA pulldown assays, RNA binding protein immunoprecipitation (RIP) assays, and mass spectrometry, RNF157-AS1 was further found to be able to bind to the HMGA1 and EZH2 proteins. Chromatin immunoprecipitation (ChIP) assays showed that RNF157-AS1 and HMGA1 bound to the ULK1 promoter and prevented the expression of ULK1. Additionally, RNF157-AS1 interacted with EZH2 to bind to the DIRAS3 promoter and diminish DIRAS3 expression. ULK1 and DIRAS3 were found to be essential for autophagy. Combination autophagy inhibitor and RNF157-AS1 overexpression or knockdown, a change in the LC3 II/I ratio was found using immunofluorescence (IF) staining and western blot (WB) analysis. The autophagy level also was confirmed by autophagy/cytotoxicity dual staining. However, the majority of advanced EOC patients require platinum-based chemotherapy, since autophagy is a cellular catabolic response to cell stress. As a result, RNF157-AS1 increased EOC cell sensitivity to chemotherapy and death under cis-platinum (DDP) treatment by suppressing autophagy, as confirmed by cell count Kit-8 (CCK8) assays, flow cytometry, and autophagy/cytotoxicity dual staining. Therefore, the OS and PPS times were longer in EOC patients with elevated RNF157-AS1 expression. RNF157-AS1-mediated autophagy has potential clinical significance in DDP chemotherapy for EOC patients.

Combined use of CA125, neutrophil/lymphocyte ratio and platelet/lymphocyte ratio for the diagnosis of borderline and malignant epithelial ovarian tumors.

BACKGROUND: The mortality rate of ovarian cancer ranks first among three common gynecological malignant tumors due to insidious onset and lack of effective early diagnosis methods. Borderline epithelial ovarian tumor (BEOT) is a type of low malignant potential tumor that is typically associated with better outcomes than ovarian cancer. However, BEOTs are easily confused with benign and malignant epithelial ovarian tumors (EOTs) due to similar clinical symptoms and lack of specific tumor biomarkers and imaging examinations. Notably, a small subset of BEOTs will transform into low-grade serous ovarian carcinoma with a poor prognosis. Therefore, searching for potential biomarkers that can be easily obtained and accurately identify malignant epithelial ovarian tumors (MEOTs) as well as BEOTs is essential for the clinician. Cancer antigen 125 (CA125) is a commonly used biomarker for the diagnosis of EOTs in the preoperative scenario but has low sensitivity and specificity. Nowadays, inflammatory biomarkers including inflammatory cell counts and derived ratios such as neutrophil/lymphocyte ratio (NLR) and platelet/lymphocyte ratio (PLR) have been proved to be associated with tumor progression and poor prognosis, and were considered to be the most economically potential surrogate biomarkers for various malignancies. The purpose of this study was to find appropriate combinations of inflammatory and tumor biomarkers to improve the diagnostic efficiency of EOTs, especially the BEOTs. RESULTS: CA125, NLR and PLR increased steadily among benign, borderline and malignant EOTs and tended to be higher in advanced (stage III-IV) and lymph node metastasis MEOT groups than in early stage (stage I-II) and non-lymph node metastasis MEOT groups. CA125, NLR and PLR could be used separately in the differentiation of EOTs but could not take into account both sensitivity and specificity. The combined use of CA125, NLR and PLR was evaluated to be more efficient, especially in the identification of BEOTs, with both high sensitivity and high specificity. CONCLUSIONS: The levels of CA125, NLR and PLR were closely related to the nature of EOTs and malignant progression of MEOTs. The combination of CA125, NLR and PLR was more accurate in identifying the nature of EOTs than either alone or double combination, especially for BEOTs.

Long noncoding RNA RFPL1S-202 inhibits ovarian cancer progression by downregulating the IFN-ß/STAT1 signaling.

BACKGROUND: RFPL1S was first identified as one of the pseudogenes located in the intrachromosomal duplications within 22q12-13. Our previous study found that one of the predicted transcripts of lncRNA RFPL1S, ENST00000419368.1 (GRCh37/hg19), also named as RFPL1S-202 in Ensembl website, is significantly downregulated in the chemoresistant ovarian cancer cells. However, its function and underlying mechanism have not been studied. METHODS: Quantitative Real-time PCR was used to analyze the expression. Cell Counting Kit-8, transwell, flow cytometry analysis and tail vein injected mouse model were used to test the function. RNA-sequencing, RNA pull down, western blot, ELISA and RNA-Binding Protein Immunoprecipitation were performed for studying the mechanism. 5' and 3' rapid amplification of complementary DNA ends were performed to analyze the full length of RFPL1S-202. RESULTS: RFPL1S-202 is significantly downregulated in epithelial ovarian cancer tissues and cell lines. Gain- and loss-of-function study indicated that RFPL1S-202 could enhance cisplatin or paclitaxel in cytotoxicity, inhibit cell proliferation, invasion and migration of ovarian cancer cells in vitro, and inhibit the liver metastasis of ovarian cancer cells in vivo. Mechanistically, RFPL1S-202 could physically interact with DEAD-Box Helicase 3 X-linked (DDX3X) protein, and decrease the expression of p-STAT1 and the IFN inducible genes by increasing the m6A modification of IFNB1. RFPL1S-202 is a spliced and polyadenylated non-coding RNA with a full length of 1071 bp. CONCLUSIONS: Our study suggested that the predicted lncRNA RFPL1S-202 exerts a tumor- suppressive function in oarian cancer chemoresistance and progression by interacting with DDX3X and down-regulating the IFN-ß-STAT1 signaling pathway.