Effect of transvaginal Lactobacillus supplementation on reversing lower genital tract dysbiosis and improving perinatal outcomes in PCOS patients after IVF-FET: a study protocol for a multicenter randomized controlled trial.

BACKGROUND: Significant lower genital tract (LGT) dysbiosis and an associated lower rate of clinical pregnancy after in vitro fertilization-frozen embryo transfer (IVF-FET) among polycystic ovary syndrome (PCOS) patients have been previously reported by our group. We aimed to assess whether transvaginal Lactobacillus supplementation can reverse LGT dysbiosis and further improve perinatal outcomes in PCOS patients after IVF-FET. METHODS/DESIGN: This is a protocol for a multicenter, open-label, randomized controlled trial in China. Women diagnosed with PCOS who are undergoing IVF-FET treatment will be recruited. Allocation to the intervention/control arms at a ratio of 1:1 will be executed by an electronic randomization system. Participants in the intervention arm will receive the live Lactobacillus capsule vaginally for 10 consecutive days before embryo transfer, while those in the control arm will receive standard individualized care. The primary outcomes will be the clinical pregnancy rate, implantation rate, and live birth rate. 16S rRNA sequencing and liquid chromatography-mass spectrometry will be conducted to evaluate the LGT microbiome and systemic metabonomics before and after the intervention. A sample of 260 participants will provide 95% power to detect a 20% increase in the rate of clinical pregnancy (α = 0.025, one-tailed test, 15% dropout rate). A total of 300 participants will be recruited. DISCUSSION: This is the first large and multicenter randomized controlled trial aimed at assessing the efficacy of transvaginal Lactobacillus supplementation on restoring the LGT microbiome and improving perinatal outcomes in PCOS patients after IVF-FET. This pragmatic trial is promising for increasing the rates of clinical pregnancy and live birth in PCOS patients after IVF-FET. ETHICS AND DISSEMINATION: Ethical review approval was obtained from the Medical Research Ethics Committees of the International Peace Maternity and Child Health Hospital of Shanghai Jiao Tong University (15 October 2020, GKLW 2020-29). To maximize dissemination, these findings will be reported in open access publications in journals with high impact, and oral and poster conference presentations will be performed. TRIAL REGISTRATION: ChiCTR ChiCTR2000036460. Registered on 13 September 2020, https://www.chictr.org.cn/showproj.html?proj=59549 .

Comparative Analysis of Lower Genital Tract Microbiome Between PCOS and Healthy Women.

Women with polycystic ovarian syndrome (PCOS) often have a history of infertility and poor pregnancy outcome. The character of the lower genital tract (LGT) microbiome of these patients is still unknown. We collected both vaginal and cervical canal swabs from 47 PCOS patients (diagnosed by the Rotterdam Criteria) and 50 healthy reproductive-aged controls in this study. Variable regions 3-4 (V3-4) were sequenced and analyzed. Operational taxonomic unit (OTU) abundance was noted for all samples. Taxa that discriminated between PCOS and healthy women was calculated by linear discriminant analysis effect size (LEFSe). Results from 97 paired vaginal and cervical canal samples collected from 97 women [mean age 30 (±4 years)] were available for analysis. Using the Rotterdam Criteria, 47 women were diagnosed with PCOS (PCOS, n = 47; control, n = 50). There was no significant difference between cervical canal microbiome and vaginal microbiome from the same individual, however, Lactobacillus spp. was less abundant in both vaginal and cervical canal microbiome of PCOS patients. Several non-Lactobacillus taxa including Gardnerella_vaginalis_00703mash, Prevotella_9_other, and Mycoplasma hominis, were more abundant in the LGT microbiota of PCOS patients. There is a difference between the microorganism in the LGT of patients with PCOS and healthy reproductive-aged women.

Novel PGK1 determines SKP2-dependent AR stability and reprograms granular cell glucose metabolism facilitating ovulation dysfunction.

BACKGROUND: Disordered folliculogenesis is a core characteristic of polycystic ovary syndrome (PCOS) and androgen receptors (ARs) are closely associated with hyperandrogenism and abnormalities in folliculogenesis in PCOS. However, whether the new AR binding partner phosphoglycerate kinase 1 (PGK1) in granulosa cells (GCs) plays a key role in the pathogenesis of PCOS remains unclear. METHODS: We identified the new AR binding partner PGK1 by co-IP (co-immunoprecipitation) in luteinized GCs, and reconfirmed by co-IP, co-localization and GST pull down assay, and checked PGK1 expression levels with qRT-PCR and western blotting. Pharmaceuticals rescue assays in mice, and metabolism assay, AR protein stability and RNA-seq of PGK1 targets in cells proved the function in PCOS. FINDINGS: PGK1 and AR are highly expressed in PCOS luteinized GCs and PCOS-like mouse ovarian tissues. PGK1 regulated glucose metabolism and deteriorated PCOS-like mouse metabolic disorder, and paclitaxel rescued the phenotype of PCOS-like mice and reduced ovarian PGK1 and AR protein levels. PGK1 inhibited AR ubiquitination levels and increased AR stability in an E3 ligase SKP2-dependent manner. Additionally, PGK1 promoted AR nuclear translocation, and RNA-seq data showed that critical ovulation-related genes were regulated by the PGK1-AR axis. INTERPRETATION: PGK1 regulated GCs metabolism and interacted with AR to regulate the expression of key ovulation genes, and also mediated cell proliferation and apoptosis, which resulted in the etiology of PCOS. This work highlights the pathogenic mechanism and represents a novel therapeutic target for PCOS. FUNDING: National Key Research and Development Program of China; National Natural Science Foundation of China grant.

Knocking down B7H3 expression enhances cell proliferation of SHEDs via the SHP1/AKT signal axis.

B7H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins associated with maintaining immune tolerance, tumor cell proliferation, migration, invasion and metabolism, drug resistance, and stem cell differentiation. Neural crest-derived Multipotent Stem Cells (MSCs) from the dental pulp has become a good choice for tissue regeneration because it is easily obtainable and has strong regeneration potentials. Although there have been many studies investigating the role of B7H3 in cancer cells and immune cells, its role in the dental pulp stem cells regeneration is unknown. In this study, we chose SHEDs (stem cells from human exfoliated deciduous teeth) as a research model to analyze the expression and function of B7H3. The result showed that SHEDs were B7H3/CD90, B7H3/CD73, B7H3/CD105 double positive, and the expression of B7H3 is primarily located within the membrane. Downregulation of B7H3 expression significantly accelerated the expansion of SHEDs through the SHP1/AKT signal axis while upregulation of B7H3 expression decreased the proliferation of SHEDs. Hence, this study indicates that B7H3 is a stem cell surface molecule and might be used as a SHEDs marker whereby its downregulation enhances the proliferation of SHEDs via the activation of B7H3/SHP1/AKT signaling pathway.

Corrigendum: Comparative Analysis of Lower Genital Tract Microbiome Between PCOS and Healthy Women.

[This corrects the article DOI: 10.3389/fphys.2020.01108.].

Quinacrine Depletes BCR-ABL and Suppresses Ph-Positive Leukemia Cells.

Drug resistance to TKIs and the existance of CML leukemia stem cells is an urgent problem. In this study, we demonstrate that quinacrine (QC) induces apoptosis in BCR-ABL positive CML and acute lymphoblastic leukemia (ALL) cells. Interestingly, QC inhibits the colony formation of primary CD34+ progenitor/stem leukemia cells from CML patients. QC targets RNA polymerase I, which produces ribosomal (r)RNA, involving in protein translation process. Also, QC treatment prolongs CML-like mice survival and inhibits K562 tumor growth in vivo. In conclusion, we demonstrate that QC depletes BCR-ABL protein and suppresses Ph-positive leukemia cells in vitro and in vivo.

Mutation allele frequency threshold does not affect prognostic analysis using next-generation sequencing in oral squamous cell carcinoma.

BACKGROUND: With the development of sequencing technologies, there may be some disputes on sequencing analysis. The aim of this study was to investigate different allele frequency thresholds of mutations in targeted genes on prognostic analyses using a panel of cancer associated gene exons (CAGE) in oral squamous cell carcinoma (OSCC). METHODS: Forty-six patients were included in this study. Twelve genes were sequenced and analyzed using next-generation sequencing from formalin-fixed paraffin-embedded tissues. Allele frequency thresholds of 10, 5, and 3% were used for prognostic analyses. RESULTS: With a mean sequence depth of 3199-fold, 99% of CAGE were represented by at least 10 reads. Ninety-four non-synonymous (missense [70.2%], nonsense [11.7%], splice site [10.6%], and insertion/deletion [7.5%]) mutations were detected in 40 OSCC patients with an allele frequency threshold of 10%. TP53 (78.3%), NOTCH1 (30.4%), CASP8 (13.0%), CDKN2A (10.9%), and CDH1 (6.5%) were the most frequently mutated genes. Using allele frequency thresholds of 10, 5, and 3%, there were no significant differences in clinical outcomes between patients with non-synonymous mutations and wild type genotypes. CONCLUSIONS: TP53, NOTCH1, CASP8, CDKN2A, and CDH1 are the most frequently mutated genes in OSCC patients. The allele frequency threshold used in this study does not affect the results of clinical outcome analysis.

Elevated growth differentiating factor 15 expression predicts long-term benefit of docetaxel, cisplatin and 5-fluorouracil induction chemotherapy in patients with oral cancer.

Our previous phase 3 trial (NCT01542931) failed to demonstrate improved survival when docetaxel, cisplatin and 5-fluorouracil (TPF) induction chemotherapy was introduced prior to surgery and postoperative radiotherapy in patients with locally advanced oral squamous cell carcinoma (OSCC). The aim of the present study was to investigate the long-term predictive value of GDF15 expression for potential personalized treatment strategies in OSCC. A total of 256 patients with stage III/IVA OSCC from our phase 3 trial were enrolled in the present study. Immunohistochemical staining against GDF15 was performed in the biopsy samples from 230/256 patients. Kaplan-Meier analysis, followed by the log-rank test, and the Cox proportional hazards model were used for outcome analysis using the statistical SPSS 18.0 software package for Windows. Among the 230 patients, low GDF15 expression was detected in 68 patients and high GDF15 expression was detected in 162 patients. With a median follow-up period of 67 months, the patients with low GDF15 expression exhibited a higher survival rate than those with high GDF15 expression, including 5-year overall survival (73.4 vs. 57.7%; P=0.059), 5-year disease-free survival (64.5 vs. 49.2%; P=0.033), 5-year locoregional recurrence-free survival (66.0 vs. 51.5%; P=0.043) and 5-year distant metastasis-free survival (73.4 vs. 56.6%; P=0.038) rates. Furthermore, the cT3/4N0M0 patients with high GDF15 expression benefited significantly from TPF induction chemotherapy, including overall survival (HR=0.233; P=0.02), disease-free survival (HR=0.296; P=0.014), locoregional recurrence-free survival (HR=0.347; P=0.035) and distant metastasis-free survival (HR=0.212; P=0.013) rates. The results of the present study suggested that elevated GDF15 expression may be used as a long-term prognostic biomarker for poor clinical outcomes in patients with locally advanced OSCC. Elevated GDF15 expression in cT3/4N0M0 patients predicts significant long-term benefit of survival from TPF induction chemotherapy.

Stathmin is overexpressed and regulated by mutant p53 in oral squamous cell carcinoma.

BACKGROUND: The aim of this study was to investigate the oncogenic function and regulatory mechanism of stathmin in oral squamous cell carcinoma (OSCC). METHODS: Two-dimensional electrophoresis and liquid chromatography-tandem mass chromatography were applied to screen differentiated proteins during carcinogenesis in OSCC. Cell Counting Kit-8 (CCK-8) assays, colony formation, migration, flow cytometry, immunofluorescence and a xenograft model were used to detect the function of stathmin. The correlation between stathmin and p53 expression was analyzed using immunohistochemistry. Mutant/wild type p53 plasmids and small interfering RNA were used to examine the regulation of stathmin. Chromatin immunoprecipitation assays and luciferase assays were performed to detect the transcriptional activation of stathmin by p53. RESULTS: Overexpression of stathmin was screened and confirmed in OSCC patients and cell lines. Silencing expression of stathmin inhibited proliferation, colony formation and migration and promoted apoptosis. Poly ADP ribose polymerase (PARP) and cyclin-dependent kinase 1 (cdc2) were activated after silencing the expression of stathmin. Suppression of tumorigenicity was also confirmed in vivo. Mutant p53 transcriptionally activated the expression of stathmin in HN6 and HN13 cancer cells, but not in HN30 cells harboring wild type p53. CONCLUSIONS: These results suggest that stathmin acts as an oncogene and is transcriptionally regulated by mutant p53, but not by wild-type p53. Stathmin could be a potential anti-tumor therapeutic target in OSCC.

Mutant GDF15 presents a poor prognostic outcome for patients with oral squamous cell carcinoma.

PURPOSE: To investigate the mutation status of growth differentiation factor 15 (GDF15) in patients with oral squamous cell carcinoma (OSCC), as well as the prognostic value of missense GDF15 mutations. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded biopsy samples from 46 OSCC patients were involved in this study. GDF15 and TP53 mutations were sequenced using the Ion Torrent Personal Genome Machine, GDF15 protein expression was detected using immunohistochemistry. Torrent Suite Software v.3.6, Integrative Genomics Viewer; v.2.3, statistical software SPSS18.0 for Windows were used for analysis. All hypothesis-generating tests were two-sided at a significance level of 0.05. RESULTS: Twenty-nine GDF15 mutations were identified in 19 out of 46 patients (41.3%), including eighteen missense mutations, two nonsense mutations and nine synonymous mutations. The patients with missense GDF15 mutations had poorer prognostic outcomes than those with wild-type GDF15, including overall survival (P = 0.035), disease-free survival (P = 0.032), locoregional recurrence-free survival (P = 0.015), and distant metastasis-free survival (P = 0.070). Missense GDF15mutations was an independent increased risk factor of overall survival (HR = 5.993, 95% CI:1.856-19.346, P = 0.003), disease-free survival (HR = 3.764, 95% CI:1.295-10.945, P = 0.015), locoregional recurrence-free survival (HR = 4.555, 95% CI:1.494-13.889, P = 0.008), and distant metastasis-free survival (HR = 4.420, 95% CI:1.145-13.433, P = 0.009). CONCLUSIONS: Patients with missense GDF15 mutations have significantly poorer outcomes than those with wild-type GDF15, missense GDF15 mutations could be used as an independent increased risk factor of poor prognosis in OSCC patients.

Targeting catalase but not peroxiredoxins enhances arsenic trioxide-induced apoptosis in K562 cells.

Despite considerable efficacy of arsenic trioxide (As2O3) in acute promyelocytic leukemia (APL) treatment, other non-APL leukemias, such as chronic myeloid leukemia (CML), are less sensitive to As2O3 treatment. However, the underlying mechanism is not well understood. Here we show that relative As2O3-resistant K562 cells have significantly lower ROS levels than As2O3-sensitive NB4 cells. We compared the expression of several antioxidant enzymes in these two cell lines and found that peroxiredoxin 1/2/6 and catalase are expressed at high levels in K562 cells. We further investigated the possible role of peroxirdoxin 1/2/6 and catalase in determining the cellular sensitivity to As2O3. Interestingly, knockdown of peroxiredoxin 1/2/6 did not increase the susceptibility of K562 cells to As2O3. On the contrary, knockdown of catalase markedly enhanced As2O3-induced apoptosis. In addition, we provide evidence that overexpression of BCR/ABL cannot increase the expression of PRDX 1/2/6 and catalase. The current study reveals that the functional role of antioxidant enzymes is cellular context and treatment agents dependent; targeting catalase may represent a novel strategy to improve the efficacy of As2O3 in CML treatment.

Important role of SUMOylation of Spliceosome factors in prostate cancer cells.

Sentrin/SUMO (small ubiquitin-like modifier)-specific proteases (SENPs) have been implicated in the development of prostate cancer. However, due to the low abundance of SUMO-modified proteins and high activity of SENPs, the SUMO substrates affected by SENPs in prostate cancer cells are largely unknown. Here, we identified SI2, a novel cell-permeable SENP-specific inhibitor, by high-throughput screening. Using SI2 as a way of inhibiting the activity of SENPs and the SUMO stably transfected PC3 cells as a prostate cancer model, in combination with the stable isotope labeling with amino acids (SILAC) quantitative proteomic technique, we identified more than 900 putative target proteins of SUMO, in which 231 proteins were further subjected to bioinformatic analysis. In the highly enriched spliceosome pathway, we validated that USP39, HSPA1A, and HSPA2 were novel target proteins of SUMO. Furthermore, we demonstrated that K6, K16, K29, K51, and K73 were the SUMOylation sites of USP39. Mutation of these SUMO modification sites of USP39 further promoted the proliferation-enhancing effect of USP39 on prostate cancer cells. This study provides the SUMOproteome of PC3 cells and reveals that SUMOylation of spliceosome factors may be implicated in the pathogenesis of prostate cancer. Optimization of SI2 for isotype-specific SENP inhibitors warrants further investigation.