Microbiome-metabolome analysis reveals cervical lesion alterations.

Cervical cancer (CC) continues to be one of the most common cancers among females worldwide. It takes a few years or even decades for CC to arise in a minority of women with cervical precancers. An increasing corpus of studies today indicates that local microecology and carcinogenesis are intimately related. To investigate the changes in cericovaginal microecology with the development of cervical cancer, we performed 16S rDNA sequencing and metabolomic analysis in cericovaginal fluid from 10 LSIL patients, 10 HSIL patients, 10 CC patients and 10 healthy controls to reveal the differential flora and metabolites during cervical carcinogenesis. Carcinogenesis is associated with alterations in microbiome diversity, individual taxa, and functions with notable changes in Lactobacillus, Prevotella and Aquabacterium, as well as in cervicovaginal metabolites that correlate with cervicovaginal microbial patterns. Increased bacterial diversity and a decline in the relative abundance of Lactobacillus, the dominant species in the cericovaginal flora, are observed when cervical lesions advance. According to KEGG pathway enrichment analysis, lipids and organic acids change as cervical cancer progresses, and the phenylalanine, tyrosine, and tryptophan biosynthesis pathway is essential for the development of cervical cancer. Our results reveal that microbic and metabolomic profiling is capable of distinguishing CC from precancer and highlights potential biomarkers for the early detection of cervical dysplasia. These differential microorganisms and metabolites are expected to become a potential tool to assist in the diagnosis of cervical cancer.

ΔNp63α attenuates tumor aggressiveness by suppressing miR-205/ZEB1-mediated epithelial-mesenchymal transition in cervical squamous cell carcinoma.

Cervical cancer is one of the most common female cancers worldwide. Although the therapeutic outcomes of patients with early-stage cervical cancer have been significantly improved in the past decades, tumor metastasis and recurrence remain the major causes of cervical cancer-related deaths. In cervical squamous cell carcinoma (SCC), the aberrant activation of epithelial-mesenchymal transition (EMT), a crucial process in invasion and metastasis of epithelial cancer, could promote lymph nodal metastasis and recurrence, and predicts poor prognosis. In this study, we show that the expression levels of EMT markers, ß-catenin and Vimentin, are associated with the p63 isoform ΔNp63α in SCC by using immunohistochemistry staining and analysis. Compared to the control SiHa cells (SiHa-NC), the expression of E-cadherin and ß-catenin are upregulated, while Vimentin and ZEB1 are downregulated in the constructed SiHa cell line with stable ΔNp63α overexpression (SiHa-ΔNp63α). Besides, the migration and invasion abilities are also suppressed in SiHa-ΔNp63α cells with a typical epithelial morphology with cobblestone-like shape, suggesting that ΔNp63α is a vital EMT repressor in SCC cells. In addition, the involvement of miR-205/ZEB1 axis in the inhibition effect of ΔNp63α on EMT program is revealed by a miRNA array and confirmed by the subsequent transfection of the miR-205 mimic and antagomir. Moreover, SCC patients with low ΔNp63α expression and high EMT level show more frequent metastasis and recurrence as well as reduced overall survival. Therefore, EMT program and its vital repressor ΔNp63α could be used as biomarkers for tumor metastasis and recurrence in cervical cancer.

Snail family proteins in cervical squamous carcinoma: expression and significance.

PURPOSE: Epithelial-mesenchymal transition (EMT) is crucial for tumor progression and metastasis. Snail family members, including Snail, Slug and Smuc, are the transcription factors that repress E-cadherin expression and induce epithelial-mesenchymal transition in some tumor tissues. In this study, the expression of snail family proteins in cervical squamous cancers was evaluated. METHODS: A series of 144 samples, comprising 28 cases of normal cervical tissues and 116 cases of squamous cell carcinoma (SCC), were used for analysis. The expression of Snail, Slug, Smuc, E-cadherin and vimentin was assessed in the tissues by immunohistochemistry and was statistically analyzed by SPSS13.0. RESULTS: The increase in nuclear expression of snail and smuc was associated with down-regulation of E-cadherin and up-regulation of vimentin. The nuclear expression of Snail and Smuc was positively associated with lymph node metastasis of the SCC, and the nuclear expression of Snail was also positively related with histological differentiation. In contrast, tumor size, histological differentiation, lymph node metastasis and stages of the SCC were not associated with the expression of Slug, cytpolasmic Smuc or cytoplasm levels of Snail. CONCLUSION: Snail and Smuc proteins, but not Slug, may contribute to the onset of EMT in SCC. Inhibiting the expression of Snail and Smuc might be a potential therapeutic target for the treatment of metastasis and invasion of cervical carcinomas.

Detailed characterization of PD-1/PD-L1 and CTLA4 expression and tumor-infiltrating lymphocytes in yolk sac tumors.

BACKGROUND: Immune checkpoint blockade (ICB) is considered as a promising approach for cancer treatment. However, the potency of ICB therapy in yolk sac tumors (YSTs) has not been confirmed, and the comprehensive analysis of tumor immune microenvironment and the expression of PD-1/PD-L1 and CTLA4 were also not thoroughly evaluated. METHODS: Immunohistochemistry was performed in formalin-fixed, paraffin-embedded tumor specimens from 23 YSTs patients to detect the density and distribution of tumor-infiltrating T cells, tertiary lymphoid structures (TLSs), as well as the expression of PD-1/PD-L1 and CTLA4. RESULTS: Overall, more than half (61 %) of all patients exhibited an immune-desert phenotype based on CD3+ T cells. PD-1 expression was identified in five tumor samples (21.7 %), and PD-L1 expression exhibited a different positive rate in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) (39.1 % and 17.4 %). Noteworthily, the rate of positive CTLA4 expression in both TCs and TILs was markedly higher (69.6 % and 56.5 %) than those of PD-1 and PD-L1 expression. Furthermore, TLSs were observed in 21.74 % of all tissues, and samples with TLSs exhibited significantly higher densities of TILs and higher expression of immune checkpoint molecules, particularly PD-1/PD-L1. In addition, tumors located in testes also exhibited a higher density of TILs and higher expression of immune checkpoint molecules. CONCLUSION: Generally a high frequency of CTLA4 expression was found, PD-1/PD-L1 expression, the immune-inflamed phenotype, and TLSs were low frequency in YSTs, however, YSTs in testes showed a higher density of TILs and higher expression of immune checkpoint molecules.

A novel ICG-labeled cyclic TMTP1 peptide dimer for sensitive tumor imaging and enhanced photothermal therapy in vivo.

TMTP1 is a polypeptide independently screened in our laboratory, which can target tumors in situ and metastases. In previous work, we have successfully developed a near-infrared (NIR) probe TMTP1-PEG4-ICG for tumor imaging. However, the limited ability to target tumor micrometastases hinders its further clinical application. Multimerization of peptides has been extensively demonstrated as an effective strategy to increase receptor binding affinity due to "multivalent effect" or "apparent cooperative affinity". In this study, a novel TMTP1 homodimer-directed NIR probe (TMTP1-PEG4)2-ICG was successfully constructed and synthesized. The cyclic TMTP1 peptides were bridged by two PEG4 linkers and then labeled with ICG-NHS for tumor imaging and photothermal therapy. In vivo biodistribution were assessed in normal BALB/c mice, and tumor targeting abilities of (TMTP1-PEG4)2-ICG and its monomer were evaluated and compared in 4T1-bearing subcutaneous tumor and lymph node metastasis model mice. Biodistribution analysis in vivo revealed that (TMTP1-PEG4)2-ICG was cleared mainly in both liver and kidney dependent way. Comparing with free ICG dye or TMTP1-PEG4-ICG probe, this improved (TMTP1-PEG4)2-ICG dimer showed more sensitive tumor imaging and could clearly identify tumors at a minimum volume of 10 mm3. Additionally, when compared to its monomer, lymph node (LN) metastases could also be apparently visualized and easily distinguished from normal LN by the novel dimer at 24 h post-injection. The blocking study revealed that the tumor accumulation of this probe was specifically medicated by receptor-ligand interaction. Furthermore, with the increase in stability and tumor targeting ability of ICG in vivo, the probe could also be an attractive photothermal agent to significantly inhibit tumor growth under 808 nm NIR laser irradiation. In conclusion, our work revealed that the novel (TMTP1-PEG4)2-ICG dimer could be a promising theranostic agent for sensitive tumor imaging and imaging-guided photothermal therapy, indicating its broad prospects for further clinical transformation.

Characterization of Vaginal Microbiota in Third Trimester Premature Rupture of Membranes Patients through 16S rDNA Sequencing.

In China, premature rupture of membranes (PROM) counts as a major pregnancy complication in China and usually results into adverse pregnancy outcomes. We analysed the vagina microbiome composition using 16S rDNA V3−V4 amplicon sequencing technology, in this prospective study of 441 women in their third trimester of pregnancy. We first divided all subjects into PROM and HC (healthy control) groups, in order to investigate the correlation of vagina microbiome composition and the development of PROM. We found that seven pathogens were higher in the PROM group as compared to the HC group with statistical significance. We also split all subjects into three groups based on Lactobacillus abundance-dominant (Lactobacillus > 90%), intermediate (Lactobacillus 30−90%) and depleted (Lactobacillus < 30%) groups, and explored nine pathogenic genera that were higher in the depleted group than the intermediate and dominant groups having statistical significance. Finally, using integrated analysis and logistics regression modelling, we discovered that Lactobacillus (coeff = −0.09, p = 0.04) was linked to the decreased risk of PROM, while Gardnerella (coeff = 0.04, p = 0.02), Prevotella (coeff = 0.11, p = 0.02), Megasphaera (coeff = 0.04, p = 0.01), Ureaplasma (coeff = 0.004, p = 0.01) and Dialister (coeff = 0.001, p = 0.04) were associated with the increased risk of PROM. Further study on how these pathogens interact with vaginal microbiota and the host would result in a better understanding of PROM development.

Gambogic Acid Induces Pyroptosis of Colorectal Cancer Cells through the GSDME-Dependent Pathway and Elicits an Antitumor Immune Response.

Pyroptosis is a recently identified form of programmed cell death (PCD) that exerts a vital influence on the antitumor immune response. GA, a xanthone structure isolated from gamboge resin, is a naturally occurring bioactive ingredient with several anticancer activities, such as activities that affect cell cycle arrest, apoptosis, and autophagy. Here, we found that GA decreased the viability of the CRC cell lines, HCT116 and CT26, in a dose- and time-dependent manner, and multiple pores and large bubbles in the membranes, which are morphological characteristics of pyroptosis, were observed by light microscopy and transmission electron microscopy (TEM). Furthermore, the cleavage of gasdermin E (GSDME) was observed after exposure to GA, along with concomitant activation of caspase-3. Additionally, GSDME-dependent pyroptosis triggered by GA could be attenuated by siRNA-mediated knockdown of GSDME and treatment with the caspase-3 inhibitor. Moreover, we found that GA induced pyroptosis and significantly inhibited tumor growth in CT26 tumor-bearing mice. Strikingly, significantly increased proportions of CD3+ T cells, cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) were observed in the tumor microenvironment in the GA-treated groups. Moreover, significantly increased proportions of CTLs and effector memory T cells (TEM) (CD8+ CD44+ CD62L-) were also detected in the spleens of the GA-treated groups, suggesting that the pyroptosis-induced immune response generated a robust memory response that mediated protective immunity. In this study, we revealed a previously unrecognized mechanism by which GA induces GSDME-dependent pyroptosis and enhances the anticancer immune response. Based on this mechanism, GA is a promising antitumor drug for CRC treatment that induces caspase-3-GSDME-dependent pyroptosis. This study provides novel insight into cancer chemoimmunotherapy.

The exon 12-containing LHX6 isoforms promote cervical cancer cell proliferation by regulating the MAPK signaling pathway.

LIM homeobox 6 (LHX6) has been reported to be downregulated and inhibits cell proliferation in various cancers. Alternative splicing of LHX6 leads to six annotated isoforms, which can be found in the NCBI database. However, the expression patterns and potential roles of these isoforms remain poorly characterized in cervical cancer. Here, we demonstrated that the LHX6 isoforms containing exon 12 (LHX6EX(+12) group) and isoforms lacking exon 12 (LHX6EX(-12) group) were differentially expressed in cervical tissue by qRT-PCR. The mRNA expression level of LHX6EX(+12) group was higher than that of LHX6EX(-12) group in cervical cancer tissue. Knockdown of LHX6EX(+12) group and all LHX6 isoforms (LHX6All group) inhibited cell growth, increased cell apoptosis, and induced cell cycle arrest from G0/G1 phase to S phase in vitro. Consistently, overexpression of the LHX6EX(+12) group promoted cervical cancer cell proliferation in vitro. In contrast, no significant differences in cell proliferation were found between LHX6EX(-12) isoform knockdown group and its control. RNA-sequencing suggested that the LHX6EX(+12) isoform group might exert its cancer-promoting effects in cervical cancer via regulating MAPK signaling pathway. Downregulation of the LHX6EX(+12) group significantly suppressed the phosphorylation of MRK, ERK, JNK, and P38 at the protein level. We also identified some unique biological processes and signaling pathways in which each isoform group might be involved. In summary, our results indicated that LHX6EX(+12) isoform group was the dominant oncogenic type of LHX6 in cervical cancer, which may be a new biomarker and a potential precise therapeutic target for cervical cancer in the future.

TMTP1-Modified, Tumor Microenvironment Responsive Nanoparticles Co-Deliver Cisplatin and Paclitaxel Prodrugs for Effective Cervical Cancer Therapy.

BACKGROUND AND PURPOSE: Cisplatin-paclitaxel (TP) combination chemotherapy as the first-line therapy for numerous cancers is hindered by its inadequate accumulation in tumors and severe side effects resulting from non-specific distribution. The aim of this study is to explore whether TMTP1-modified, cisplatin and paclitaxel prodrugs co-loaded nanodrug could improve cervical cancer chemotherapy and relieve its side effects through active and passive tumor targeting accumulation and controlled drug release. METHODS: TDNP, with capacities of active targeting for tumors and controlled drug release, was prepared to co-deliver cisplatin and paclitaxel prodrugs. The characteristics were investigated, including the diameter, surface zeta potential, stability and tumor microenvironment (TME) dependent drug release profiles. Cellular uptake, cytotoxicity, drug accumulation in tumors, antitumor effects and safety analysis were evaluated in vitro and in vivo. RESULTS: The oxidized cisplatin and the paclitaxel linked to the polymer achieved a high loading effciency of over 80% and TME-dependent sustained drug release. Moreover, TMTP1 modification enhanced cellular uptake of TDNP and further improved the cytotoxicity of TDNP in vitro. In vivo, TDNP showed an extended blood circulation and increased accumulation in SiHa xenograft models with the aid of TMTP1. More importantly, TDNP controlled tumor growth without life-threatening side effects. CONCLUSION: Our study provided a novel TP co-delivery platform for targeted chemotherapy of cervical cancer, which was promising to improve the therapeutic effcacy of TP and may also have application in other tumors.

Integrated metagenomics and metabolomics analysis of third-trimester pregnant women with premature membrane rupture: a pilot study.

BACKGROUND: Premature rupture of membranes (PROM) is a major pregnancy complication in China and usually leads to adverse pregnancy outcomes. The major aim of this study was to search for microorganisms and their related metabolites that have direct relationship with PROM. METHODS: For vaginal discharge samples, metagenomics sequencing was applied to identify microorganisms that were enriched in PROM subjects, and untargeted metabolomics was applied to characterize the metabolites changes in PROM subjects compared to healthy controls (HC). Correlation analysis was then used to explore the relationship between these microorganisms and metabolites changes. RESULTS: Two upstream metabolites of glycolysis, N-acetyl-D-galactosamine (GalNAc) and sucrose, were found downregulated in the PROM group (P=0.04 and P=0.041, respectively). Higher percentages of conditional pathogens, such as of Streptococcus (8.4% vs. 6.1% in HC group, P=0.15) and Chlamydia (4.3% vs. 2.3% in HC group, P=0.07) were found in PROM group. Other common conditional pathogens including Prevotella, Staphylococcus, Mycobacterium and Enterobacter, were also higher in PROM group, although their absolute percentages were low and the differences did not reach statistical significance due to relative small sample size. Correlation analysis further demonstrated a positive correlation of downregulation of glycolysis metabolites with higher percentage of conditional pathogens. CONCLUSIONS: Integrated metagenomics and metabolomics analysis can be used to track the subtle changes in the vaginal microenvironment. Downregulation of glycolysis substrates (GalNAc and sucrose) and increase of related pathogenic microorganisms (Streptococcus and Chlamydia) could serve as early warning biomarkers of PROM.

Detection of Vaginal Metabolite Changes in Premature Rupture of Membrane Patients in Third Trimester Pregnancy: a Prospective Cohort Study.

Premature rupture of membranes (PROM) is usually associated with pregnant and neonatal complications. Most of the PROM cases are caused by ascending asymptomatic genital infection. In China, PROM (15.3%) is more common than spontaneous preterm labor (7.3%) and leads to more adverse pregnancy outcomes. Here, we designed a prospective cohort study to measure the metabolomics changes in vaginal swab samples and explored their potential contribution to PROM. A total of 260 differentially expressed metabolites were identified and further analyzed. In the PROM group, N-acetyl-D-galactosamine and sucrose were downregulated (P = 0.0025, P = 0.0195, respectively), both of which are the upstream metabolites of the glycolysis pathway. Furthermore, estriol 3-sulfate 16-glucuronide (P = 0.0154) and 2-methoxy-17beta-estradiol 3-glucosiduronic acid (P = 0.004), two final metabolites in steroid hormone biosynthesis, were both downregulated in the PROM group. Finally, we found two catechin metabolites (epigallocatechin-7-glucuronide, P = 0.0009; 4'-methyl-epigallocatechin-7-glucuronide, P = 0.01) as well as DL-citrulline (P = 0.0393) were also significantly downregulated in the PROM group compared with the healthy control (HC) group, which are related to important antioxidant and anti-inflammatory activities in the human body. Altogether, metabolite changes in glycolysis, steroid hormone biosynthesis, and antioxidant/anti-inflammatory pathways may contribute to (or be a consequence of) vaginal dysbiosis and PROM. Metabolite pathway analysis is a new and promising approach to further investigate the mechanism of PROM and help prevent its unfavorable pregnant outcomes at a functional level. Trial registration number: ChiCTR2000034721.

LncRNA FAL1 Upregulates SOX4 by Downregulating miR-449a to Promote the Migration and Invasion of Cervical Squamous Cell Carcinoma (CSCC) Cells.

This study aimed to investigate the role of lncRNA FAT1 in cervical squamous cell carcinoma (CSCC). We found that FAT1 was upregulated in CSCC tissues. The expression of FAT1 was not affected by clinical stages. High levels of FAT1 predicted poor survival. The expression of miR-449a was inversely correlated with the expression of FAT1. SOX4 mRNA expression was positively correlated with the expression of FAT1 in CSCC tissues. FAT1 over-expression led to an upregulated SOX4 expression and downregulated miR-449a expression. MiR-449a over-expression failed to affect FAT1 expression but downregulated SOX4 expression. FAT1 and SOX4 over-expression led to increased rates of CSCC cell migration and invasion, miR-449a over-expression led to decreased rates of CSCC cell migration and invasion and attenuated the effects of SOX4 over-expression. Therefore, FAL1 can upregulate SOX4 by downregulating miR-449a to promote the migration and invasion of CSCC cells.

Confirmation of chronic endometritis in repeated implantation failure and success outcome in IVF-ET after intrauterine delivery of the combined administration of antibiotic and dexamethasone.

PROBLEM: The aim of this prospective study was to investigate the prevalence of chronic endometritis (CE) in repeated implantation failure (RIF) patients and to determine whether intrauterine delivery of the combined administration of antibiotic and dexamethasone improves the clinical pregnancy outcome. METHOD OF STUDY: The combination of hysteroscopy and histology was used to diagnose CE in the patients with RIF, and the diagnosed patients were treated with the intrauterine delivery of the combined antibiotic and dexamethasone therapy. The prevalence of CE in the RIF patients was recorded, and the therapeutic effect was also evaluated in the first IVF-ET cycle. RESULTS: In a total number of 298 patients with RIF, 109 cases (36.58%) were diagnosed as CE. Intrauterine infusion treatment resulted in CE resolution in 77.98% of patients. After the treatment, 85 cases with no signs of CE (Group 2) had a significantly higher implantation rate (31.33%) and clinical pregnancy rate (51.76%) as compared with both 126 cases without CE diagnosis (Group 1; 16.30% and 30.15%, respectively) and 24 cases with persistence of CE (Group 3; 14.89% and 25.00%, respectively). The live birth rate in the first IVF-ET cycle following treatment in Group 2 was significantly higher than that of both Group 1 and Group 3 (all P < .05). CONCLUSION: Chronic endometritis is highly prevalent in patients with RIF. To the best of our knowledge, we for the first time reported that intrauterine delivery of the combined administration of antibiotic and dexamethasone as a treatment option of CE could achieve expected therapeutic effects and successful pregnancy outcomes.

Alantolactone induces apoptosis of human cervical cancer cells via reactive oxygen species generation, glutathione depletion and inhibition of the Bcl-2/Bax signaling pathway.

Alantolactone is the active ingredient in frankincense, and is extracted from the dry root of elecampane. It has a wide variety of uses, including as an insect repellent, antibacterial, antidiuretic, analgesic and anticancer agent. In addition, alantolactone induces apoptosis of human cervical cancer cells, however, its mechanism of action remains to be elucidated. Therefore, the present study investigated whether alantolactone was able to induce apoptosis of human cervical cancer cells, and its potential mechanisms of action were analyzed. Treatment of HeLa cells with alantolactone (0, 10, 20, 30, 40, 50 and 60 µM) for 12 h significantly inhibited growth in a dose-dependent manner. Cells treated with 30 µM of alantolactone for 0, 3, 6 and 12 h demonstrated marked induction of apoptosis in a time-dependent manner. Treatment of HeLa cells with 30 µM of alantolactone for 0, 3, 6 and 12 h significantly induced the generation of reactive oxygen species (ROS) and inhibited glutathione (GSH) production in HeLa cells in a dose-dependent manner. Alantolactone additionally markedly inhibited the Bcl-2/Bax signaling pathway in HeLa cells. Therefore, administration of alantolactone induced apoptosis of human cervical cancer cells via ROS generation, GSH depletion and inhibition of the Bcl-2/Bax signaling pathway.