Hyperglycemia Inhibits Hepatic SHBG Synthesis Through the NGBR-AMPK-HNF4 Pathway in Rats with Polycystic Ovary Syndrome Induced by Letrozole in Combination with a High-Fat Diet.

SCOPE: Polycystic ovary syndrome (PCOS) is closely related to non-alcoholic fatty liver disease (NAFLD), and sex hormone-binding globulin (SHBG) is a glycoprotein produced by the liver. Hepatic lipogenesis inhibits hepatic SHBG synthesis, which leads to hyperandrogenemia and ovarian dysfunction in PCOS. Therefore, this study aims to characterize the mechanism whereby liver lipogenesis inhibits SHBG synthesis. METHODS AND RESULTS: This study establishes a rat model of PCOS complicated by NAFLD using a high-fat diet in combination with letrozole and performs transcriptomic analysis of the liver. Transcriptomic analysis of the liver shows that the expression of neurite growth inhibitor-B receptor (NgBR), hepatocyte nuclear factor 4α (HNF4α), and SHBG is low. Meantime, HepG2 cells are treated with palmitic acid (PA) to model NAFLD in vitro, which causes decreases in the expression of NgBR, HNF4α, and SHBG. However, the expression of HNF4α and SHBG is restored by treatment with the AMP-activated protein kinase (AMPK) agonist AICAR. CONCLUSIONS: NgBR regulates the expression of HNF4α by activating the AMPK signaling pathway, thereby affecting the synthesis of SHBG in the liver. Further mechanistic studies regarding the effect of liver fat on NGBR expression are warranted.

CircPAPPA2 plays a role in preeclampsia pathogenesis via regulation of the miR-942/miR-5006-3p.

CircRNAs are a class of endogenous non-coding RNAs implicated in the pathogenesis of many pregnancy related diseases, one of which is pre-eclampsia (PE). This study aims to investigate the role of CircPAPPA2 (circbase ID: hsa_circ_0015382) in regulating the migration and invasion of trophoblast cells. RNA sequencing was used to identify the differentially expressed circRNAs in placenta of PE and normal pregnant women. Quantitative polymerase chain reaction (qRT-PCR) was used to verify the expression of circPAPPA2 and two miRNAs (miR-942-5p, 5006-3p) in placenta of PE and normal pregnant women. CCK8 and transwell experiments were performed to assess the function of circPAPPA2 in PE development.The interaction between circPAPPA2 and miR-942-5p/miR-5006-3p was verified by dual-luciferase reporter assay. Finally, bioinformatics analyzed with gene ontology, Kyoto Encyclopedia of the target genes. The results showed that the expression of circPAPPA2 was increased in placenta of PE pregnant women. Also, circPAPPA2 impedes trophoblasts cell proliferation and invasion. Moreover, the expression of circPAPPA2 was positively correlated with systolic blood pressure and urine protein. In addition, circPAPPA2 serves as a sponge of miR-942-5p and miR-5006-3p. In conclusion, CircPAPPA2 regulates trophoblasts cell proliferation and invasion by mediating the miR-942/miR-5006-3p.

Efficacy of Pgp3 vaccination for Chlamydia urogenital tract infection depends on its native conformation.

Urogenital tract infections with Chlamydia trachomatis have frequently been detected among patients diagnosed with sexually transmitted infections, and such infections lead to inflammatory complications. Currently, no licensed chlamydial vaccine is available in clinical practice. We previously reported that immunization with recombinant C. trachomatis plasmid-encoded virulence factor Pgp3 provided cross-serovar protection against C. muridarum genital tract infection. Because Pgp3 is a homotrimer and human antisera only recognize the trimeric form of Pgp3, we compared the effects of the native conformation of Pgp3 (trimer) and heat-denatured Pgp3 (monomer) to determine whether the native conformation is dispensable for the induction of protective immunity against chlamydial vaginal challenge. Both Pgp3 trimer and monomer immunization induced corresponding specific antibody production, but only trimer-induced antibody recognized endogenous Pgp3, and trimer-immunized mouse splenocytes showed the highest IFN-γ production upon restimulation with the chlamydial elementary body or native Pgp3 in vitro. Importantly, only Pgp3 trimer-immunized mice showed shortened lower genital tract chlamydial shedding and decreased upper genital tract pathology. Thus, Pgp3-induced protective immunity against Chlamydia urogenital tract infection is highly dependent on the native conformation, which will guide the design of Pgp3-based polypeptides and multi-subunit chlamydial vaccines.

The diagnostic value of miR-145 and miR-205 in patients with cervical cancer.

OBJECTIVE: To investigate the diagnostic value of miRNA-145 (miR-145) and miRNA-205 (miR-205) in cervical cancer patients. METHODS: Cervical tissue samples were collected from 144 patients diagnosed with and suspected to have cervical cancer in our hospital. Confirmed by pathology, 84 samples were obtained from cervical cancer patients and 60 samples were from patients with cervical intraepithelial neoplasia. Meanwhile, 30 patients with cervicitis were also selected, and the expression levels of miR-145, miR-205 and human papillomavirus (HPV) were detected in cervical lesions and normal cervical tissue. RESULTS: In comparison to normal cervical tissue, cervicitis and cervical intraepithelial neoplasia groups, the relative expression level of miR-145 was significantly lower, whereas the relative expression level of miR-205 was notably higher in the cervical cancer group, respectively (P<0.001). The area under the receiver operating characteristic (ROC) curve of miR-145 for diagnosis of cervical cancer in patients was 0.878, of which the sensitivity and the specificity were 0.905 and 0.822, respectively. The area under the ROC curve of miR-205 was 0.881, of which the sensitivity and the specificity was 0.869 and 0.889, respectively. Among all patients, the relative expression level of miR-145 was significantly lower while the relative expression level of miR-205 was considerably higher in HPV-positive patients than those of HPV-negative groups (P<0.001). Parauterine invasion, FIGO stage III-IV and lymphatic metastasis were considered as independent factors that affect the expression of miR-145. FIGO stage III-IV and lymphatic metastasis were independent factors affecting the expression of miR-205. CONCLUSION: The low expression level of miR-145 and the high expression level of miR-205 in patients with cervical cancer demonstrate a certain diagnostic value in cervical cancer. The expression level of miR-145 and miR-205 is correlated with HPV infection and cervical tumor malignancy.

Silencing of hsa_circ_0009035 Suppresses Cervical Cancer Progression and Enhances Radiosensitivity through MicroRNA 889-3p-Dependent Regulation of HOXB7.

Circular RNAs (circRNAs), a novel type of endogenous noncoding RNAs, have been identified as critical regulators in human carcinogenesis. Here, we investigated the precise actions of hsa_circ_0009035 in the progression and radioresistance of cervical cancer (CC). The levels of hsa_circ_0009035, microRNA 889-3p (miR-889-3p), and homeobox B7 (HOXB7) were detected by quantitative real-time PCR (qRT-PCR) or Western blotting. RNase R and actinomycin D assays were used to assess the stability of hsa_circ_0009035. Cell proliferation, cell cycle progression, apoptosis, migration, and invasion were gauged with Cell Counting Kit-8 (CCK-8), flow cytometry, and transwell assays. Cell colony formation and survival were determined by the colony formation assay. Targeted correlations among hsa_circ_0009035, miR-889-3p, and HOXB7 were examined by the dual-luciferase reporter, RNA immunoprecipitation (RIP), or RNA pulldown assay. Animal studies were performed to evaluate the impact of hsa_circ_0009035 on tumor growth. We found that hsa_circ_0009035 was highly expressed in CC tissues and cells, and it was associated with the radioresistance of CC patients. Moreover, the silencing of hsa_circ_0009035 inhibited CC cell proliferation, migration, invasion, and it enhanced apoptosis and radiosensitivity in vitro and weakened tumor growth in vivo. Mechanistically, hsa_circ_0009035 directly targeted miR-889-3p by binding to miR-889-3p, and hsa_circ_0009035 modulated HOXB7 expression through miR-889-3p. HOXB7 was a functional target of miR-889-3p in regulating CC progression and radioresistance in vitro, and hsa_circ_0009035 modulated CC progression and radioresistance in vitro by miR-889-3p. Our current study first identified hsa_circ_0009035 as an important regulator of CC progression and radioresistance at least in part through targeting the miR-889-3p/HOXB7 axis, highlighting its significance as a potential therapeutic target for CC treatment.

[Association of EGFR gene G719S and T790M mutations with cervical cancer].

OBJECTIVE: To establish a rapid and accurate "on/off" switch technique consisted of 3'-phosphorothioate-modified allele-specific primers and exo+ polymerase to screen the G719S and T790M mutations of epidermal growth factor receptor (EGFR) gene. The switch was used to identify cervical cancer patients who are sensitive to tyrosine kinase inhibitor (TKI). METHODS: Allele-specific primers targeting recombinant wild-type and mutation-type templates were designed with 3' terminal phosphorothioate modification. Two-directional primer extension was carried out using Pfu polymerase. The G719S and T790M mutations were detected by the technique among cervical cancer tissues. The results were verified by Sanger sequencing. RESULTS: No mutation was detected among the 80 cervical cancer cases, and the results were consistent with that of Sanger sequencing. No significant difference was found between the frequencies of the G719S and T790M mutations between the patient and the control groups (P> 0.05). CONCLUSION: A sensitive "on/off" switch technique for detecting the two EGFR mutations was established. The G719S and T790M mutations are not associated with cervical cancer.

Chrysin Inhibits Proinflammatory Factor-Induced EMT Phenotype and Cancer Stem Cell-Like Features in HeLa Cells by Blocking the NF-κB/Twist Axis.

BACKGROUND/AIMS: TNF-α and TGF-ß associated epithelial-mesenchymal transition (EMT) occurs via NF-κB-dependent transcriptional upregulation of Twist1.Chrysin (ChR) is a major active ingredient ofpropolis, which inhibits various cancer cells and possesses anti-inflammatory activities. This study aimed to assess whether and how ChR inhibits proinflammatory cytokine-induced EMT phenotype and cancer stem-like cell (CSLC) features in the HeLa cell line. METHODS: HeLa cells were co-administered TNF-α (10.0 ng/mL) and TGF-ß (5.0 ng/mL) for 24h following TGF-ß (5.0 ng/mL) alone for 6 d in the presence or absence of ChR (5.0, 10.0 and 20.0µM). Then, the levels of EMT-related factors, multi-potential transcription factors, and stem cell markers were analyzed by immunoblot. Wound healing and tumor sphere formation assays were performed to assess the migration and self-renewal capabilities of cells, respectively. Overexpression and/or knockdown of NF-κBp65 and/or Twsit1 were used to explore the molecular mechanisms. RESULTS: The results showed that ChR inhibited EMT and CSLC properties in HeLa cells administered TNF-α after prolonged TGF-ß treatment, in a concentration-dependent fashion. NF-κBp65 knockdown and ChR(10.0µM) cooperatively enhanced the inhibition of NF-κBp65 and Twist1 expression, EMT, and CSLC properties. Conversely, overexpression of NF-κBp65 combined with ChR(10.0µM) antagonized such activities. Meanwhile, Twist1silencing or overexpression combined with ChR treatment did not affect NF-κBp65 levels, but also reduced or enhanced EMT and CSLC properties. Importantly, overexpressing Twist1 combined with ChR reversed the effects of NF-κBp65 knockdown and ChR. CONCLUSION: ChR inhibits proinflammatory cytokine-induced EMT and CSLC features in HeLa cells by blocking the NF-κB/Twist axis.

Exposure to TNF­α combined with TGF­ß induces carcinogenesis in vitro via NF-κB/Twist axis.

Persistent human papilloma virus (HPV) infection induces chronic inflammation resulting in human cervical cancer. However, the mechanisms underlying carcinogenesis via chronic inflammation remain largely unclear. We investigated the role of pro-inflammatory factors in epithelial-mesenchymal transition (EMT) and cancer stem cell-like (CSCL) characteristics of HeLa cells exposed to TNF­α with or without TGF­ß. We then determined the role of NF-κB/Twist signal axis in the pathogenesis of cervical cancer. We found that HeLa cells exposed to TNF­α following chronic treatment with TGF­ß exhibited EMT, self-renewal and high mobility. Knockdown of NF-κBp65 inhibited NF-κB and Twist1 expression, and EMT and CSCL properties of HeLa cells following co-treatment with TNF­α and TGF­ß. Conversely, overexpression of NF-κBp65 potentiated the above effects. However, knockdown or overexpression of Twist1 had no effect on NF-κBp65 expression, but inhibited or promoted EMT and CSCL features. Notably, overexpression of Twist1 rescued NF-κBp65 knockdown. Our results demonstrate the role of NF-κB/Twist signaling axis in which HeLa cells treated with TNF­α following chronic exposure to TGF­ß induce EMT and CSCL properties. The NF-κB/Twist signal axis may represent an effective therapeutic target in cervical cancer.