MiR-223-3p attenuates M1 macrophage polarization via suppressing the Notch signaling pathway and NLRP3-mediated pyroptosis in experimental autoimmune uveitis.

Autoimmune uveitis is an intraocular inflammatory disease with a high blindness rate in developed countries such as the United States. It is pressing to comprehend the pathogenesis of autoimmune uveitis and develop novel schemes for its treatment. In the present research, we demonstrated that the Notch signaling pathway was activated, and the level of miR-223-3p was significantly reduced in rats with experimental autoimmune uveitis (EAU) compared with the level of normal rats. To investigate the relationship between miR-223-3p and Notch signaling, EAU rats received miR-223-3p-carrying lentivirus, miR-223-3p vector-carrying lentivirus (miR-223-3p-N), and γ-secretase inhibitor (DAPT), respectively. The results of Q-PCR, immunological experiments, and flow cytometry analysis all support the hypothesis that both miR-223-3p and DAPT, a Notch signaling pathway inhibitor, had similar inhibitory effects on the EAU pathological process. That is to say, they could both inhibit the activation of the Notch signaling pathway via modulating recombination signal binding protein-Jκ (RBPJ) to restore the polarization imbalance of M/M2 macrophages in EAU rats. In addition, miR-223-3p could also inhibit NLRP3 inflammasome activation and inflammasome-induced pyroptosis in ocular tissues. Taken together, our findings indicate that miR-223-3p serves as an important regulator in M1 macrophage polarization and pyroptosis, thereby alleviating the inflammatory response in uveitis.

Glucocorticoids improve the balance of M1/M2 macrophage polarization in experimental autoimmune uveitis through the P38MAPK-MEF2C axis.

Uveitis is a common ocular disease that can induce serious complications and sequelae. It is one of the major causes of blindness. Currently, mounting evidence suggests that glucocorticoids (GCs) can suppress ocular inflammation and promote the healing of damaged ocular tissues, but the underlying mechanism remains unclear. The present study aimed to elucidate the mechanism by which GCs modulate the homeostasis of M1/M2 macrophage polarization in experimental autoimmune uveitis (EAU) through the p38MAPK-MEF2C axis. Female Lewis rats were randomly divided into four groups: a normal control (NC) group, an EAU group, an EAU + glucocorticoid (EAU + GC) group, and an EAU + p38MAPK inhibitor (EAU + SB) group. The EAU model was induced in EAU, EAU + GC, and EAU + SB groups, followed by the treatments of normal saline, GC (predisione), and SB203580, respectively. The findings demonstrated that the rats in GC and SB groups had much less ocular inflammation, and the clinical and pathological scores decreased. Further research revealed that GC and SB treatment could inhibit iNOS and CD86 expression while promoting Arg-1 and CD206 secretion in IRBP-induced uveitis rats. Moreover, we found that the role of GC was similar to the results of SB203580, but the role of GC was masked by the C16-PAF (a p38MAPK activator) treatment. Molecular docking and western blot results confirmed that GC's therapeutic action against EAU is mediated via the p38MAPK-MEF2C axis. It regulates macrophage polarization by encouraging M1 to M2 transition and releasing anti-inflammatory factors.

Double vitrification-warming cycles, coupled with blastocyst biopsy, impair live birth but do not affect neonatal outcomes.

OBJECTIVE: To identify whether the transfer of blastocysts that have been vitrified, thawed, biopsied, revitrified, and subsequently rethawed affects clinical outcome and neonatal outcome. METHODS: A retrospective study was conducted in a single assisted reproduction technology center from September 2016 to March 2021. Women undergoing single frozen euploid blastocysts transfer were stratified into two groups based on number of vitrification-thawing cycles: single vitrification coupled with single biopsy (group A, n = 177) and double vitrification coupled with single biopsy (group B, n = 30). Pregnancy and perinatal outcomes of the two groups were compared. RESULTS: Clinical pregnancy rates were similar between the two groups. Group B was associated with an increased likelihood of live birth when compared with group A by different multivariable analysis models (model 1: odds ratio, 0.42 [95% confidence interval, 0.18-0.97], P = 0.041; model 2: odds ratio, 0.38 [95% confidence interval, 0.16-0.92], P = 0.033). No major obstetrical complication was reported in the two groups and only one malformation live birth was reported in group A. CONCLUSION: The procedure of double vitrification-warming cycles, coupled with single biopsy, increases pregnancy loss and ultimately diminishes live birth but does not affect perinatal outcome. Future studies with a larger sample size would help to validate the results.

Natural cycle increases the live-birth rate compared with hormone replacement treatment for frozen-thawed single euploid blastocyst transfer.

Background: A number of studies have compared the clinical outcomes between the two endometrial preparation methods: natural cycles (NCs) and hormone replacement treatment (HRT) before frozen embryo transfer, but the results were conflicting. In order to mitigate the potential effect of embryos per se, several researchers have worked on this subject for euploid blastocyst transfer, but the results were still inconsistent. Therefore, the present study was aimed to investigate the clinical outcomes between HRT and NC for autologous single vitrified-warmed euploid blastocyst transfer based on our data. Methods: A total of 598 frozen-thawed single euploid blastocyst transfer cycles in the assisted reproductive center of Northwest Women's and Children's Hospital from January 2014 to May 2021 were retrospectively analyzed. Women were stratified into the NC (n = 125) or HRT (n = 473) group according to the patient's preference and the physician's guidance. Multivariate regression models and subgroup analysis were constructed to analyze the association between endometrial preparation and live birth. Results: Women in the NC group had a higher live birth rate (68.80% versus 58.35%, P = 0.034) and a lower risk of total pregnancy loss (8.51% versus 21.14%, P = 0.005) when compared with women in the HRT group. The biochemical pregnancy rate (75.20% versus 74.00%, P = 0.784) and clinical pregnancy rate (74.40% versus 69.98%, P = 0.334) were similar between the two groups (NC versus HRT). NC was associated with an increased odds of live birth compared with HRT by different multivariable analysis models (Model 1: adjusted odds ratio [aOR], 95% confidence interval [CI]: 0.57, 0.36 - 0.90; Model 2: aOR, 95%CI: 0.57, 0.35 - 0.92). In addition, the increased chance of live birth in the NC group was found in all subgroups. No major obstetrical complications and two malformation livebirths were reported. Conclusions: In women undergoing single euploid frozen blastocyst transfers, the NC group was associated with a lower pregnancy loss rate and an ultimately higher live birth rate than the HRT group. Although HRT is convenient for both clinicians and patients, the lower live birth rate should be taken into account and NC might be the first choice of endometrial preparation method.

Is duration of estrogen supplementation associated with clinical outcomes in frozen-thawed autologous single-blastocyst transfer cycles?

PURPOSE: To investigate the relationship between different duration of estrogen administration and live birth rate (LBR) after autologous single frozen blastocyst transfer with hormone replacement therapy. METHODS: A total of 2026 frozen blastocyst transfer cycles in the assisted reproductive center of northwest women and children's hospital from January, 2017, to August, 2020, were retrospectively analyzed. All the cycles were allocated into 3 groups according to the duration of estrogen administration: group A, 11-14 days (n = 346); group B, 15-18 days (n = 1191), and group C, ≥ 19 days (n = 489). Baseline data, clinical, and perinatal outcomes of the three groups were compared. A multivariate regression model was constructed to analyze the association between duration of estradiol administration and clinical outcomes. RESULTS: We did not observe a significant association between duration of estrogen supplementation and LBR in group B (adjusted odds ratio [aOR] 1.14; 95% confidence interval [CI], 0.89-1.45) or group C (aOR 1.16; 95% CI, 0.86-1.56) patients with group A as the reference group, through logistic regression analysis. No statistical differences were observed in perinatal outcomes among the three groups. CONCLUSION: The duration of estrogen administration was not associated with the likelihood of live birth in women undergoing frozen-thawed autologous single-blastocyst transfer.

Epigallocatechin gallate enhances human lens epithelial cell survival after UVB irradiation via the mitochondrial signaling pathway.

The aim of the present study was to explore the mechanism underlying the ultraviolet B (UVB) irradiation­induced apoptosis of human lens epithelial cells (HLECs), and to investigate the protective effect of epigallocatechin gallate (EGCG) against the UVB­induced apoptosis of HLECs. HLECs were exposed to different concentrations of EGCG plus UVB (30 mJ/cm2). Cell viability was determined using the MTT assay. Furthermore, mitochondrial membrane potential (Δψm) and apoptosis were assessed by flow cytometry with JC­1 and Annexin V/PI staining, respectively. Moreover, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH­Px), as well as the levels of GSH, hydrogen peroxide (H2O2) and hydroxyl free radicals were determined using biochemical assay techniques. Reverse transcription­quantitative PCR and western blotting were used to detect the mRNA and protein expression levels of Bcl­2, Bax, cytochrome c, caspase­9 and caspase­3, respectively. The results revealed that UVB irradiation reduced the Δψm of HLECs and induced apoptosis. Notably, EGCG significantly attenuated the generation of H2O2 and hydroxyl free radicals caused by UVB irradiation in HLECs, and significantly increased CAT, SOD and GSH­Px activities, however, the GSH levels were not significantly increased. EGCG also reduced UVB­stimulated Bax, cytochrome c, caspase­9 and caspase­3 expression, and elevated Bcl­2 expression, suggesting that EGCG may possess free radical­scavenging properties, thus increasing cell viability. In conclusion, EGCG may be able to protect against UVB­induced HLECs apoptosis through the mitochondria­mediated apoptotic signaling pathway, indicating its potential application in clinical practice.

Promoter hypermethylation of SOX11 promotes the progression of cervical cancer in vitro and in vivo.

The development of cervical cancer (CC) is a multi­gene, multi­step carcinogenic process that involves complex genetic and epigenetic mechanisms. SRY­related HMG­box gene 11 (SOX11) is a member of the SOX family of transcription factors with an emerging crucial role in the development of various tumor types. To elucidate the function of SOX11 in cervical carcinogenesis, the expression level of SOX11 during the development of human CC was analyzed by immunohistochemistry and western blot analysis. Additionally, the methylation status of the SOX11 was examined using bisulfite sequencing and methylation­specific polymerase chain reaction. The SOX11 expression and promoter methylation in human CC cell lines were also determined. The effect of SOX11 expression restoration after 5­aza­2'­deoxycytidine (5­Aza­dC) treatment on the CC cell proliferation ability was evaluated in CC cell lines. SOX11 was highly expressed in normal cervix (NC) and precancerous low­grade squamous intraepithelial lesions, but weakly expressed or virtually absent in precancerous high­grade squamous intraepithelial lesions and CC, which is consistent with the result of the western blot analysis. Hypermethylation of the SOX11 promoter was detected in CC, which was significantly higher than that in NC samples at each CpG site. The expression level of SOX11 in the CC cell lines was downregulated compared with the positive control, Tera­1human teratoma cell line. Upon 5­Aza­dC treatment, SOX11 expression was significantly upregulated in the CC cell lines at the mRNA and protein levels, and cell proliferation was inhibited. The results indicated that the downregulation of SOX11 in CC is due to the hypermethylation of the SOX11 promoter region. Thus, SOX11 methylation may have a role in the growth of CC cells and cervical carcinogenesis.

Identification of Significant Gene Signatures and Prognostic Biomarkers for Patients With Cervical Cancer by Integrated Bioinformatic Methods.

Cervical cancer is the leading cause of death with gynecological malignancies. We aimed to explore the molecular mechanism of carcinogenesis and biomarkers for cervical cancer by integrated bioinformatic analysis. We employed RNA-sequencing details of 254 cervical squamous cell carcinomas and 3 normal samples from The Cancer Genome Atlas. To explore the distinct pathways, messenger RNA expression was submitted to a Gene Set Enrichment Analysis. Kyoto Encyclopedia of Genes and Genomes and protein-protein interaction network analysis of differentially expressed genes were performed. Then, we conducted pathway enrichment analysis for modules acquired in protein-protein interaction analysis and obtained a list of pathways in every module. After intersecting the results from the 3 approaches, we evaluated the survival rates of both mutual pathways and genes in the pathway, and 5 survival-related genes were obtained. Finally, Cox hazards ratio analysis of these 5 genes was performed. DNA replication pathway ( P < .001; 12 genes included) was suggested to have the strongest association with the prognosis of cervical squamous cancer. In total, 5 of the 12 genes, namely, minichromosome maintenance 2, minichromosome maintenance 4, minichromosome maintenance 5, proliferating cell nuclear antigen, and ribonuclease H2 subunit A were significantly correlated with survival. Minichromosome maintenance 5 was shown as an independent prognostic biomarker for patients with cervical cancer. This study identified a distinct pathway (DNA replication). Five genes which may be prognostic biomarkers and minichromosome maintenance 5 were identified as independent prognostic biomarkers for patients with cervical cancer.

Identification and validation of the role of matrix metalloproteinase-1 in cervical cancer.

Lymph node (LN) metastasis at an early stage of cervical cancer is often an indicator of poor prognosis and is critical for subsequent adjuvant therapy. The current study aimed to identify aberrant gene signatures and biomarkers of metastasis for patients with cervical cancer. RNA-sequencing data of 132 LN negative (N0) and 60 LN positive (N1) cervical cancer samples obtained from The Cancer Genome Atlas database were analyzed. Differentially expressed genes were identified using R packages 'edgeR' and 'limma'. Kyoto Encyclopedia of Genes and Genomes pathway enrichment and Gene Set Enrichment Analysis (GSEA) were conducted. The GSE9750 dataset obtained from Gene Expression Omnibus was analyzed to identify genes that are persistently aberrantly expressed during the development of cervical cancer. The peroxisome proliferator-activated receptor (PPAR) signaling pathway was screened out to be significant during LN metastasis. In the two analyzed datasets, 11 genes were aberrantly expressed, while matrix metalloproteinase 1 (MMP1) was the only gene that was persistently overexpressed. Cell viability, wound healing and Transwell assays were performed to evaluate the effects of MMP1 knockdown in cervical cancer cell lines, and the expression of epithelial mesenchymal transition (EMT) markers was detected. Finally, the clinical significance of MMP1 was investigated. The current study identified that MMP1 was overexpressed and the PPAR signaling pathway was associated LN metastasis in patients with cervical cancer. Following knockdown of MMP1, the proliferation, migration and invasion of cervical cancer cell lines were weakened, the expression of epithelial marker E-cadherin was increased, and the expression of metastasis-associated gene vimentin was decreased. MMP1 was an independent prognostic factor for cervical cancer. The current study indicated that MMP1 has a key role in the regulation of cervical tumor growth and LN metastasis via EMT to a certain extent. The results suggest that MMP1 may be a biomarker for LN metastasis of cervical cancer, and further validation should be performed.

Identification of a histone family gene signature for predicting the prognosis of cervical cancer patients.

Heterogeneity in terms of tumor characteristics, prognosis, and survival among cancer patients is an unsolved issue. Here, we systematically analyzed the aberrant expression patterns of cervical cancer using RNA-Seq data from The Cancer Genome Atlas (TCGA). We incorporated gene profiling, molecular signatures, functional and pathway information with gene set enrichment and protein-protein interaction (PPI) network analysis, to identify sub-networks of genes. Those identified genes relating to DNA replication and DNA repair-mediated signaling pathways associated with systemic lupus erythematosus (SLE). Next, we combined cross-validated prognostic scores to build an integrated prognostic model for survival prediction. The combined approach revealed that the DNA repair-mediated including the functional interaction module of 18 histone genes (Histone cluster 1 H2A, B and H4), were significantly correlated with the survival rate. Furthermore, five of these histone genes were highly expressed in three cervical cancer cohorts from the Oncomine database. Comparison of high and low histone variant-expressing human cervical cancer cell lines revealed different responses to DNA damage, suggesting protective functions of histone genes against DNA damage. Collectively, we provide evidence that two SLE-associated gene sets (HIST1H2BD and HIST1H2BJ; and HIST1H2BD, HIST1H2BJ, HIST1H2BH, HIST1H2AM and HIST1H4K) can be used as prognostic factors for survival prediction among cervical cancer patients.

MicroRNA-494 promotes cervical cancer proliferation through the regulation of PTEN.

The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway appears to be a key regulator in cervical carcinogenesis. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein is principally involved in the homeostatic maintenance of PI3K/Akt signaling and PTEN has been identified to play an important role in the occurrence and development of cervical cancer. MicroRNA (miRNA)-494 has been proven to be involved in the carcinogenesis and development of various types of cancer by directly targeting PTEN. However the role, mechanism and clinical significance of miR-494 in cervical cancer have not been further reported. In the present study, we analyzed the expression of miR-494 in -with PTEN expression and clinicopathological data of cervical cancer patients. The results showed that miR-494 expression was significantly upregulated in human cervical cancer cell lines and tissues. miR-494 upregulation was significantly associated with PTEN downregulation, adverse clinicopathological characteristics, poor overall and progression-free survival and poor prognosis. In vitro experiments showed that inhibition of miR-494 suppressed cell proliferation and growth by directly targeting the 3'-untranslated region (3'-UTR) of PTEN mRNA. These findings identified a novel molecular mechanism involved in the regulation of PTEN expression and cervical cancer progression. Results of the present study indicated that miR-494 may have an essential role in the carcinogenesis and progression of cervical cancer and targeting miR-494 may be a promising therapeutic strategy for the treatment of cervical cancer.

Relations between GPR4 expression, microvascular density (MVD) and clinical pathological characteristics of patients with epithelial ovarian carcinoma (EOC).

G-protein coupled receptor 4 (GPR4) is a G protein-coupled receptor (GPCR) activated by sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC). Later studies indicated that GPR4 can serve as a proton sensor. GPR4 has been known to play a critical role in the tube formation of vascular endothelial cells, and GPR4 overexpression is observed in various types of malignancies, suggesting its involvement in the cancer-related angiogenesis. In this study, we examined the GPR4 expression levels in blood vessels of ovarian cancer, and analyzed the relationship between GPR4 expression and the clinical and pathological characteristics of patients with epithelial ovarian carcinomas (EOC). Results from immunohistochemistry showed that GPR4 is detectable in the endothelium of vessels of both EOC and benign ovarian tumor tissue, but the expression levels were significantly increased in EOC. Moreover the increased expression is accompanied by a higher microvascular density (MVD) in EOC compared to that in the benign ovarian tumors. We demonstrated a positive correlation between GPR4 expression density and MVD in EOC, but not benign ovarian tumor tissues. Further analyses indicated that GPR4 expression and MVD in EOC were correlated to the status of lymph node metastasis and clinical stage, but not significantly correlated to the pathological classifications, histopathological grades, the amounts of ascites, status of peritoneal cytology, tumor sizes, or patients' ages. These results suggested that GPR4 may play an important role in the development of EOC, and its overexpression might be required for the angiogenesis, tumor growth, and metastasis of EOC.

Epigenetic interventions increase the radiation sensitivity of cancer cells.

Epigenetic changes including DNA methylation, histone modifications, chromatin remodeling and microRNAs play critical roles in tumorigenesis and tumor development. Reversal of epigenetic changes sensitizes some tumor cells to radiation. DNMT-I enhances the response of tumor cells to radiotherapy. AZA demethylated promoters of genes related to ionizing radiation response, such as p16 and hMLH1. The genes expression of the p53, RASSF1, and DAPK gene families was increased by 5-aza-CdR, which induces G2-M phase arrest and increased apoptosis. HDAC-I has both anti-tumor activity and radiation sensitization activity. HDAC-I disrupts both DNA damage sensing and repair processes: HDAC-I disrupts the association between HDAC enzyme and DNA sensor proteins 53BP1 and ATM. HDAC-I changes the acetylation status of both proteins involved in homologous recombination (HR) repair pathway which include BRCA1, Rad51, and Rad50, and proteins involved in non-homologous end joining (NHEJ) repair pathway which include Ku70, and DNA-PK. HDACs are also implicated as essential components in the DNA repair process itself. Besides the radiosensitizing mechanism of intervention of DNA repair, other possible mechanisms including cell cycle redistribution, acetylation of Hsp90, increased apoptosis, and decreased survival signals are also suggested. Some miRNAs also regulate the radiosensitivity of tumor cells. Inhibition of miR-34 expression or function, downregulation of miR-155, upregulation of miR-18a, Overexpression let-7g or knocking down LIN28B, and ectopically overexpressed miR-10 in cells with low endogenous miR-101 level increase the response of cells to irradiation. For radiation-resistant cancer cells, miR-7 sensitizes the radiation for cells which activated EGFR-PI3K-AKT signaling pathway.

HDAC as a therapeutic target for treatment of endometrial cancers.

Accumulating evidence suggested that epigenetic changes such as promoter-specific DNA hypermethylation and histone deacetylation cause tumor suppressor gene silencing and contribute to malignant transformation. Treatment of cancer cells with HDAC inhibitors can reactivate the expression of silenced genes, block the cell cycle, and induce cell apoptosis. In vitro experiments in cancer cell cultures and in vivo studies using mouse xynograft model have shown that HDAC inhibitors deliver potent anti-cancer effects. Clinical trials have led to approval of SAHA (Vorinostat) for treatment of lymphoma. Endometrial cancer (EC) is the most frequent malignancy in women's reproductive tract. EC is known for extensive epigenetic alterations, including overexpression of HDAC and DNMT enzymes, and the frequent epigenetic silencing of DNA repair genes such as MLH1, tumor suppressor genes PTEN, and progesterone receptor, which suggests a potentially high sensitivity of this type of cancer to HDAC inhibitors. Indeed, studies from many laboratories using various models have shown that HDAC inhibitors are promising chemotherapy reagents for endometrial cancers. This review summarizes the results from these studies, with an emphasis to provide an update on the new findings from new drugs. Background information on HDAC expression in EC, and features of HDAC inhibitors are presented based on their relevance to our focused topic. The combined application of HDAC inhibitors with radiation therapy and other conventional therapeutic reagents are also discussed.

Facile assembly of an unusual lead borate with different cluster building units via a hydrothermal process.

Pb(BO2)2·H2O as sources of B and Pb via a simple hydrothermal process provided the first binodal 5,9-connected lead borate, Pb6B4O11(OH)2 (1). Compound 1 crystallizes in the orthorhombic space group Pnma. The crystal structure is composed of different cluster building units of B4O9 and Pb6O4. Compound 1 has an optical band gap of 3.24 eV.

[Expression purification and clinical application of high-risk HPV16 E4 gene].

OBJECTIVE: To determine the relationship between serum antibody against HPV16 E4 and cervical cancer, and to construct HPV 16 E4 protein expression vector as an antigen to detect its corresponding serum antibody among different populations. METHODS: HPV16 E4 early gene was ligated into pRSET-A expression vector. The constructed plasmids were transformed into BL21 (DE3)cells, and induced to express HPV 16 E4 protein by isopropylthio-beta-D-galactoside (IPTG). The expressed E4 inclusions were denatured, purified through Ni-column, and renatured. After the activity was revealed, antibodies against HPV 16 E4 in the sera from healthy women and patients with chronic cervicitis and cervical cancer were respectively determined by enzyme-linked immunosorbent assay (ELISA) using the fusion protein as the antigen. RESULTS: HPV 16 E4 fusion protein of Mr 15*10(3) was expressed by pRSET-16E4 after IPTG induction. The fusion protein accounted for 30% of the total bacterial proteins and expressed as inclusive body. After purification with Ni-NTA agarose resin, the recombinant protein revealed purity of 95%, and activity of the renatured protein was identified by ELISA. The serum antibody-positive rate of HPV 16 E4 was 10.00%, 39.13% and 28.13%, respectively in 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients. The antibody-positive rate in cervical cancer patients and chronic cervicitis patients were significantly higher than that in healthy women (P<0.01), while the difference between the antibody-positive rate in cervical cancer patients and chronic cervicitis patients was not significant. CONCLUSION: HPV 16 E4 protein expressed from pRSET-A/BL21 can be used in serological studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E4 is present in a significantly higher percentage in cervical cancer and chronic cervicitis patients than in healthy women.

[Expression, purification, and serum antibody detection of HPV16 E6 in cervical cancer patients].

BACKGROUND & OBJECTIVE: Human papillomavirus type 16 (HPV16) is the predominant high-risk type of HPV in cervical cancer tissues. Serum antibody responded to HPV16-related proteins is associated with the development of cervical cancer. This study was to construct and purify recombinant HPV16 E6 protein, detect its corresponding serum antibody among different populations, and explore the correlation of HPV16 E6 serum antibody reaction to cervical cancer. METHODS: HPV16 E6 early gene was constructed into pRSET-A expression vector. The plasmids were transfected into BL21 (DE3) cells, which were induced to express HPV16 E6 protein by isopropylthio-beta-D-galactoside (IPTG). E6 inclusions were denatured, purified through Ni column, and renatured. After the activity of HPV16 E6 protein being identified, the antibodies against HPV16 E6 in serum samples from 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients were determined by ELISA using the fusion protein as antigen. HPV DNA genotype was estimated in cervical cancer tissues by fluorescence polarization. RESULTS: HPV16 E6 fusion protein of Mr 24x10(3) was expressed in pRSET-16E6 after induction of IPTG. The fusion protein was accounted for 22.3% of total bacterial proteins, and expressed as inclusive body. After purification with Ni-NTA agarose resin, the purity of the recombinant protein was over 95%, and its activity was identified by ELISA. The antibody-positive rate was significantly higher in cervical cancer patients than in healthy women and chronic cervicitis patients (31.2% vs. 5.0% and 6.5%, P<0.01). In the 32 cervical cancer patients, the positive rates of HPVs DNA and HPV16 DNA in cancer tissues were 90.61% and 46.88%. The antibody-positive rate of HPV16 E6 was higher in HPV16 DNA-positive cervical cancer patients than in HPV16 DNA-negative patients (46.7% vs. 17.6%), but the difference was not significant. CONCLUSIONS: HPV16 E6 fusion protein obtained from pRSET-A/BL21 can be used in serologic studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E6 is more common in cervical cancer patients than in healthy women and chronic cervicitis patients.

[Detection of HPV 58 and cloning of its E7 gene in cervical cancer].

OBJECTIVE: To detect HPV 58, a common type of human papillomavirus (HPV), clone and express its E7 gene from biopsy specimens of cervical cancer. METHODS: HPV 58 from 58 biopsy tissues of cervical cancer was detected by GP5+/GP6+ PCR followed by template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP). HPV 58 E7 gene was amplified from one HPV 58-positive sample, and then cloned into pGEM-T Easy vector. The recombinant plasmid, HPV58E7-pGEM-T was confirmed by sequencing. Subsequently, E7 gene was cloned into prokaryotic expression vector pRSET-A. The constructed pRSET-58E7 plasmids were transfected into BL21(DE3) cells, and induced to express 58 E7 protein by IPTG. RESULTS: Among the 58 biopsy tissues of cervical cancer, 10 were HPV 58-positive, accounting for 19.2% of 52 HPV-positive cases. HPV 58 E7 gene was amplified from one HPV 58-positive sample. The constructed plasmids were identified containing HPV58 E7 gene by restriction enzyme analysis and sequencing. SDS-PAGE analysis showed that HPV58 E7 His6 fusion protein of M(r) 16 x 10(3) was expressed by pRSET-58E7 after induction by IPTG. The fusion protein accounted for 30% of total bacterial proteins. CONCLUSION: HPV 58 is not uncommon in Chinese women with cervical cancer in Shaanxi province. Constructed HPV58 E7 recombinant plasmids can be effectively expressed in E.coli, which may provide a tool in diagnosis and vaccine design for HPV of HPV58-associated tumors.

Detection and genotyping of human papillomavirus DNA in cervical cancer tissues with fluorescence polarization.

To evaluate the type-specific prevalence of eight common types of human papillomavirus (HPV) in patients with cervical cancer living in Shanxi, China, with fluorescence polarization detection, crude DNA extracted from 137 samples of early-stage cervical cancer (within stage IIa) and chronic cervicitis was subjected to HPV L1 consensus GP5+/GP6+ system. Then, the HPV-positive products identified by GP5 + /GP6+ PCR were genotyped based on template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP): the PCR products were respectively hybridized with designed type-specific probes within the GP5+/GP6+ amplicons for eight common HPV types (HPV 6, 11, 18, 16, 31, 33, 35, and 58), and specific fluorescence-labeled ddNTPs (TAMRA-ddTTP or R110-ddGTP) were directly incorporated to the ends of the corresponding probes under directing of the corresponding template in PCR products, which was reflected and read by high FP values for TAMRA or R110. HPV DNA was detected in 38.89% (28/72) cases of chronic cervicitis, and 87.69% (57/65) cases of cervical cancer. There was a significant difference in HPV prevalence between these two groups. The four commonly identified types in patients with cervical cancer were HPV 16 (45.6%), HPV 18 (22.8%), HPV 58 (17.5%), and HPV 31 (7.02%), and in those with chronic cervicitis were HPV 16 (35.7%), HPV 11 (32.1%), HPV 6 (21.4%), and HPV 18 (10.7%). 57.14% of HPV types detected in patients with chronic cervicitis were high-risk types. HPV 16 was the most common viral type identified in both groups. Type-specific prevalence of HPV DNA has some characteristics in patients with chronic cervicitis and cervical cancer living in Shanxi, China and the worldwide uncommon type HPV 58 is relatively common in both kinds of cases. The high prevalence of HPV 58 in Chinese women should been considered in diagnosis and vaccine designs of HPV.

A high throughout assay for human papillomavirus genotypes with fluorescence polarization.

OBJECTIVE: To develop a simple, cheap, quick, accurate and practical method for a high throughout genotypes assay of human papillomavirus (HPV) DNA. METHODS: Crude DNA was extracted by a simplified proteinase K digesting method. HPV common conservative primers: GP5+/6+ system was used to amplify HPV DNA in 127 samples of condylomata acuminatum (CA) and cervical scrapes by PCR, then the PCR product was assayed using a template directing terminator incorporation (TDI) and genotypes were detected with fluorescence polarization (FP). Major HPVs type-specific probes (HPV6, 11, 16, 18, 31, 33, 35 and 58) designed by us were hybridized with the specific PCR products and a special fluorescent ddNTP terminator was directly added to the end of the probe under direction of specific PCR products. The results were measured with FP and compared with the results of the DNA sequence. RESULTS: Compared with the results of DNA sequencing, the results detected with fluorescence polarization were all correct. The proposed method could detect more than one type of HPV infection, but DNA sequencing method could not. The positive rate of HPV was 100% in 78 CA biopsies. Among them, there were 14 HPV double infections [HPV6B and 11 (9 cases), HPV11 and 16 (4), HPV11 and 18 (1)], 5 HPV triple infections [HPV6B, 11 and 16 (4), HPV11, 16 and 18 (1)], and one HPV quadruple infection (HPV6B, 11, 16 and 18). The positive rate of HPV was 77% in the 49 cervical scrapes. Six HPV double infections [HPV6B and 11 (2), HPV11 and 16 (1), HPV6B and 16 (1), HPV16 and 18 (1), HPV18 and 58 (1)], 3 HPV triple infections [HPV6B, 11 and 16 (2), HPV11, 16 and 18 (1)] and one HPV quadruple infection (HPV6B, 11, 16 and 18) were detected in cervical cancer scrapes. CONCLUSIONS: The proposed method allowed a high throughout, special, simple, rapid, automatic and economical detection of HPV-DNA genotyping without a use of labeling probes. It can detect multiple HPV genotype infection and will be and useful tool in HPV genotype screening.