SOX21-AS1 Augmented Cervical Cancer Growth by Triggering FZD3 to Activate the Wnt/ß-Catenin Signaling Pathway.

Long non-coding RNA (LncRNA) SOX21-AS1 has been reported that it plays an important role in biological processes of several cancers. However, how it functions in cervical cancer (CC) still remain unclear. This investigation seeks to explore the impact of SOX21-AS1 on CC cell proliferation, invasion and migration and its association to the FZD3 and the Wnt/ß-catenin signaling pathway. SOX21-AS1 expression levels were detected using real-time quantitative PCR in 20 cases of cervical cancer together with its adjacent tissues and several cervical cancer cell lines. Transgenic technology and functional experiments were conducted to confirm the carcinogenic properties of SOX21-AS1, and western blot was utilized to analyze the regulatory network composed of SOX21-AS1, FZD3 and the Wnt/ß-catenin signaling pathway in CC. Through bioinformatics analysis, we found that the expression of SOX21-AS1 in CC was the highest among 16 kinds of tumor tissues. Moreover, clinical specimens confirmed that both CC tissues and cell lines possessed elevated SOX21-AS1 expressions (P < 0.01). CC cells which stably expressed upregulated SOX21-AS1 were noted to possesses higher rates of metastasis, invasion and proliferation, lower apoptotic rates and higher expression of FZD3,ß-catenin and c-myc (P < 0.01). Conversely, the use of small interfering RNA to inhibit the expression of SOX21-AS1 yielded the opposite results (P < 0.01). SOX21-AS1 functions as an oncogenic LncRNA which enhances CC cell metastasis, invasion and proliferation through FZD3 upregulation via Wnt/ß-catenin-signaling pathway activation. This LncRNA may represent an important biomarker for CC patients.

Aberrant Methylation of the SOX21-AS1 Promoter Region Promotes Gene Expression and Its Clinical Value in Cervical Cancer.

Cervical cancer is the fourth most common female cancer worldwide. Long non-coding RNAs (lncRNAs), such as SOX21-AS1, play pivotal roles in the progression and metastasis of cancer. We previously described that SOX21-AS1 was hypomethylated in cervical cancer (CC) and aimed to further explore the relationship between methylation of the SOX21-AS1 promoter and CC using clinical cervical samples. Pyrosequencing was performed to detect the methylation status of the SOX21-AS1 promoter in 33 cervical specimens. Additionally, expression levels of related genes in 43 clinical cervical specimens were measured using quantitative real-time PCR (qRT-PCR). The SOX21-AS1 promoter was significantly hypomethylated in CC (P < 0.01). SOX21-AS1 hypomethylation was also significantly associated with an advanced Federation of Gynecology and Obstetrics (FIGO) stage (P < 0.01). The expression levels of SOX21-AS1 and SOX21 were noted to be higher in cancer vs. normal cervix (all P < 0.001). Moreover, the expression of SOX21-AS1 was positively correlated with SOX21 in all samples (r = 0.891, P < 0.001). Methylation statue of the SOX21-AS1 promoter region was negatively correlated with the expression levels of SOX21-AS1 and SOX21 (SOX21-AS1, r = - 0.628; SOX21, r = - 0.648; both P < 0.001). The methylation status of SOX21-AS1 displayed promising diagnostic potential for CC, exhibiting good sensitivity (100.0%) and specificity (69.2%), with an area under the curve of 0.846. In addition, bioinformatic analyses identified a potential link between SOX21-AS1 and the Wnt signaling pathway. Furthermore, methylation status of SOX21-AS1 was negatively correlated with ß-catenin/c-myc/cyclin D1 mRNA levels (rs = - 0.529, - 0.462 ,and - 0.383, respectively, P < 0.05). Our findings illuminated that lncRNA SOX21-AS1 showed hypomethylation in cervical cancer and SOX21-AS1 could serve as a novel biomarker for CC diagnosis or a potential therapeutic target.

Study on the relationship between methylation status of HPV 16 E2 binding sites and cervical lesions.

BACKGROUND: The aim of this study was to investigate the methylation status of E2BSs in the HPV 16 long control region (LCR) in clinical cervical samples. METHODS: Methylation status of the four E2BSs in 43 clinical cervical samples with HPV 16 infection was quantitatively detected using pyrosequencing. Meanwhile, Quantivirus® HPV E6/E7 RNA 3.0 assay (bDNA) was used to detect E6/E7 mRNA levels in the corresponding specimens. RESULTS: Our results showed that methylation status of E2BS1, 2 and 4 sites in high-grade squamous intraepithelial lesions (HSIL) and cervical cancer were significantly higher than that of asymptomatic HPV 16 infection and low-grade squamous intraepithelial lesions (LSIL) (all P < .05). Furthermore, methylation status of HPV 16 E2BS1 and 2 was positively correlated with E6/E7 mRNA levels (rs = 0.529 and 0.512 respectively, P < .01). Receiver operating characteristic curve analysis was used to compare the diagnostic performance of E2BSs methylation. When the Youden index was the maximum value, the methylation level of E2BS1 and E2BS2 all demonstrated optimum diagnostic sensitivity of 77.8%, and specificity of 80% and 92%, respectively. CONCLUSIONS: The methylation status of E2BS1 and 2 may have utility as diagnostic markers for the severity of cervical lesions in the future.

An improved method for primary culture of normal cervical epithelial cells and establishment of cell model in vitro with HPV-16 E6 gene by lentivirus.

Normal human cervical epitheliums infected with HPVs gene in vitro are underlying molecular models to investigate physiological mechanisms of cervical epithelia and cervical disease. The current study aimed to establish a modified culture method for cervical epithelium and explore the feasibility of transfection with HPV-16 E6 gene mediated by lentivirus in primary cervical cells. The cells were dissociated enzymatically using Dispase II combined with 0.25% Trypsin-0.01% ethylenediamine tetracetic acid (EDTA) or Collagenase I. The detached effectiveness of Dispase II at different times was compared. Isolated cells were cultured and subcultured in modified keratinocyte serum-free medium (K-SFM) supplemented with 5% fetal bovine serum (FBS) or K-SFM alone. Cytokeratin was used as the identification of cervical epitheliums. Proliferative capacity and growth curve of cervical epitheliums were evaluated by cell counting kit-8 (CCK-8). The cells at passages 3 were used to infect with HPV-16 E6 gene by lentivirus. The expression of green fluorescent protein (GFP) presented in the infected cells was observed via fluorescence microscopy and the levels of E6 mRNA were detected by quantitative real-time PCR (qRT-PCR). The results indicate that cervical epithelial cells can be isolated successfully by Dispase II combined with 0.25% Trypsin-0.01% EDTA method for 20 hr and maintained for five or six passages in K-SFM medium with 5% FBS. The present study proposed a brief and high-yield protocol for isolation and culture of human cervical epitheliums. Moreover, an infected cell model with HPV-16 E6 gene mediated by lentivirus was established which can do duty for studies in vitro on the carcinogenic mechanism of HR-HPVs.

Detection of cervical intraepithelial neoplasia with HPVE6/E7 mRNA among women with atypical squamous cells of unknown significance.

OBJECTIVE: To compare E6/E7 mRNA and HPV DNA assays for evaluating women with atypical cells of undetermined significance (ASCUS). METHODS: The present prospective study enrolled patients with ASCUS undergoing HPV testing at Third Affiliated Hospital of Zhengzhou University, China, between September 1, 2013, and January 31, 2016. Patients with positive HPV DNA test results underwent screening by E6/E7 mRNA assay, and the accuracy of HPV DNA and E6/E7 mRNA testing were compared, with histology used for definitive diagnoses. RESULTS: In total, 591 patients with ASCUS underwent HPV DNA screening, with 455 and 136 having positive and negative results, respectively; 252 patients with positive results and 66 with negative results underwent biopsy and histology testing and were included in the study. The sensitivity of the E6/E7 mRNA assay was similar to that of HPV DNA testing (88.2%, 95% confidence interval [CI] 77.6-94.4 vs 90.7%, 95%CI 81.2-95.9; P=0.636) for the detection of cervical intraepithelial neoplasia grade 2+, and the specificity was higher (36.4%, 95%CI 29.6-43.9 vs 24.3%, 95%CI 19.1-30.3; P=0.006). The area under the receiver operating characteristic curve was greater for E6/E7 mRNA testing compared with HPV DNA testing (0.658 vs 0.588). CONCLUSION: The higher specificity of the E6/E7 mRNA assay means it could be a promising technique in the management of women with ASCUS.

Performance of the HPV-16 L1 methylation assay and HPV E6/E7 mRNA test for the detection of squamous intraepithelial lesions in cervical cytological samples.

HPV-16 L1 methylation and E6/E7 mRNA have suggested that they had close relationship with cervical neoplastic progression. This study aimed to evaluate the clinical performance of the HPV-16 L1 methylation assay and E6/E7 mRNA test for detecting high-grade cervical lesions (CIN2+). A total of 81 women with liquid-based cytology (LBC) samples, histological results, and positive HPV-DNA test for HPV type 16 only were included in this study. HPV-16 L1 methylation and E6/E7 mRNA levels were measured using methylation-sensitive high resolution melting (MS-HRM) analysis and Quantivirus®HPV E6/E7 RNA 3.0 assay (bDNA), respectively, in the same residue of LBC samples. The current date showed a positive correlation between the HPV-16 L1 methylation and the E6/E7 mRNA levels. The L1 methylation and mRNA levels both increased with disease severity. The mRNA test method showed higher sensitivity and NPV (98.0 and 91.7% vs. 89.8 and 80.8%), while lower specificity and PPV (34.4 and 69.6% vs. 65.6 and 80.0%), than the L1 methylation assay for detecting histology-confirmed CIN2+. When using the detection method of mRNA test combined with L1 methylation assay, we obtained a sensitivity of 89.8% and a specificity of 71.9%. These findings suggest that assessment of HPV-16 L1 methylation testing combined with E6/E7 mRNA testing may be a promising method for the triage of women with HPV type 16 only.

High-resolution melting analysis of HPV-16L1 gene methylation: A promising method for prognosing cervical cancer.

OBJECTIVE: Methylation-sensitive high-resolution melting (MS-HRM) is a new technique for DNA methylation analysis, but it is rarely used for the detection of viral DNA methylation. In this study, we investigated the HPV-16L1 gene methylation that is detected by MS-HRM as a potential biomarker for prognosing cervical dysplasia and cancer. DESIGN AND METHODS: A total of 114 HPV-16 infected patients (normal (17), CIN1 (25), CIN2 (29), CIN3 (32), SCC (11)) who underwent liquid-based cytology test and biopsy were included in this study. 17 cases with HPV-16 infection and negative cytologic and histologic results served as the control group. The HPV-16L1 gene methylation statuses of these samples were investigated using a methylation-sensitive high-resolution melting (MS-HRM) assay after bisulfite modification. RESULTS: The HPV-16L1 gene methylation statuses of all the 114 specimens were successfully detected by MS-HRM, and we observed increasing methylation levels in severe lesions, as determined using histologic assays. In addition, the methylation levels of CIN2+ (CIN2, CIN3 and SCC) were significantly higher than that of CIN2- (normal and CIN1, P<0.001). When taking CIN2+ as the reference, our HPV-16L1 DNA methylation assay achieved 91.7% sensitivity and 59.5% specificity, respectively. CONCLUSIONS: The results of the present work demonstrated that HPV-16L1 gene methylation was closely associated with cervical precancerosis and cancer. Moreover, using MS-HRM to detect HPV-16L1 gene methylation may be a powerful assay for the triage of HPV-16-positive females, which could identify patients with high risk of invasive cancer.

Hypermethylation of estrogen receptor-alpha gene in atheromatosis patients and its correlation with homocysteine.

OBJECTIVE: To investigate the aberrant DNA methylation in promoter region of estrogen receptor alpha (ERalpha) in atherosclerosis (As) and the possible involvement of homocysteine (Hcy) in its pathogenesis. METHODS: The blood samples were collected from 54 patients with As approved by carotid colorized ultrasound examination and 28 healthy control subjects. The methylation status of CpG islands in ER-alpha gene promoter region of genome DNA was analyzed by nested-methylation-specific PCR (nMSP). tHcy was examined by fluorescent-biochemical method. Spearman rank correlation was used to analyse the relationship between the degree of methylation in ER-alpha gene and the level of tHcy. Cultured smooth muscle cells of Homo sapiens were treated by Hcy with different concentrations and different treating time, again the DNA methylation status was assayed by nMSP, and the proliferation of SMC was assayed by MTT. RESULTS: Hypermethylation of ER-alpha gene promoter region was found in 38 cases of atherosclerosis patients, and the methylation frequency was 70.4%. While in healthy controls, just 8 of 28 samples hypermethylation was found, only 28.6% methylation frequency was detected, much lower than the one in atherosclerosis group (p<0.05). Meanwhile, the level of tHcy in atherosclerosis group was significantly higher than that in control group (p<0.05). The spearman rank correlation analysis explored an obvious correlation between the degree of methylation in ER-alpha gene and the level of tHcy (r=0.809, p<0.05), and the severity of atherosclerotic lesion was also heightened along with the increment of plasma level of tHcy. The cultured SMCs treated by Hcy resulted in de novo methylation in promoter region of ERalpha gene with a concentration and treating time-dependent manner, and a dose-dependent promoting effect on SMC proliferation. The in vivo and in vitro data coincidently showed that the Hcy could promote the hypermethylation of ERalpha gene, which may be an important mechanism for the pathogenesis of As. CONCLUSION: Hypermethylation of CpG islands in ER-alpha gene promoter region was found in much higher frequency in atherosclerosis patients, which is positively correlated with the increased level of plasma tHcy and the severity of atherosclerotic lesion, and the in vitro experimental results further extended above clinical data that HHcy can lead to the hypermethylation of ER-alpha gene, and hence to promote the occurrence and development of As.

[Hypermethylation of estrogen receptor-alpha gene and high homocysteine in atheromatosis patients].

OBJECTIVE: To investigate the methylation status in promoter region of ER-alpha gene and the relationship beteen the degree of methylation in ER-alpha gene and the level of total plasma homocysteine(tHcy) in atherosclerosis patients. METHODS: 82 cases including 54 atherosclerosis patients and 28 healthy control subjects were selected under carotid colorized ultrasound examination. The methylation status of CpG islands in ER-alpha gene promoter region were analyzed by nested-methylation-specific PCR (nMSP). tHcy was examined by fluorescent-biochemical method. Using spearman rank correlation to analyse the relatinship between the degree of methylation in ER-alpha gene and the level of tHcy. RESULTS: Hypermethylation of ER-alpha gene promoter region was found in 38 cases of atherosclerosis patients, the methylation frequency was 70.4%. While the only 28.6% methylation frequency was detected in healthy controls, just 8 of 28 samples were found hypermethylation, much lower than the one in atherosclerosis group (P < 0.05). Meanwhile, the level of tHcy in atherosclerosis group was significantly higher than that in control group (P < 0.05). The spearman rank correlation analysis explored an obviously correlation between the degree of methylation in ER-alpha gene and the level of tHcy (r = 0.809, P < 0.05), and the severity of atherosclerotic lesion was also heightened along with the increment of plasma level of tHcy. CONCLUSION: Hyermethylation of CpG islands in ER-alpha gene promoter region was found in high frequency in atherosclerosis patients, which is positive correlated with the increased level of plasma tHcy and the severity of atherosclerotic lesion. This clinical data further extended our in vitro experimental result that HHcy can lead to the hypermethylation of ER-alpha gene, and hence to promote the occurrence and development of AS.

[Clinical investigation of the protective effects of potassium magnesium aspartate against arrhythmia and its possible anti-oxidative mechanism].

OBJECTIVE: To investigate the protective effects of potassium magnesium aspartate against oxidative stress status and lipid oxidative damage in the patients with angina and arrhythmia due to coronary artery disease, its therapeutic effect on arrhythmia and its possible mechanism. METHODS: With single blind protocol, 98 patients with angina and arrhythmia due to coronary artery disease were randomly divided into (1)Experiment group (n = 65), who received routine remedy for coronary heart disease plus potassium magnesium aspartate. (2)Control group (n = 33), who received only routine therapy for coronary heart disease without potassium magnesium aspartate. Reduced glutathione (GSH), oxidized glutathione (GSSG), malondialdehyde (MDA) and oxidized low density lipoprotein (ox-LDL) in plasma of all patients were examined before and one week after treatment, all patients with arrhythmia were equipped with Holter for continuous monitoring of cardiac rhythm. RESULTS: After one week's treatment, the GSH level in plasma of experiment group and the ratio of GSH and GSSG (GSH/GSSG) were significantly increased comparing with control group (both P<0.01), while GSSG, MDA and ox-LDL levels significantly lowered comparing with control group(all P<0.01). The premature beats diminished 86.5% in experiment group, but the decrease rate in control group was only 47.4% (P<0.01). The improvement in indexes of oxidative stress status (including GSH/GSSG, MDA and ox-LDL) and the reduction of premature beats showed close correlation with each other (all P<0.01). No adverse effects of the drug were found after one week of administration of Potassium magnesium aspartate. CONCLUSION: Potassium magnesium aspartate can strikingly improve oxidative stress status and decrease lipid oxidative damage in the patients with coronary heart disease, and the frequent premature beats were also significantly reduced by potassium magnesium aspartate. The analysis of above results reveals an intrinsic relationship between the improvement of oxidative stress status and the good therapeutic effects on frequent premature beats by potassium magnesium aspartate, which may suggest an involvement of oxidative stress in the pathogenesis of arrhythmias.

[Plasma glutathione of patients with coronary heart disease measured by fluorospectrophotometer].

The standard samples of reduction form glutathione (GSH) and oxidization form glutathione (GSSG) were scanned with full-wavelength range to determine the excitation wavelength lambda(ex) 334.4 nm, the emission wavelength lambda(em) 424 nm, and the spectral bandwidth 5 nm respectively. Phosphate buffer saline (PBS) of pH 8. 3 served as buffer solution. GSH was incubated for 30 min with 100 microL o-phthaldehyde (OPA) of 10 mmol x L(-1) methyl alcohol solution for derivatization, and then fluorescence intensities were measured. With standard glutathione concentration being independent variable and fluorescence intensity being dependent variable, the linear equations for GSH and GSSG were deduced: Y(GSH) = 6.9 + 8.6X (r2 = 0.994) and Y(GSSG) = 6.2 + 17.2X (r2 = 0.999). Standard curves were done hereby. The plasma glutathione of three groups was then measured, and GSH/ GSSG redox potential was calculated according to Nernst equation. The results indicated that, from normal control group to prophase coronary heart disease group, then to coronary heart disease group, the GSH and GSH/GSSG ratio gradually reduced, GSSG and GSH/GSSG redox potential gradually increased (more positive) (all P < 0.05), and the redox potential shifted to oxidization direction along with the development of coronary heart disease. This fluorospectrophotometry method showed simple operation, and fine precision and accuracy.

[The relationship between oxidized form glutathione, coenzyme II and carotid artery atherosclerosis].

OBJECTIVE: To explore the relationship between plasma redox status and atherosclerosis. METHODS: IVUS was performed in common carotid in the neck of 167 patients with heart diseases. Patients were divided into three groups: plaque-forming group (A, n = 79), intima-thickening group (B, n = 52) and control group (C, n = 36). Plasma glutathione (reduced form GSH and oxidized form GSSG), nicotinamide adenine dinucleotide phosphate (reduced form NADPH and oxidized form NADP(+)), oxidized low density lipoprotein (ox-LDL) and malondialdehyde (MDA) were measured in all patients. The GSH/GSSG and NADPH/NADP(+) redox potential were calculated according to Nernst equation, and correlation analysis performed. RESULTS: GSH and GSH/GSSG gradually reduced and GSH/GSSG redox potential gradually increased in proportion to the thickening of artery intima (from Group C to Group A, P < 0.05). Similar but milder results were shown for NADPH and NADPH/NADP(+) redox status. The products of oxidative stress ox-LDL and MDA also increased significantly (P < 0.05) in proportion to the thickening of artery intima. GSH/GSSG redox potential is positively correlated to ox-LDL (P < 0.05). The redox status shifted to oxidizing direction in proportion to the intima thickness. CONCLUSION: The imbalance of plasma redox status deviating to oxidation might be implicated in oxidized injury of lipid, intima thickening and atherosclerosis progress.

[Effects of xuezhikang capsules on vascular endothelial function and redox status in patients with coronary heart disease].

OBJECTIVE: To explore the effects of Xuezhikang Capsules (ZXKC) and probucol on blood lipids, vascular endothelial functions and redox status in patients with coronary heart disease. METHODS: One hundred and twelve patients with coronary heart disease were randomly divided into XZKC-treated group and probucol-treated group, 56 in each. Before and after 8-week treatment, the blood levels of total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C), nitric oxide (NO), endothelin-1 (ET-1), reduced glutathione (GSH) and oxidized glutathione (GSSG) were all measured in both groups. The GSH/GSSG redox potential (Eh) was calculated according to the Nernst equation. RESULTS: In the XZKC-treated group, the blood levels of TC, LDL-C and TG were significantly decreased after 8-week treatment as compared with those before treatment. The blood levels of TC and LDL-C were also significantly decreased in the probucol-treated group as compared with those before treatment. In the XZKC-treated group, the blood levels of ET-1 and GSSG and the GSH/GSSG Eh after treatment were all significantly lower than those before treatment, whereas the blood levels of GSH and NO, the NO/ET-1 ratio, and the GSH/GSSG ratio after treatment were all significantly higher than those before treatment. CONCLUSION: The XZKC or probucol treatment can yield a significant decrease in blood lipids in patients with coronary heart disease. Furthermore, XZKC exerts effective protection on vascular endothelial function, and can make GSH/GSSG redox status shift towards deoxidation.