Analysis of somatic mutations and key driving factors of cervical cancer progression.

We investigated the somatic mutations and key driving factors of cervical cancer by whole exome sequencing . We found 22,183 somatic single nucleotide variations (SNVs) in 52 paired samples. Somatic SNVs in cervical cancer were significantly higher than those in high-grade intraepithelial lesion and low-grade squamous intraepithelial lesion groups (P < 0.05). C → T/G accounted for 44.12% of base substitution. Copy number variation (false discovery rate < 0.05) was found in 57 chromosome regions. The three regions with significant differences between cervical cancer and non-cervical cancer groups were 1q21.1, 3q26.33, and 13q33.1, covering genes related to tumor proliferation, differentiation, and apoptosis. The frequency of human papillomavirus (HPV) insertion/integration and the number of "tCw" mutations in the cervical cancer group were significantly higher than those in the non-cervical cancer group (P < 0.05). The total number of mutations was positively correlated with the number of "tCw" mutations (R 2 = 0.7967). HPV insertion/integration (OR = 2.302, CI = 1.523-3.589, P = 0.0005), APOBEC enrichment (OR = 17.875, CI = 2.117-150.937, P = 0.001), and HLA-B*39 in HLA-I (OR = 6.435, CI = 0.823-48.919, P = 0.0042) were risk factors for cervical cancer, while HLA-DQB1*05 in HLA-II was a protective factor (OR = 0.426, CI = 0.197-0.910, P = 0.032). Conclusively, HPV insertion/integration, APOBEC mutagenesis, and HLA polymorphisms are high-risk factors for cervical cancer and may be causes of genome instability and somatic mutations. This study provides experimental data for revealing the molecular mechanism of cervical cancer.

Correlation of APOBEC3G Polymorphism with Human Papillomavirus (HPV) Persistent Infection and Progression of Cervical Lesions.

BACKGROUND We studied the effect of APOBEC3G on persistent human papillomavirus (HPV) infection and the correlation between APOBEC3G polymorphism and HPV persistent infection and cervical disease progression in Uygur women in China. MATERIAL AND METHODS From January 2015 to December 2017, we enrolled 529 Uygur ethnic group patients with HPV infection. SIHA cells were transfected with APOBEC3G. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to detect mRNA and protein expression levels of APOBEC3G and HPV E6 and p53. Exon 3 of APOBEC3G was sequenced by first-generation sequencing. RESULTS The mRNA and protein expression levels of APOBEC3G in the cervical cancer group were significantly higher than in the cervical intraepithelial neoplasia (CIN) group (p<0.05). The mRNA and protein expression levels of APOBEC3G in the CIN group were significantly higher than in the non-cervical lesions group (p<0.05). The mRNA and protein expression levels of HPV E6 in SIHA cells transfected with APOBEC3G were significantly lower than in the control group and the no-load group (p<0.05), and the mRNA and protein expression levels of p53 were significantly higher than in the control group and the no-load group (p<0.05). There was a polymorphic locus rs5757465 on exon 3 of APOBEC3G in Uygur women, and this rare CC type was a risk factor for cervical lesions and cervical cancer (OR=3.714, 95%CI: 1.916-7.202, p<0.05). CONCLUSIONS APOBEC3G is involved in continuous HPV infection, cervical prelesions, and the development of cervical cancer, and the rare genotype (CC) of APOBEC3G may be one of the factors causing cervical lesions in Uygur women who have HPV infection.

Prognosis and related factors of HPV infections in postmenopausal Uyghur women.

With the aim to explore the characteristics of persistent HPV infections in postmenopausal Uyghur women and analyse the possible related risk factors, from September 2012 to September 2013; postmenopausal Uyghur women with HPV positive and pathologically diagnosed as non-cervical intraepithelial neoplasia (CIN) lesions and non-cervical cancer were recruited. Their clinical course was closely followed up for 24-36 months, and the risk factors were analysed by a logistic regression model. One hundred and sixteen positive women were followed for 36 months. The total persistent HPV infection rate was 67.9%, and the type-specific persistent infection rate was 73.7% at 36 months. Nine (32.1%) women were naturally cleared of their HPV infection at 36 months. We found that an HPV16 infection and an HPV58 infection, and time since menopause over 2 years were closely related with a persistent HPV infection. More attention should be paid to the women above 2 years of menopause who were infected with HPV16 and HPV58 in their further cervical carcinoma screening. Impact statement What is already known on this subject? Previous study revealed that menopause was a risk factor for a persistent HPV infection in Uyghur women. What do the results of this study add? The present study presented the characteristics of HPV persistent infection and the risk factors in Uyghur postmenopausal women. More attention should be paid to the women above 2 of years of menopause who are infected with HPV16 and HPV58. What are the implications of these findings for clinical practice and/or further research? This study would offer a theoretical basis for a better screening design, especially the women above 2 years' menopause who have been infected with HPV16 and HPV58 in the Xinjiang region.

MicroRNA-505 predicts prognosis and acts as tumor inhibitor in cervical carcinoma with inverse association with FZD4.

PURPOSE: We investigated the expression and mechanisms of microRNA-505 (miR-505) and its downstream target gene Frizzled-4 (FZD4) in cervical cancer. METHODS: miR-505 expression was evaluated by qRT-PCR in cervical cancer cell lines and human carcinomas. Cancer patients' clinicopathological factors and survival were analyzed based on their tumorous miR-505 levels. Ca-Ski and HeLa cells were transduced with lentivirus to upregulate or downregulate miR-505. Their impacts on cervical cancer were evaluated by in vitro proliferation, invasion and in vivo tumorigenicity assays, respectively. Target gene of miR-505, FZD4, was evaluated by dual-luciferase reporter assay. Its expression in cervical cancer cell was evaluated by qRT-PCR. FZD4 was either upregulated or downregulated in cervical cancer cells to further assess its impact on modulating cervical cancer development in vitro. RESULTS: MiR-505 is lowly expressed in cervical cancer cell lines and human carcinomas. Low tumorous miR-505 expression was associated with patients' advanced tumor stage and short survival. In Ca-Ski and HeLa cells, lentivirus-mediated miR-505 upregulation suppressed cancer proliferation and invasion in vitro, and tumorigenicity in vivo, whereas miR-505 downregulation had no functional effects. FZD4 was confirmed to be a downstream target of miR-505, and found to be upregulated in cervical cancer. Genetic modification of FZD4 in cervical cancer cells yielded a significant change in cancer growth, as FZD4 upregulation suppressed whereas FZD4 downregulation promoted cervical cancer proliferation and invasion In vitro. CONCLUSION: MiR-505 may act as a cancer inhibitor and prognostic factor in cervical cancer. FDZ4 is reversely expressed as miR-505, and has dramatic regulatory function in cervical cancer.

Correlation between Methylation of Human Papillomavirus-16 L1 Gene and Cervical Carcinoma in Uyghur Women.

BACKGROUND: This study aims at determining the correlation between CpG methylation in human papillomavirus (HPV)-16 L1 and the persistent infections and development of cervical carcinoma in Uyghur women. METHODS: Among the 4,364 Uyghur women, specimens were collected from 145 (3.3%) HPV-16 single infected cases, which were divided into 5 groups: transient infection (n = 32), persistent infection (n = 21, 12 months), cervical intraepithelial neoplasia (CIN) grade 1 (CIN1, n = 21), CIN2-3 (n = 33) and invasive cervical cancer (n = 38) groups. Methylation level in HPV-16 L1 was quantified by pyrosequencing, and values in the prediction and diagnosis of CIN2+ lesions were evaluated with receiver operating characteristic curves. RESULTS: With the progression of the disease, increased methylation was detected at 13 CpG sites, and a high methylation level was associated with the risk of CIN2+. The strongest related site was 6650 (OR 9.89, 95% CI 3.57-27.44). The area under ROC curve (AUC) of methylation at each CpG site to differentiate between CIN2+ and

[Death-associated protein kinase promoter (DAPK) hypermethylation in uterine cervical cancer and intraepithelial neoplasia in Uyghur nationality women].

OBJECTIVE: To investigate the methylation levels of death-associated protein kinase (DAPK) in Uyghur female patients with different cervical lesions in Xinjiang, and to discuss the relationship of the expression and significance of DAPK in normal cervix, chronic cervicitis, cervical intraepithelial neoplasia (CINI, CIN II/III) and invasive cervical squamous cell carcinoma. METHODS: 30 cases of normal cervix and chronic cervicitis, 30 cases of CINI, 30 cases of CINII/III and 30 cases of cervical squamous cell carcinoma were tested by methylation specific PCR (MSP). Expressions of DAPK in 30 cases of normal cervix and chronic cervicitis, 30 cases of CINI, 30 cases of CINII/III and 30 cases of cervical squamous cell carcinoma were assayed using immunohistochemical SP staining. RESULTS: The methylation rate of DAPK gene in normal cervix and chronic cervicitis was 3.33%, 10% in cervical intraepithelial neoplasia CINI, 36.7% in CINII/III, and 63.3% in invasive cervical squamous cell carcinoma. The methylation rate of DAPK in the SCC group was significantly higher than that in the other groups (P < 0.05). Aberrant promoter methylation of the DAPK gene was positively correlated with the degree of cervical lesions. The positive rate of DAPK protein in normal cervix and chronic cervicitis was 93.3%, 83.3% in cervical intraepithelial neoplasia CINI, 60.0% in CINII/III, and 33.3% in invasive cervical squamous cell carcinoma. The expression of DAPK in the SCC group was significantly lower than that in the other groups (P < 0.05). The positive rate of DAPK protein was negatively correlated with the degree of cervical lesions (r(s) = -0.603, P < 0.001). CONCLUSIONS: Methylation of DAPK is involved in the cervical carcinogenesis and DAPK gene promoter methylation occurs in the early development of cervical cancer in Uyghur women in Xinjiang. Detection of DAPK gene methylation may provide a basis for use in early detection of cervical cancer. DAPK protein expression is decreasing even disappears along with the progression of cervical lesions.

Correlation between TCRCalpha -560 C/T polymorphism and the clinical presentation of Uygur IgA nephropathy patients in XinJiang.

OBJECTIVE: To investigate the relationship between T cell receptor alpha chain constant gene (TCRCalpha) -560 C/T polymorphism and the clinical presentation of Uygur IgA Nephropathy patients in XinJiang. METHODS: TCRCalpha -560 C/T genotypes were determined by PCR-RFLP in 300 Chinese Uygur IgAN patients and 600 healthy Chinese Uygur control subjects. All subjects were classified, based on their genotype, into TT, CT and CC groups and their corresponding clinical presentation was analyzed. RESULTS: No significant difference was observed in the frequency of CC/CT/TT genotypes in patients and control subjects (chi2 = 0.904, P = 0.636). However, the incidence of intermittent microscopic hematuria and proteinuria is significantly higher in patients with CT genotype than CC and TT genotypes (chi2 = 33.978, P < 0.05). CONCLUSION: TCRCalpha-560 C/T gene polymerphism may be associated with the occurrence of intermittent microscopic hematuria and proteinuria in Chinese Uygur IgAN patients.

Prevalence and risk factors associated with chronic kidney disease in a Uygur adult population from Urumqi.

Evaluating the prevalence of kidney damage according to population-based studies in different communities has been limited in developing countries. We conducted a population-based screening study in Uygur people of Urumqi, aiming to identify the prevalence and associated risk factors of chronic kidney disease (CKD) in Uygur populations. A total of 2576 residents (>18 years) from four districts of Urumqi were interviewed from June 2007 to January 2009 and tested for haematuria, albuminuria and reduced renal function. Associations between age, gender, smoking, diabetes mellitus, hypertension, hyperuricaemia and kidney damage were examined. There were 2576 subjects enrolled in this study. After age correction, the prevalence of albuminuria, haematuria and reduced estimated glomerular filtration rate (eGFR) was 3.58%, 2.26% and 1.03%, respectively. Approximately 5.65% of the sample population had at least one indicator of kidney damage. Age, diabetes mellitus, hypercholesteremia, hyperuricaemia and hyperlipidaemia were independently associated with CKD. In the general Uygur adult population from Urumqi, 5.65% had either proteinuria, haematuria or reduced eGFR, indicating the presence of kidney damage, with an awareness of only 1.05%. The high prevalence and low awareness of CKD in this population suggest an urgent need for CKD prevention programs in Uygur people.

Prevalence and Risk Factors Associated with Chronic Kidney Disease in a Uygur Adult Population from Urumqi / 华中科技大学学报（医学）（英德文版）

Evaluating the prevalence of kidney damage according to population-based studies in different communities has been limited in developing countries. We conducted a population-based screening study in Uygur people of Urumqi, aiming to identify the prevalence and associated risk factors of chronic kidney disease (CKD) in Uygur populations. A total of 2576 residents (＞18 years)from four districts of Urumqi were interviewed from June 2007 to January 2009 and tested for haematuria, albuminuria and reduced renal function. Associations between age, gender, smoking,diabetes mellitus, hypertension, hyperuricaemia and kidney damage were examined. There were 2576subjects enrolled in this study. After age correction, the prevalence of albuminuria, haematuria and reduced estimated glomerular filtration rate (eGFR) was 3.58%, 2.26% and 1.03%, respectively. Approximately 5.65% of the sample population had at least one indicator of kidney damage. Age, diabetes mellitus, hypercholesteremia, hyperuricaemia and hyperlipidaemia were independently associated with CKD. In the general Uygur adult population from Urumqi, 5.65% had either proteinuria,haematuria or reduced eGFR, indicating the presence of kidney damage, with an awareness of only 1.05%. The high prevalence and low awareness of CKD in this population suggest an urgent need for CKD prevention programs in Uygur people.

[Relationship between human papillomavirus 16 infection and the expression of p33(ING1b), human telomerase reverse transcriptase in cervical squamous cell carcinoma of Uygur female in Xinjiang Uygur Autonomous Region].

OBJECTIVE: To explore the relationship between human papillomavirus (HPV) 16 infection and the expression of p33(ING1b), human telomerase reverse transcriptase (hTERT) in cervical squamous cell carcinoma of Uygur Female in Xinjiang Uygur Autonomous Region. METHODS: Polymerase chain reaction (PCR) and immunohistochemical methods were used to detect HPV16 infection and the expression of p33(ING1b) and hTERT in the normal control group (n=12), the patients with cervical intraepithelial neoplasm (CIN) (n=34), and the patients with cervical squamous cell carcinoma (SCC) (n=50). RESULTS: In the cervical tissues of Uygur female, the HPV16 infection rate was 0 in control group, 22.2% in the CIN 1 group, 44.0% in CIN 2 & CIN 3 group, and 74.0% in SCC group (P = 0.000). The expression rate of p33(ING1b) decreased was 91.7% in control group, 77.7% in CIN 1 group, 68.0% in CIN 2 & CIN 3 group, and 36.0% in SCC group (P = 0. 000). The expression rate of hTERT was 50.0% in control group, 66.6% in CIN 1 group, 88.0% in CIN 2 & CIN 3 group, and 94.0% in SCC group (P = 0.000). In the cervical tissues of Uygur female, the HPV16 infection rate was negatively correlated with the expression of p33(ING1b) (r = -0.294, P = 0.004), and was positively correlated with the expression of hTERT (r = 0.286, P = 0.005). The expression of p33(ING1b) was negatively correlated with the expression of hTERT (r = -0.361, P = 0.000). CONCLUSION: The infection of HPV 16 correlates with the decreased expression of p33(ING1b) and increased expression of hTERT in the cervical squamous cell carcinoma of Uygur female in Xinjiang.

A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis.

This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, a modification of the basic boiling lysis technique, to simplify the processing of large volumes of Escherichia coli cultures. Fermented bacteria are harvested using a hollow fiber-membrane module and pre-treated with lysozyme prior to passing through a thermal exchange coil set at 70 degrees C to lyse the cells, and into a juxtaposed cooling coil on ice. The lysed and cooled bacteria are subsequently separated from the lysate by centrifugation and plasmid DNA is precipitated from the supernatant for further purification. The use of peristaltic pumps and two heating coils at constant temperature without the use of centrifugation enable the lysis process to become constant and controllable, providing a flow-through protocol for cell lysis and plasmid DNA extraction. Large volumes of bacterial cultures (20 l) can be processed in 2 h, yielding approximately 100 mg plasmid DNA l(-1) culture, making this an attractive protocol for consistent and large-scale preparation of plasmid DNA.

DNA prime followed by protein boost enhances neutralization and Th1 type immunity against FMDV.

Prime-boost strategy has been exhibited its potency to enhance immune responses, which would be important to the success to develop a vaccine against the foot-and-mouth disease virus (FMDV). An eukaryotic expression construct encoding the FMDV capsid VP1 protein with a recombinant VP1 protein or a commercial FMDV vaccine were tested in the prime-boost strategy in mice and cattle trials. The levels of induced specific antibodies, T cell proliferations, and DTH activities were significantly higher in the prime-boost groups than in those vaccinated with DNA, protein or FMDV vaccine alone. More importantly, the levels of neutralizing antibodies in the former groups were significantly higher than others and could last for at least four months in cattle trials. This study suggests that the prime-boost strategy significantly improves the effective immunity and may provide a longer protection against FMDV infection.

A continuous thermal lysis procedure for the large-scale preparation of plasmid DNA.

There is an increasing interest and need for the development of scaleable process for the preparation of plasmid DNA for vaccines and gene therapy. In this report, we describe a streamline modified process of plasmid extraction based on boiling lysis in order to simplify the operation and process large volumes of Escherichia coli cultures. The bacteria, harvested using a hollow fiber cartridge after fermentation, were treated with lysozyme at 37 degrees C prior to passing through a heat-exchanger coil. Subsequently, the supernatant was separated from lysed bacteria using a 65 microm nylon filter. The employment of a peristaltic pump and two heating coils at constant temperature without the use of centrifugation enabled the process protocol to be constant and controllable. A relatively low lysis temperature of approximately 70-80 degrees C and a buffer modified for the high-density cultures were also optimized for the process. Prior to thermal lysis, a pre-treatment step with the lysozyme for 20 min at 37 degrees C was one of the crucial steps contributing to the high plasmid quantity and quality from batch to batch. After harvesting 17 L of E. coli cultures (OD600 = 50), the plasmid can be extracted within 45 min with this streamline protocol. The plasmid yields are approximately 100mg/L culture, which makes it attractive and promising for the large-scale preparation of plasmid.

Effects of multiple copies of CpG on DNA vaccination.

It has previously been demonstrated that a dose-dependent enhancement of immune response is derived from immunization with several copies of the CpG motif. Following that lead, we sought to incorporate a higher copy number of CpG motifs into an expression construct to evaluate the augmentation of immune responses. By multiple insertions, 30 copies of the CpG motif were cloned into the backbone of an expression construct encoding the foot-and-mouth disease virus (FMDV) capsid protein VP1. After intramuscular immunization, an augmented immune response with significantly increased levels of the specific antibody, T-cell proliferation, and IFN-gamma in Balb/c mice was observed. Compared to chemically synthesized CpG ODN, application of such a multicopy of CpG sequences within the expression backbone for DNA vaccination strategy is feasible and warranted.

Induction of active immune suppression by co-immunization with DNA- and protein-based vaccines.

Although immunization has been used for eliciting immune response, here we show that it can also induce immune suppression. When a DNA vaccine encoding a viral antigen such as the VP1 protein from the foot and mouth disease virus is administered together with its recombinant protein antigen or a viral preparation containing the same antigen, the immunized animals developed significantly reduced antigen-specific T cell-mediated responses and became impaired to subsequent rechallenge with the same antigen. The induction of immune suppression is mediated by suppressor T cells, as demonstrated by an adoptive transfer experiment and mixed lymphocyte reactions. The induction of immune suppression in immunized animals is also correlated with a shift of cytokine balance, as reflected by an elevated level of IL-10 and reduced level of IFN-gamma or IL-2. Hence, co-immunization with DNA- and protein-based vaccines may represent a novel means for inducing active suppression against untoward immunity.

Induction of Th1 type response by DNA vaccinations with N, M, and E genes against SARS-CoV in mice.

Vaccination against the SARS-CoV infection is an attractive means to control the spread of viruses in public. In this study, we employed a DNA vaccine technology with the levamisole, our newly discovered chemical adjuvant, to generate Th1 type of response. To avoid the enhancement antibody issue, genes encoding the nucleocapsid, membrane, and envelope protein of SARS-CoV were cloned and their expressions in mammalian cells were determined. After the intramuscular introduction into animals, we observed that the constructs of the E, M, and N genes could induce high levels of specific antibodies, T cell proliferations, IFN-gamma, DTH responses, and in vivo cytotoxic T cells activities specifically against SARS-CoV antigens. The highest immune responses were generated by the construct encoding the nucleocapsid protein. The results suggest that the N, M, and E genes could be used as the targets to prevent SARS-CoV infection in the DNA vaccine development.