Abulizi Abudula

Post-transcriptional and epigenetic regulation of antigen processing machinery (APM) components and HLA-I in cervical cancers from Uighur women.

Normal function of human leukocyte antigen class I (HLA-I) and antigen processing machinery (APM) proteins is required for T cell-mediated anti-tumor or antiviral immunity, whereas the tumor survival indicates a failure of the host in immune surveillance associated with the dysfunction in antigen presentation, mainly due to the deregulation in HLA-I and APM expression or function. The posttranscriptional regulation of HLA-I and APM expression may associate with epigenetic modifications in cancer development which was not described so far. Here we showed that the development of cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) in Uighur women was accompanied with the partial or total loss of protein expression of HLA-I, ß2-m and APM components, including the transporter associated with antigen processing (TAP1/2), low molecular mass protein (LMP2, LMP7), endoplasmic reticulum aminopeptidase 1(ERAP1), chaperone molecules include calreticulin (CLR), calnexin (CNX) and ERp57, and this was proved again by analysis of transcription of the same genes in addition to three genes HLA-A, B and C coding for HLA-I. By bisulfite sequencing approach, we identified target CpG islands methylated at the gene promoter region of TAP1, TAP2, LMP7, tapasin and ERp57 in cervical carcinoma cells. Further analysis of CpG site specific methylation of these genes in cases of CSCC and CIN demonstrated an inverse correlation of altered CpG island methylation of TAP1, LMP7, and ERp57 with changes in protein expression. Moreover, promoter methylation of these genes was significantly higher in cases positive for human papillomavirus 16 (HPV 16) than negative ones. Our results suggested that epigenetic modifications are responsible for the aberrant expression of certain HLA-I and APM genes, and may help to understand unrevealed mechanisms of tumor escape from immune surveillance in cervical carcinogenesis.

Potential predictive plasma biomarkers for cervical cancer by 2D-DIGE proteomics and Ingenuity Pathway Analysis

The current methods available for screening and detecting cervical squamous cell carcinoma (CSCC) have insufficient sensitivity and specificity. As a result, many patients suffered from erroneous and missed diagnosis. Because CSCC is usually asymptomatic at potentially curative stages, identification of biomarkers is an urgent need for the early detection of CSCC. Comparative proteomics based on two-dimensional differential in-gel electrophoresis (2D-DIGE) was employed to quantitatively analyze plasma proteins of healthy Uyghur women and with early stage cervical carcinoma. The 2D-DIGE image were analyzed statistically using DeCyder™ 2D software. The statistical analysis of proteomic data revealed that 43 protein spots showed significantly different expression (ratio > 1.5, P < 0.01). A further identification of these protein spots by MALDI-TOF-MS found out 16 different proteins. Bioinformatic analysis within the framework of Ingenuity Pathway Analysis (IPA@) showed that 10 plasma proteins as candidate biomarker were screened, mainly including lipid metabolism-related proteins (APOA4, APOA1, APOE), complement (EPPK1, CFHR1), metabolic enzymes (CP, F2, MASP2), glycoprotein (CLU), and immune function-related proteins (IGK@). Networks involved in lipid metabolism, molecular transport, and small molecule biochemistry were dysfunctional in CSCC. Acute phase response signaling and JAK/Stat signaling and IL-4 signaling, etc., were identified as the canonical pathways that are overrepresented in CSCC. Furthermore, the expression of three proteins (APOA1, APOE, CLU) were validated using ELISA in plasma of patients with different stage cervical lesion. With the combined proteomic and bioinformatic approach, this study was successful in identifying biomarker signatures for cervical cancer and might provide new insights into the mechanism of CSCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.

TLR9 Expression in Uterine Cervical Lesions of Uyghur Women Correlate with Cervical Cancer Progression and Selective Silencing of Human Papillomavirus 16 E6 and E7 Oncoproteins in Vitro

BACKGROUND Cervical cancer is listed as one of high-incidence endemic diseases in Xinjiang. Our study aimed to evaluate the expression of TLR9 in uterine cervical tissues of Uyghur women and examine associations with clinicopathological variables. We further characterized the direct effects of TLR9 upon the selective silencing of human papillomavirus (HPV) E6 and E7 oncoprotein expression in HPV 16-positive human cervical carcinoma cells treated with siRNA in vitro. MATERIALS AND METHODS Immunohistochemistry was applied to evaluate TLR9 expression in 97 formalin-fixed paraffin-embedded cervical samples from Uyghur women; 32 diagnosed with cervical squamous cell carcinomas (CSCC) , 14 with low-grade cervical intraepithelial neoplasias (CINI), 10 medium-grade (CINII), 24 high-grade (CINIII), and 17 chronic cervicitis. BLOCK-iTTM U6 RNAi Entry Vector pENTRTM/U6-E6 and E7 was constructed and transfected the entry clone directly into the mammalian cell line 293FT. Then the HPV 16-positive SiHa human cervical carcinoma cell line was infected with RNAi recombinant lentivirus. RT-PCR and Western blotting were used to determine the expression of TLR9 in both SiHa and HPV 16 E6 and E7 silenced SiHa cells. RESULTS Immunohistochemical staining showed that TLR9 expression was undetectable (88.2%) or weak (11.8%) in chronic cervicitis tissues. However, variable staining was observed in the basal layer of all normal endocervical glands. TLR9 expression, which was mainly observed as cytoplasmic staining, gradually increased in accordance with the histopathological grade in the following order: chronic cervicitis (2/17, 11.8%) <CINI (4/19, 28.6%) <CINII (3/10, 30.0%) <CINIII (12/24, 50.0%) <CSCC (17/32, 53.1%) (p<0.05), but not with tumor differentiation. RT-PCR and Western blotting showed that TLR9 expression was up-regulated in HPV16 E6 and E7 silenced SiHa cells at both mRNA and protein levels. CONCLUSIONS TLR9 expression increases according to the histopathological grade of cervical pathological process. HPV E6 and E7 oncoprotein have negative effects on the expression and function of TLR9.

Proteomic identification of potential biomarkers for cervical squamous cell carcinoma and human papillomavirus infection.

It is known that high-risk human papillomavirus infection is the main etiological factor in cervical carcinogenesis. However, human papillomavirus screening is not sufficient for early diagnosis. In this study, we aimed to identify potential biomarkers common to cervical carcinoma and human papillomavirus infection by proteomics for human papillomavirus–based early diagnosis and prognosis. To this end, we collected 76 cases of fresh cervical tissues and 116 cases of paraffin-embedded tissue slices, diagnosed as cervical squamous cell carcinoma, cervical intraepithelial neoplasia II–III, or normal cervix from ethnic Uighur and Han women. Human papillomavirus infection by eight oncogenic human papillomavirus types was detected in tissue DNA samples using a quantitative polymerase chain reaction. The protein profile of cervical specimens from human papillomavirus 16–positive squamous cell carcinoma and human papillomavirus–negative normal controls was analyzed by proteomics and bioinformatics. The expression of candidate proteins was further determined by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. We identified 67 proteins that were differentially expressed in human papillomavirus 16–positive squamous cell carcinoma compared to normal cervix. The quantitative reverse transcriptase-polymerase chain reaction analysis verified the upregulation of ASAH1, PCBP2, DDX5, MCM5, TAGLN2, hnRNPA1, ENO1, TYPH, CYC, and MCM4 in squamous cell carcinoma compared to normal cervix (p < 0.05). In addition, the transcription of PCBP2, MCM5, hnRNPA1, TYPH, and CYC was also significantly increased in cervical intraepithelial neoplasia II–III compared to normal cervix. Immunohistochemistry staining further confirmed the overexpression of PCBP2, hnRNPA1, ASAH1, and DDX5 in squamous cell carcinoma and cervical intraepithelial neoplasia II–III compared to normal controls (p < 0.05). Our data suggest that the expression of ASAH1, PCBP2, DDX5, and hnRNPA1, and possibly MCM4, MCM5, CYC, ENO1, and TYPH, is upregulated during cervical carcinogenesis and potentially associated with human papillomavirus infection. Further validation studies of the profile will contribute to establishing auxiliary diagnostic markers for human papillomavirus–based cancer prognosis.

Differential integrative omic analysis for mechanism insights and biomarker discovery of abnormal Savda syndrome and its unique Munziq prescription.

Research has shown that many cancers have acommon pathophysiological origin and often present with similar symptoms. In terms of Traditional Uighur Medicine (TUM) Hilit (body fluid) theory, abnormal Savda syndrome (ASS) formed by abnormal Hilit is the common phenotype of complex diseases and in particular tumours. Abnormal Savda Munziq (ASMq), one representative of TUM, has been effective in the treatment of cancer since ancient times. Despite the physiopathology of ASS, the relationship between causative factors and the molecular mechanism of ASMq are not fully understood. The current study expanded upon earlier work by integrating traditional diagnostic approaches with others utilizing systems biology technology for the analysis of proteomic (iTRAQ) and metabolomic (1H-NMR) profiles of Uighur Medicine target organ lesion (liver) tumours. The candidate proteins were analyzed by enrichment analysis of the biological process and biomarker filters. Subsequently, 3Omics web-based tools were used to determine the relationships between proteins and appropriate metabolites. ELISA assay and IHC methods were used to verify the proteomic result; the protein von Willebrand factor (vWF) may be the “therapeutic window” of ASMq and biomarkers of ASS. This study is likely to be of great significance for the standardization and modernization of TUM.

Upregulation of stem cell markers ALDH1A1 and OCT4 as potential biomarkers for the early detection of cervical carcinoma

Previous studies have reported the upregulation of stem cell biomarkers that are associated with tumorigenesis, in particular with cancer infiltration, recurrence and metastasis. Infection by human papilloma virus (HPV) is the main etiopathological factor of cervical carcinogenesis, but the expression of stem cell markers in cervical carcinoma and HPV infection have yet to be investigated so far. A total of 94 cases of fresh cervical tissues, 116 cases of paraffin-embedded cervical specimens and 72 cases of peripheral blood samples were collected from Uighur women who were either diagnosed with cervical squamous cell carcinoma (SCC) or cervical intraepithelial neoplasia (CIN) II–III, or from healthy subjects (negative controls, NC). HPV infection was detected in tissue DNA by polymerase chain reaction (PCR) with a HPV genotyping kit. The mRNA expression levels of aldehyde dehydrogenase 1 family member A1 (ALDH1A1), nanog homeobox (NANOG), POU class 5 homeobox 1 (OCT4), SRY-box 2 (SOX2) and twist family BHLH transcription factor 1 (Twist1) were determined using reverse transcription-quantitative PCR (RT-qPCR). Histological analysis was performed in order to examine the protein expression of ALDH1A1 and OCT4 in paraffin-embedded tissue specimens by immunohistochemical staining and the plasma levels of those two proteins was measured by ELISA. RT-qPCR analysis indicated a significant increase in the mRNA expression of ALDH1A1 and OCT4 in CIN II–III and SCC tissue specimens compared with NC (P<0.05). Although the expression levels of NANOG, SOX2 and Twist1 were significantly higher in SCC compared with NC (P<0.05), no significant difference was revealed in CIN II–III tissues compared with SCC or NC (P>0.05). Subsequent analysis by immunohistochemistry staining confirmed that the upregulation of ALDH1A1 and OCT4 was also significantly increased in SCC and CIN II–III compared with controls at the protein level. Notably, ELISA analysis detected significantly higher levels of ALDH1A1 and OCT4 in the peripheral blood (plasma) of patients with SCC compared with healthy subjects. The upregulation of stem cell markers ALDH1A1 and OCT4 in cervical carcinoma and its precursor lesions, in particular in the peripheral blood, indicates that ALDH1A1 and OCT4 may serve as biomarkers for the early detection of cervical carcinoma or for the monitoring of treatment of patients.