OBJECTIVE: To investigate paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples. METHODS: p16 immunohistochemical staining was performed in 150 cases of chronic cervicitis, 126 cases of low-grade squamous intraepithelial lesions(LSIL), 96 cases of high-grade squamous intraepithelial lesions (HSIL) and 78 cases of cervical cancer from January 2014 to March 2018 in Zhongshan Boai Hospital. The results of p16 protein expression in paraffin sections with thickness of 2, 3, 4, 5 and 6 μm were compared using Logistic regression analysis. RESULTS: With the increase of slice thickness, the staining of p16 protein in nucleus gradually increased. The thickness of cervical slices in chronic cervicitis and cervical cancer samples had no significant effect on the positive rate of p16 protein(=7.817 and 1.332, both >0.05), while the thickness of slices in LSIL and HSIL samples had significant effect on the positive rate of p16 protein (=17.688 and 10.182, <0.05 or <0.01). The stable and reliable results were obtained when the slices were between 3 and 5 μm thick. CONCLUSIONS Paraffin sections with thickness of 3.0-5.0 μm are recommended for immnohistochemical staining of p16 protein in cervical tissue samples.
Biomarkers, Tumor ; genetics ; metabolism ; Cervical Intraepithelial Neoplasia ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Female ; Histocytological Preparation Techniques ; standards ; Humans ; Immunohistochemistry ; Paraffin ; Squamous Intraepithelial Lesions of the Cervix ; Uterine Cervical Neoplasms ; physiopathology

OBJECTIVE: To investigate the expression of claudin 4 (CLDN4) in cervical tissues from patients with different cervical lesions, and to explore the value of combined detection of CLDN4 and high risk human papilloma virus (HR-HPV). METHODS: The cervical tissue specimens of low-grade squamous intraepithelial lesion (LSIL, =30), high-grade squamous intraepithelial lesion (HSIL, =30), squamous cell carcinoma (SCC, =30) as well as chronic cervicitis (control, =30) were collected from the Sir Run Run Shaw Hospital of Zhejiang University during June 2015 and December 2016. The expression of CLDN4 protein in tissue specimens was detected by immunohistochemistry, HR-HPV was detected by real-time quantitative PCR, and the cervical exfoliated cells were examined by thinprep cytologic test (TCT). The ROC curve was applied to analyze the diagnostic value of TCT combined with HR-HPV and CLDN4 combined with HR-HPV tests for HSIL and SCC of the cervix. RESULTS: With the increase of the severity of cervical lesions, the positive rate of CLDN4 expression rose (=0.832, <0.05). Positivity of both HR-HPV infection and CLDN4 expression was found mainly in the HSIL and SCC groups. The areas under curve (AUC) of TCT combined with HR-HPV and CLDN4 combined with HR-HPV tests for diagnosis of HSIL and SCC were 0.683 and 0.633, respectively; the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of TCT combined with HR-HPV test for diagnosis of HSIL and SCC were 100.0%, 36.7%, 61.2%, 100.0% and 46.7% respectively; those of CLDN4 combined with HR-HPV test were 96.7%, 30.0%, 58.0%, 90.0% and 55.0%, respectively. CONCLUSIONS CLDN4 expression may be related to the occurrence and development of cervical carcinoma and precancerous lesions. CLDN4 combined with HR-HPV test may be used for diagnosis of HSIL and SCC of the cervix clinically.
Carcinoma, Squamous Cell ; diagnosis ; virology ; Cervical Intraepithelial Neoplasia ; diagnosis ; virology ; Claudin-4 ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunochemistry ; Papillomaviridae ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Squamous Intraepithelial Lesions of the Cervix ; virology ; Uterine Cervical Neoplasms ; diagnosis

OBJECTIVETo investigate the expression of cyclin D1 in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma and its relationship with human papillomavirus 16 (HPV16) E7 gene expression.

METHODSBoth SiHa and Hcc94 cell lines were obtained from cervical epithelial cells of squamous cell carcinoma. E6/E7 gene was silent in Hcc94 cell line.Expression levels of cyclin D1 mRNA and protein in CIN and squamous cell carcinoma were detected by QT-PCR and immunohistochemistry (IHC) respectively. SiRNA was constructed for targeting the promoter of HPV16 E7 and then transfected into SiHa cells to establish cm-16 line with stable silencing of E7. Control cell line B3 was obtained by blank plasmid transfection into SiHa cells. RT-PCR and Western blot were used to detect cyclin D1 mRNA and protein expression in the SiHa, B3, and cm-16 cells, respectively.

RESULTSCyclin D1 was expressed in the basal cells of normal cervical squamous epithelia and the expression gradually decreased in the progression from CIN1 to CIN3. Squamous cell carcinoma showed negative or scattered expression of cyclin D1 (P<0.05). Both mRNA and protein of cyclin D1 in E7(+) SiHa cells were lower than those in cm-16 and Hcc94 cells.

CONCLUSIONSquamous cell carcinoma with high HPV E7 expression shows low level of cyclin D1, suggesting that HPV16 E7 gene inhibits the expression of cyclin D1.

Carcinoma, Squamous Cell ; metabolism ; virology ; Cell Line, Tumor ; Cervical Intraepithelial Neoplasia ; metabolism ; virology ; Cyclin D1 ; genetics ; metabolism ; Female ; Human papillomavirus 16 ; Humans ; Immunohistochemistry ; Papillomavirus E7 Proteins ; genetics ; Promoter Regions, Genetic ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Uterine Cervical Neoplasms ; metabolism ; virology

OBJECTIVETo study the expression and significance of tumor suppressor in lung cancer 1 (TSLC1) gene methylation, the expression of TSLC1 protein in cervix cancer and precancerous lesions as well as their relationship with HR-HPV DNA infection.

METHODSThe clinicopathological data of 92 cases of different cervical lesions during March 2011 to August 2012 treated in our hospital were collected. There were pathologically confirmed 10 cases of normal cervix, 26 cases of cervical intraepithelial neoplasia (CIN) I, 20 cases of CIN II, 15 cases of CIN III, and 21 cases of cervical cancer. Methylation-specific polymerase chain reaction (MSP) was used to detect the TSLC1 gene methylation status in cervical lesions, immunohistochemistry (SP) was used to detect the expressions of TSLC1 protein in cervical lesions, and the second generation hybrid capture (HC2) method was used to detect the high-risk HPV in cervical lesions.

RESULTSThe expression rate of TSLC1 gene methylation in normal cervical tissue, CIN I, CIN II, CIN III and SCC were 10.0%, 30.8%, 55.0%, 60.0%, 66.7%, respectively, showing a statistically significant difference (P = 0.004). The positive expression rate of TSLC1 protein in normal cervical tissue, CIN I, CIN II, CIN III and SCC were 100.0%, 80.8%, 65.0%, 33.3%, and 23.8%, respectively, with a significant difference (P = 0.004). In the progression from CIN to invasive cervical cancer, there was no significant correlation between TSLC1 gene methylation and HR-HPV DNA infection (P = 0.919), TSLC1 protein expression and HR-HPV DNA infection (P = 0.664). The correlation analysis showed a negative correlation between TSLC1 gene methylation and TSLC1 protein expression (r = -0.674, P < 0.001).

CONCLUSIONSTSLC1 gene promoter methylation may be an early event in the cervical carcinogenesis, become an early sensitive marker, and serve the early prevention and prognostic prediction for cervical cancer.

Cell Adhesion Molecule-1 ; Cell Adhesion Molecules ; genetics ; metabolism ; Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; DNA Methylation ; Disease Progression ; Female ; Humans ; Immunoglobulins ; genetics ; metabolism ; Immunohistochemistry ; Methylation ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Uterine Cervical Neoplasms ; genetics ; metabolism

OBJECTIVETo investigate the differential expressions of nucleolin in invasive cervical squamous cell carcinoma, cervical intraepithelial neoplasms (CIN) and normal cervical epithelial tissues and explore the role of nucleolin in the carcinogenesis and progression of cervical squamous cell carcinoma.

METHODSFifty specimens of invasive cervical squamous cell carcinoma, 65 specimens of CIN, and 60 adjacent normal cervical epithelial tissue specimens were examined immunohistochemically for nucleolin expression. The correlation of nucleolin expression levels with histological grades of invasive cervical squamous cell carcinoma and CIN were analyzed.

RESULTSThe specimens of invasive cervical squamous cell carcinoma showed a significantly higher positivity rate for nucleolin expression than CIN and normal cervical epithelial tissues, and the rate in CIN tissues was significantly higher than that in normal cervical epithelial tissues (P<0.01). The expression level of nucleolin was significantly higher in invasive cervical squamous cell carcinoma than in CIN and normal cervical epithelia tissues, and higher in CIN than in normal cervical epithelia tissues, whose immunostaining scores were 7.6±0.3, 6.1±0.2, and 3.0±0.2, respectively (P<0.01). The mean nucleolin immunostaining score was significantly higher in poorly and moderately differentiated than in highly differentiated cervical squamous cell carcinoma (7.9 vs 7.1, P<0.01), and higher in high grade CIN than in low grade CIN tissues (6.0 vs 4.0, P<0.01).

CONCLUSIONSOverexpression of nucleolin plays an important role during carcinogenesis of cervical squamous cell carcinoma and is positively correlated with tumor progression of CIN and cervical squamous cell carcinoma.

Carcinogenesis ; Carcinoma in Situ ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; Disease Progression ; Female ; Humans ; Phosphoproteins ; metabolism ; RNA-Binding Proteins ; metabolism ; Uterine Cervical Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate the expression of SOX2 in cervical intraepithelial neoplasia (CIN) and cervical cancer and explore its association with the clinical features.

METHODSSOX2 expressions were examined using immunohistochemical method in 10 normal cervical tissue specimens, 36 cervical intraepithelial neoplasia specimens (including 10 cases of grade I, 12 of grade II, and 14 grade III) and 40 cervical cancer specimens (including 21 cases of stage I and 19 of stage II). The correlation between the immunohistochemical results and the clinical features of the patients was analyzed.

RESULTSSOX2 expression was negative in normal cervical tissues, and was positive in 41.6% of CIN specimens (10.0% in CIN I, 41.7% in CIN II, and 64.3% in CIN III) in 82.5% of cervical cancer specimens (78.2% in stage I and 88.2% in stage II). The patients with cervical cancer had a significantly higher positivity rate of SOX2 than normal control group (P<0.05). The positivity rate of SOX2 increased with the evolution of cervical disease. SOX2 protein expression was significantly correlated with the histological grade and lymph node metastasis (P<0.05), but not with the age or clinical stage of the patients (P<0.05).

CONCLUSIONSOX2 expression may serve as a useful indicator for evaluating metastasis and malignancy of cervical cancer.

Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; pathology ; Humans ; Lymphatic Metastasis ; Neoplasm Grading ; Neoplasm Staging ; SOXB1 Transcription Factors ; genetics ; metabolism

OBJECTIVETo explore the expression of Snail and Slug in primary cervical squamous cell carcinoma (CSCC) and their relationship with KAI1 expression.

METHODSThe expressions of Snail, Slug, and KAI1 proteins were examined by immunohistochemistry in 154 specimens of CSCC tissues, 50 specimens of cervical intraepithelial neoplasm (CIN), and 40 specimens of normal cervical tissues.

RESULTSThe positivity rates of Snail, Slug, and KAI1 expression were 0%, 2.5%, and 95.0% in normal cervical tissues, 32.0%, 34.0% and 64.0% in CIN tissues, and 66.2%, 66.9%, and 43.5% in CSCC tissues, respectively, showing significant differences in the rates among the 3 groups (P<0.05). The expressions of Snail, Slug, and KAI1 were significantly correlated with the histological grades of the tumor, depth of invasion, lymph node metastasis, International Federation of Gynecology and Obstetrics (FIGO) stages, and postoperative survival time (P<0.05). The expressions of Snail and Slug were positively correlated (r=0.752, P<0.001), and both of them were negatively correlated with the expression of KAI1 (P<0.001). Kaplan-Meier analysis showed that patients positive for Snail and Slug had significantly lower survival rates than the negative patients (P<0.001), while a positive expression of KAI1 was associated with a higher survival rate of the patients. Cox regression analysis identified Snail, KAI1, and FIGO stage as independent factors that affected the outcomes of CSCC (P<0.05).

CONCLUSIONThe expressions of Snail, Slug, and KAI1 are related to the tumor grade, FIGO stage, invasive depth, lymph node metastasis, and prognosis of CSCC, and their combined detection can help estimate the outcomes of the patients.

Carcinoma, Squamous Cell ; metabolism ; pathology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Kangai-1 Protein ; metabolism ; Kaplan-Meier Estimate ; Lymphatic Metastasis ; Neoplasm Staging ; Prognosis ; Snail Family Transcription Factors ; Survival Rate ; Transcription Factors ; metabolism ; Uterine Cervical Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate the clinicopathological significance of Twist and YB-1 up-regulation in cervical cancer, and to correlate the expression of the two genes with E-cadherin, a marker of epithelial-mesenchymal transition (EMT).

METHODSA total of 202 tissue samples were collected during January 2008 to December 2013, including 50 cases of normal cervical tissues, 100 cases of cervical intraepithelial neoplasia (CIN) and 52 cases of squamous cell carcinoma (SCC). Twist, YB-1 and E-cadherin expression was investigated by MaxVision.

RESULTSIncreased expression levels of Twist and YB-1 were found and correlated with the malignant transformation of cervical epithelium, histological progression and metastasis of cervical cancer. In addition, Twist and YB-1 overexpression was also associated with aberrant expression of E-cadherin. Regression analysis revealed that Twist expression was an independent factor for the histological progression of cervical cancer.

CONCLUSIONSIt is suggested that Twist and YB-1 overexpression is significantly linked to cervical cancer tumorigenesis and progression, likely related to EMT through (YB-1)-Twist-(E-cadherin) pathway. Twist and YB-1 may be markers for determining the metastatic potential of cervical cancer.

Biomarkers, Tumor ; genetics ; metabolism ; Cadherins ; genetics ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Transformation, Neoplastic ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; Disease Progression ; Epithelial-Mesenchymal Transition ; Epithelium ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Nuclear Proteins ; genetics ; metabolism ; Twist-Related Protein 1 ; genetics ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; metabolism ; pathology ; Y-Box-Binding Protein 1 ; genetics ; metabolism

OBJECTIVE: To explore the role of apoptosis stimulating proteins of p53 2 (ASPP2) and inhibitory member of the ASPP family (iASPP) in the occurrence of cervical cancer and the relation between these two proteins and p53. METHODS: We used immunohistochemical method to detect the expression of ASPP2, iASPP, p53 in 51 patients with early cervical cancer tissue, 53 patients with cervical intraepithelial neoplasia (CIN) II-III, 48 patients with CIN I and 45 patients with normal cervical tissue. The relation among ASPP2, iASPP and p53 was analyzed. RESULTS: When p53 was negative, the positive expression rate of ASPP2 in the cervical cancer group, the CIN II-III group, the CIN I group and the normal cervix group was gradually increased. There was significant difference between the CIN II-III group or the cervical cancer group and the normal cervix (P<0.05), but no statistical difference was found among the other groups (P>0.05). The positive expression rate of iASPP in the 4 groups gradually reduced, and the difference was significant difference between the cervical cancer group or the CIN II-III group and the normal cervix group (P<0.05). When the p53 was positive, there was no significant change in the expression of ASPP2 and iASPP in every group (P>0.05). CONCLUSION ASPP2 and iASPP may play an important role in the occurrence of early stage of cervical cancer by regulating the ability of wild type p53 in induction of apoptosis. ASPP2 and iASPP gene might be a potential molecular target for cervical carcinoma.
Apoptosis ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Uterine Cervical Neoplasms ; genetics ; metabolism

OBJECTIVETo investigate the expression and clinical significance of enhancer of zeste homolog 2 (EZH2) and p53 proteins in cervical squamous cell carcinoma (SCC) and cervical intraepithelial neoplasia (CIN).

METHODSThe expression and distribution of EZH2 and p53 were determined with reference to clinicopathological features and patient survival. 168 cervical SCC, 19 CINII, 35 CINIII patients and 30 normal control cases were collected for immunohistochemical analysis.

RESULTSThe expression of EZH2 in the normal cervix, CIN and SCC was 6.7% (2/30), 37.0% (20/54) and 75.6% (127/168), respectively, with a significant difference between them (P < 0.05). The expression of p53 was 3.3% (1/30), 20.4% (11/54) and 39.3% (66/168), respectively, also (P < 0.05). In the 168 SCC cases, the positive rate of EZH2 in the cases with lymph node metastasis was 82.9%, and that of p53 was 45.7%; the positive rate of EZH2/p53 protein expression in the cases with negative lymph nodes was 70.4%, and that of p53 was 34.7%, with a significant difference between the two subgroups (P < 0.05). Among the 143 followed-up SCC patients, the EZH2(+) p53(+) cases had a progression-free survival of (51.3 ± 3.8) months, that of EZH2(+) or p53(+) cases was (66.1 ± 2.0) months, and that of EZH2(-) p53(-) cases was (71.3 ± 1.9) months, with a very significant difference among the three subgroups (P < 0.001). The overall survival times of EZH2(-) p53(-), EZH2(+) or p53(+), and EZH(2+) p53(+) cases were (72.9 ± 1.1), (68.6 ± 1.8), and (57.4 ± 3.4) months, respectively, with a significant difference among the three subgroups (P < 0.001). The multivariate analysis showed that EZH2 expression, lymph node metastasis and tumor staging were independent prognostic factors.

CONCLUSIONSBoth EZH2 and p53 proteins may play important roles in the occurrence and development of cervical squamous cell carcinoma. There is a close relationship between the expression of both EZH2 and p53 proteins and the prognosis of SCC patients.

Carcinoma, Squamous Cell ; diagnosis ; metabolism ; Cervical Intraepithelial Neoplasia ; diagnosis ; metabolism ; Disease-Free Survival ; Enhancer of Zeste Homolog 2 Protein ; Female ; Humans ; Lymphatic Metastasis ; Neoplasm Staging ; Polycomb Repressive Complex 2 ; genetics ; metabolism ; Prognosis ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Uterine Cervical Neoplasms ; diagnosis ; metabolism