[Clinical Value of Peripheral Blood Circulating Tumor Cells Detection in Early Cervical Infiltrating Carcinoma Screening].

OBJECTIVE: To observe the difference of circulating tumor cells (CTC) in peripheral blood of patients with different degrees of cervical lesions, and to evaluate the effectiveness of CTC detection in screening early invasive cervical cancer. METHODS: From December 2015 to October 2017, 63 cases of cervicitis, low-grade and high-grade intraepithelial lesions (LSIL, HSIL) and early invasive cervical cancer were confirmed by histopathological and clinical stages in Zhongshan Boai Hospital and Zhongshan Hospital Affiliated to Zhongshan University. The immunomagnetic bead negative enrichment technique combined with immunofluorescence was used. In situ hybridization (imFISH) was used to detect CTC in peripheral blood of patients. The positive rate and quantity of CTC in four groups were analyzed. The diagnostic efficacy of CTC in early invasive cervical cancer was evaluated based on the results of histopathological diagnosis and clinical staging. RESULTS: ①The positive rates of CTC in cervicitis group, LSIL group, HSIL group and early invasive cervical cancer group were 0, 0, 19.05% and 84.13% respectively. The overall difference was statistically significant ( χ 2=504.00， P<0.05). The positive rate of CTC in early invasive cervical cancer group was higher than that in other groups ( P<0.008 3). The positive rates of CTC in HSIL group were significantly different from those in LSIL group and cervicitis group ( P<0.008 3). ②The average number (median) of CTC positive in cervicitis group, LSIL group, HSIL group and early invasive cervical cancer group was 0, 0, 1/4 mL, 3/4 mL, respectively. The average number of positive CTC in early invasive cervical cancer group was higher than that in other groups, and the difference was significant compared with other groups ( P<0.008 3). The average number of CTC positive in HSIL group was significantly different from that in LSIL and cervicitis group ( P<0.008 3). ③The sensitivity, specificity, coincidence rate, positive predictive value and negative predictive value of CTC positive results in the diagnosis of early invasive cervical cancer were 84.13%, 93.65%, 91.27%, 81.54% and 94.65%, respectively. CONCLUSION: CTC exists in patients with HSIL and early invasive cervical cancer. With the aggravation of cervical lesions, the positive rate and number of CTC test results increase. CTC detection in early invasive cervical cancer screening has a certain practical value in clinic.

[Paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples].

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(χ2=7.817 and 1.332, both P>0.05), while the thickness of slices in LSIL and HSIL samples had significant effect on the positive rate of p16 protein (χ2=17.688 and 10.182, P<0.05 or P<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.

[Expression pattern of hsa-miR-9 and its association with BCL6 in EBV-positive and EBV-negative Burkitt lymphoma cell lines].

OBJECTIVE: To investigate the differential expression pattern of hsa-miR-9 between EBV-positive and -negative Burkitt lymphoma cell lines and its association with BCL-6. METHODS: The expression of hsa-miR9 and BCL-6 mRNAs in EBV(+) Raji and EBV-Ramous cells in mRNA levels were detected using fluorescence quantitative PCR (QRT-PCR). The two cells lines were transiently transfected with hsa-mir9-inhibitor and hsa-mir9-minicsvia Oligofectamine 2000, and the changes in BCL6 expressions was detected using QRT-PCR and Western blotting. Annexin V/PI staining was used to analyze the apoptosis and morphological changes of the transfected cells. RESULTS: The expression of Hsa-miR9 and BCL-6 was significantly higher in EBV(+) Raji cells than EBV(-) Ramous cells (P<0.01). BCL-6 mRNA and protein expression was reduced in EBV(+) Raji cells after transfection with hsa-miR9-inhibitor but up-regulated in EBV(-) Ramous cells transfected with hsa-miR9-minics. Flow cytometry revealed a significantly decreased apoptosis rate in EBV(+) Raji cells transfected with hsa-miR9-inhibitor but an increased rate in EBV(-) Ramous cells transfected with hsa-miR9-minics, and the results were confirmed by microscopic observations. CONCLUSION: Hsa-miR9 positively regulate the expression of BCL-6 and apoptosis of EBV(+) Raji cells and EBV(-) Ramous cells.

Re-expression of microRNA-150 induces EBV-positive Burkitt lymphoma differentiation by modulating c-Myb in vitro.

Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma that includes two forms of BL differing in Epstein-Barr virus (EBV) infection status, EBV-positive and EBV-negative. Although many efforts, such as high-intensity, short-duration combination chemotherapy, have been devoted to improving therapy for this rapidly proliferating neoplasm, there are still significant treatment-associated toxicities. Therefore, there remains a need for novel effective therapeutic strategies. MicroRNAs play a role in "fine tuning" the physiological and pathological differentiation process, by which cells can rapidly regulate dynamic events such as cell-lineage decisions and morphogenesis. This unique miRNA feature shifts the traditional one drug target paradigm to a novel one drug multiple targets paradigm. Here, we found that BL cell lines showed an extremely low expression of microRNA-150 (miR-150), and then restored miR-150 expression at physiologic levels in BL cell lines Daudi, Raji, BJAB, and Ramos. The results showed that re-expression of miR-150 reduced proliferation of Daudi and Raji cells. Furthermore, Daudi and Raji, both of which are of EBV-positive germinal center B-cell origin, transduced with miR-150 can be rescued to differentiate toward B-cell terminal stage. However, no significant changes were observed in BJAB or Ramos cells, which are of EBV-negative germinal center B-cell origin. Of note, re-expression of miR-150 also resulted in decreasing c-Myb protein levels. Additionally, c-Myb knockdown in Daudi and Raji cell lines recapitulated the partial characteristics similar to that caused by re-expression of miR-150. Taken together, our findings show that miR-150 can induce EBV-positive BL differentiation by targeting c-Myb.