Objective To detect the expression of CD99 in breast cancer and its effects on proliferation and migration of MDA-MB-435S cell.Methods Expression of CD99 was detected by immunohistochemical examination in tissues from 80 cases of breast cancer,40 cases of ductal carcinoma in situ,60 cases of benign breast hyperplasia,and 20 cases of normal breast.A cell line MDA-MB-435S-SiCD99 and the negative control cell line MDA-MB-435S-SiNC were constructed,which were transfected with SiCD99 and its negative control.Western blot was used to detect the protein levels of CD99 after CD99 silencing in MDA-MB-435S cell.The effects of CD99 on MDA-MB-435S cell proliferation and migration were evaluated using cell counting kit-8(CCK-8),transwell and wound healing assays via silencing CD99 in MDA-MB-435S cell.Results The expression of CD99 in breast cancer tissue was higher than that in tissue of ductal carcinoma in situ,benign breast hyperplasia tissue and normal breast tissue(P＜0.01).Western blot result showed that the expression of CD99 was decreased after CD99 silencing in MDA-MB-435S cell(P＜0.01).Compared with the MDA-MB-435S-SiNC,the proliferation rate was lower(P＜0.01,CCK-8 test),the scratch distance was shorter(P＜0.01,wound healing assay),and the migration abilities was obviously decreased(P＜0.01,tr-answell assay)in MDA-MB-435S-SiCD99.Conclusion CD99 is upregulated in the breast cancer and silencing CD99 obviously inhibited the proliferation and migration of breast cancer cells,providing evidence for diagnosis and potential treatment of breast cancer.

Objective To detect the expression of CD99 in breast cancer and its effects on proliferation and migration of MDA-MB-435S cell.Methods Expression of CD99 was detected by immunohistochemical examination in tissues from 80 cases of breast cancer,40 cases of ductal carcinoma in situ,60 cases of benign breast hyperplasia,and 20 cases of normal breast.A cell line MDA-MB-435S-SiCD99 and the negative control cell line MDA-MB-435S-SiNC were constructed,which were transfected with SiCD99 and its negative control.Western blot was used to detect the protein levels of CD99 after CD99 silencing in MDA-MB-435S cell.The effects of CD99 on MDA-MB-435S cell proliferation and migration were evaluated using cell counting kit-8(CCK-8),transwell and wound healing assays via silencing CD99 in MDA-MB-435S cell.Results The expression of CD99 in breast cancer tissue was higher than that in tissue of ductal carcinoma in situ,benign breast hyperplasia tissue and normal breast tissue(P＜0.01).Western blot result showed that the expression of CD99 was decreased after CD99 silencing in MDA-MB-435S cell(P＜0.01).Compared with the MDA-MB-435S-SiNC,the proliferation rate was lower(P＜0.01,CCK-8 test),the scratch distance was shorter(P＜0.01,wound healing assay),and the migration abilities was obviously decreased(P＜0.01,tr-answell assay)in MDA-MB-435S-SiCD99.Conclusion CD99 is upregulated in the breast cancer and silencing CD99 obviously inhibited the proliferation and migration of breast cancer cells,providing evidence for diagnosis and potential treatment of breast cancer.

Objective To explore the significance of human papilloma virus(HPV)L1 capsid protein and p16 protein in the clinical outcomes of cervical low-grade squamous intraepithelial lesions(LSIL)with persistent high-risk HPV infection.Methods Immunohistochemical analysis of HPV L1 and p16 was conducted on cervical tissues from 114 patients with persistent high-risk HPV infection and pathologically confirmed LSIL who were treated at the Cervical Disease Center of Department of Gynecology,Bo'ai Hospital of Zhongshan from January to July 2022,and follow-up was conducted.Results The cumulative progression rate of 114 LSIL patients was 21.93%,and the positive rates of HPV L1 and p16 were 25.44%and 39.47%,respectively.In the single detection,the regression rate of HPV L1(+)expression was as high as 68.97%,which was significantly different from the progression group and the persistent group(P<0.05);In the combined detection,the cumulative regression rates of HPV L1(+)/p16(+),HPV L1(+)/p16(-),and HPV L1(-)/p16(+),were 60.00%,78.57%,and 43.64%,respectively.There were statistically significant differences in the cumulative progression rate and persistence rate(P<0.05).Stratified analysis revealed that among individuals with HPV L1(+)expression,the regression rate at 24 months was higher than that of individuals with p16(+)expression,with a statistically significant difference(P<0.05).In the combined detection,the progression rate of HPV L1(-)/p16(+)expression in the 6th and 12th months was higher than that of other groups;The regression rate of HPV L1(+)/p16(-)expression in the 24th month was higher than that of other groups,and the differences were statistically significant(P<0.05).The results of the binary Logistic regression analysis indicate that the presence of HPV16/18 and p16 is a risk factor for the progression of LSIL,with OR values of 3.242(95%CI:1.261-8.336)and 2.714(95%CI:1.055-6.980),respectively.Conclusion The analysis of persistent infection types of high-risk HPV,as well as immunohistochemical detection of HPV L1 capsid protein and p16 protein,have certain clinical value in the clinical outcomes of LSIL patients with persistent infection of high-risk HPV,which can help guide clinical physicians to conduct individualized follow-up and triage management of LSIL patients with persistent infection of high-risk HPV.

Objective To explore the significance of human papilloma virus(HPV)L1 capsid protein and p16 protein in the clinical outcomes of cervical low-grade squamous intraepithelial lesions(LSIL)with persistent high-risk HPV infection.Methods Immunohistochemical analysis of HPV L1 and p16 was conducted on cervical tissues from 114 patients with persistent high-risk HPV infection and pathologically confirmed LSIL who were treated at the Cervical Disease Center of Department of Gynecology,Bo'ai Hospital of Zhongshan from January to July 2022,and follow-up was conducted.Results The cumulative progression rate of 114 LSIL patients was 21.93%,and the positive rates of HPV L1 and p16 were 25.44%and 39.47%,respectively.In the single detection,the regression rate of HPV L1(+)expression was as high as 68.97%,which was significantly different from the progression group and the persistent group(P<0.05);In the combined detection,the cumulative regression rates of HPV L1(+)/p16(+),HPV L1(+)/p16(-),and HPV L1(-)/p16(+),were 60.00%,78.57%,and 43.64%,respectively.There were statistically significant differences in the cumulative progression rate and persistence rate(P<0.05).Stratified analysis revealed that among individuals with HPV L1(+)expression,the regression rate at 24 months was higher than that of individuals with p16(+)expression,with a statistically significant difference(P<0.05).In the combined detection,the progression rate of HPV L1(-)/p16(+)expression in the 6th and 12th months was higher than that of other groups;The regression rate of HPV L1(+)/p16(-)expression in the 24th month was higher than that of other groups,and the differences were statistically significant(P<0.05).The results of the binary Logistic regression analysis indicate that the presence of HPV16/18 and p16 is a risk factor for the progression of LSIL,with OR values of 3.242(95%CI:1.261-8.336)and 2.714(95%CI:1.055-6.980),respectively.Conclusion The analysis of persistent infection types of high-risk HPV,as well as immunohistochemical detection of HPV L1 capsid protein and p16 protein,have certain clinical value in the clinical outcomes of LSIL patients with persistent infection of high-risk HPV,which can help guide clinical physicians to conduct individualized follow-up and triage management of LSIL patients with persistent infection of high-risk HPV.

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 compare two different methods to detect the differences of gene mutation rate, sensitivity, specificity and coincidence rate of epidermal growth factor receptor (EGFR) in non-small-cell lung cancer (NSCLC) so as to assess the clinical value of qRT-PCR method and its environmental-friendly technologyplatforms.One uses environmental fixative poly hydroxyl acrylic acid and green transparent liquid dewaxing Van-clear alone or in combination to replace the traditional fixative 4％ (volume fraction) neutral buffered formalin and the traditional transparent dewaxing liquid xylene in application of quantitative real-time polymerase chain reaction (qRT-PCR).The other uses traditional reagents in direct sequencing.Methods We selected 91 cases of primary NSCLC specimens resected between May 2013 and March 2016 in Zhongshan Bo`ai Hospital and Zhongshan Hospital of Traditional Chinese Medicine.Five samples were taken from the same tumor lesion.We used a random number table to randomly divide these samples into Groups A, B , C, D, and E.Group A received direct sequencing method in detection of EGFR gene mutations.Besides, during the experiment, 4％ neutral buffered formalin was used for fixing, and xylene transparent dewaxing was used to make slices for DNA extraction dewaxing.Group B received qRT-PCR method to detect EGFR gene mutations.Meanwhile, during the experiment, 4％ neutral buffered formalin was used for fixing, and xylene transparent dewaxing was used to make slices for DNA extraction dewaxing.Group C received qRT-PCR method in detection of EGFR gene mutations.At the same time, during the experiment, polyhydroxy acrylic acid was used for fixing, and xylene transparent dewaxing was used to make slices for DNA extraction dewaxing.Group D received qRT-PCR method to detect EGFR gene mutations.In the meantime, 4％ neutral buffered formalin was used for fixing, Van-clear transparent dewaxing was used to make slices for DNA extraction dewaxing.Group E received qRT-PCR method in detection of EGFR gene mutations.In addition, during the experiment, polyhydroxy acrylic acid was used for fixing, and Van-clear transparent dewaxing was used to make slices for DNA extraction dewaxing.In addition, during the experiment, polyhydroxy acrylic acid was used for fixing, and Van-clear transparent dewaxing was used to make slices for DNA extraction dewaxing.The mutations of Exons 18, 19, 20, and 21 in EGFR genes were respectively determined in the five groups of NSCLC.Results ① Groups B, C, D, E and A did not significantly differ in the percentage of people with mutations or target site mutation rates of EGFR genes in NSCLC (P> 0.05).② The detection results of EGFR target site mutation in Groups B, C, D, E and A had good sensitivity, strong specificity, and high compliance rate.Conclusion The green transparent liquid dewaxing Van-clear alone or in combination to replace the traditional fixative 4％ neutral buffered formalin and the traditional transparent dewaxing liquid xylene in the application of qRT-PCR so as to detect EGFR gene mutations in NSCLC has good consistent results compared with the method that uses traditional reagents in direct sequencing.It has the significance and value in clinical application.

Objective:To observe the difference of the human telomeres RNA component (hTERC) genes’amplification in the cervical tissue by applying the environment-friendly fixative poly hydroxy acrylic acid and the transparent dewaxing solution Van-clear separately or jointly to replace the traditional fixative 4% (volume fraction)neutral buffered formalin and the conventional transparent dewaxing solu-tion xylene in the use of fluorescence in situ hybridization (FISH)for detection.Methods:In the study, 255 cases of cervical tissue specimens submitted by the Department of Gynecology in Zhongshan Boai Ho-sipital were collected from Mar.2013 to Apr.2015.Four samples were taken from the same lesion site. All the cases were divided into 4 groups and named group A,B,C,and D.Group A used 4% neutral buffered formalin fixed and xylene dewaxing to make slices.Group B used poly hydroxy acrylic fixed and xylene dewaxing to make slices.Group C used 4% neutral buffered formalin fixed and Van-clear trans-parent to make slices.Group D used poly hydroxy acrylic fixed and Van-clear transparent dewaxing to make slices.The amplification of hTERC genes in the four groups of cervical specimens was also detected by FISH technique.Results:When the hTERC genes were detected by FISH method under the fluore-scence microscope,it was obvious that the tissue profile and the background of group A,B,C and D were all clear.The probe was fixed in the accurate position so that the bright red or green fluorescence signals were easily found in these four groups.Compared with the positive rate of group A,there was no statistical significance in that of group B,C and D (P>0.05).At the same time,the coincidence rate of the FISH results was high,which showed that the new environment-friendly reagent had no significant difference in the detection of cervical hTERC genes by FISH technique.Conclusion:It is possible for the environment-friendly reagent poly hydroxy acrylic acid and Van-clear to replace 4%neutral buffered for-malin and xylene separately or jointly to detect the cervical hTERC genes by adopting FISH technique.

Background and purpose: Adequate tissue ifxation, transparent dewaxing is an important step of hematoxylin eosin (HE) staining and lfuorescence in situ hybridization (FISH) in detection of breast cancerHER-2 gene. The purpose of this study was to make a comparison between poly hydroxyl acrylic acid which is an environmen-tally friendly ifxation liquid and 4% neutral buffered formaldehyde in tissue ifxation for HE staining and FISH to detect theHER-2 gene in the breast cancer tissue sections. The study aimed to evaluate the feasibility of replacing 4% buffered formaldehyde, a traditional ifxation liquid, with the poly hydroxyl acrylic acid, an environmentally friendly ifxation lfuid.Methods:This project was performed on tissue samples collected from 69 cases of breast cancer, 41 cases of breast ifbroadenoma, 40 cases of uterine leiomyoma, 25 cases of cervical tissue, 25 cases of placenta obtained from the outpatient and inpatient departments of Zhongshan Boai Hospital from Mar. 2011 to Jan. 2015, from each of which two samples were drawn and two blocks of each specimen were divided into two groups randomly. Then one group was ifxed with 4% neutral buffered formaldehyde and made into 200 sections by HE while the other group was ifxed with poly hydroxyl acrylic acid and made into another 200 sections. The slice level of the two groups was determined by the staining condition of the sections, and SPSS 19.0 was employed to compare the excellent and good rate of HE staining. Additional 69 sections were produced with two groups of breast invasive ductal carcinoma tissues, and SPSS 19.0 was used to detect the ampliifcation ofHER-2 gene by FISH.Results:First, the number of best-quality slices stained with HE ifxed separately by poly hydroxyl acrylic acid and 4% neutral buffered formaldehyde was 155 and 166, respectively. The number of excellent pieces was 41 and 33, respectively, while the number of mediocre pieces was 3 and 1 with bad pieces being 1 and 0, respectively. The excellent and good rates of HE staining were 98% and 99.5%, respectively. There was no significant difference between the two groups (χ2=1.33,P>0.05).Second, the positive rates of the tis-sue slices by FISH ifxed separately by poly hydroxyl acrylic acid and 4% neutral buffered were 26.09% and 23.19%, respectively. There was no signiifcant difference between the two groups (χ2=0.50,P>0.05).Conclusion:The results obtained with HE staining and FISH using poly hydroxyl acrylic acid as a ifxation liquid are not signiifcantly different from those using 10% neutral buffered formaldehyde. Therefore, poly hydroxyl acrylic acid meets the requirements of environmental protection, and thus has the potential to be promoted and widely used.

OBJECTIVETo investigate the differential expression pattern of hsa-miR-9 between EBV-positive and -negative Burkitt lymphoma cell lines and its association with BCL-6.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONHsa-miR9 positively regulate the expression of BCL-6 and apoptosis of EBV(+) Raji cells and EBV(-) Ramous cells.

Apoptosis ; Burkitt Lymphoma ; genetics ; pathology ; virology ; Cell Division ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Herpesvirus 4, Human ; Humans ; MicroRNAs ; metabolism ; Proto-Oncogene Proteins c-bcl-6 ; Transfection

Objective To explore the expression of miR-9 in H/RS cells and its regulation on target PRDM1.Methods miR-9 expression in normal CD19+ B-cell subsets and eight lymphoma cell lines was detected by fluorescence quantitative RT-PCR and in situ hybridization (ISH),for quantification and location,respectively.Chemically synthesizcd antisense oligonucleotide of miR-9 was transiently transfected into L428 for its silence,and the PRDM1 expression was tested.Results Fluorescence quantitative RT-PCR showed that the expression of miR-9 in L428 cells was marked higher than that of normal CD19+ B-cell subsets and other lymphoma cell lines (the expression of miR-9 in L428 cells was 47-fold of OCI-Ly1,50-fold of Raji cells,7-fold of EBV+ immortalized B cell line,and 6-fold of ALCL cell line).ISH indicated that miR-9 located in cytoplasm,it was a diffuse and strong positive in L428,scattered and weak in DLBCL and Burkitt' s lymphoma cell lines,while negative in KARPAS-299 or Jurkat cell lines.Transient down-regulation of miR-9 in L428 leded to the increase of PRDMI protein.Conclusion miR-9 plays the role of cancer gene in cHL,and may exert a potential function in regulating terminal B cell differentiation through a post transcription regulation of PRDM1 gene.