Silencing of MGMT with small interference RNA reversed resistance in human BCUN-resistant glioma cell lines / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Our previous study had cloned two glioma cell lines SWOZ1 and SWOZ2 isolated from parental glioma cell line SWO38. The 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance of SWOZ1 was higher than that of SWOZ2. Since O6-methylguanine-DNA methyltransferase (MGMT) was thought to be closely related to BCNU resistance in glioma, this study aimed to explore the function of MGMT in glioma resistant to BCNU.</p><p><b>METHODS</b>A BCNU resistant glioma cell line SWOZ2-BCNU was established. The expression of MGMT was detected in SWOZ1, SWOZ2 and SWOZ2-BCNU. Small interferencing RNA targeting MGMT was used to silence the expression of MGMT in resistant cell lines SWOZ1 and SWOZ2-BCNU. The cytotoxicity of BCNU to these cells was measured using the cell counting kit-8 assay. Statistical analysis was carried out by one-way analysis of variance in statistical package SPSS 13.0.</p><p><b>RESULTS</b>The resistance of SWOZ1 and SWOZ2-BCNU against BCNU was 4.9-fold and 5.3-fold higher than that of SWOZ2. The results of quantitative RT-PCR and Western blotting confirmed that MGMT was both significantly increased in SWOZ1 and SWOZ2-BCNU compared to SOWZ2. After transfection with small interferencing RNA targeting MGMT, a decreased level of MGMT mRNA expression in SWOZ1 and SWOZ2-BCNU for more than 75% compared to negative control was found and confirmed by Western blotting. As a result, the resistance against BCNU was reversed for about 50% both in the BCNU-resistant cell lines SWOZ1 and SWOZ2-BCNU.</p><p><b>CONCLUSIONS</b>Silencing MGMT with specific small interferencing RNA can reverse the BCNU resistant phenotype in these glioma cell lines. MGMT may play an important role both in intrinsic and acquired BCNU-resistance in glioma.</p>

Expression and significance of tumor drug resistance related proteins and beta-catenin in esophageal squamous cell carcinoma / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>As chemotherapy is generally used in the clinical treatment of cancer, the problem of multidrug resistance (MDR) of tumors against the chemotherapeutic agents becomes more and more serious. It has been the major cause for the failure of the chemotherapy. We detected the expressions of beta-catenin and tumor drug resistance related proteins, MRP2, P-gp, and Bcl-2, in esophageal squamous cell carcinoma (ESCC) to explore their function and correlation in the occurrence and development of MDR in ESCC.</p><p><b>METHODS</b>We used the tissue microarray technique, immunohistochemistry, and image analysis methods to detect the expressions of MRP2, P-gp, beta-catenin, and Bcl-2 proteins and analyze their relationships with clinical data in a ESCC tissue microarray consisting of 582 specimens of ESCC, 294 specimens of normal mucosa, 92 specimens of basal cell hyperplasia, and 87 specimens of dysplasia adjacent to cancer tissue.</p><p><b>RESULTS</b>The integral optical density (IOD) of MRP2 and Bcl-2, which was 195.7 +/- 175.9 x 10(3)) and 90.5 +/- 112.5 x 10(3)), respectively, was significantly higher in ESCC than in normal mucosa, which was 104.8 +/- 86.1 x103) and 25.2 +/- 46.6 x 10(3)), respectively (P < 0.01). The IOD of P-gp and beta-catenin, which was 57.7 +/- 75.5 x 10(3)) and 32.0 +/- 47.0 x 10(3)) respectively, was significantly lower in ESCC than in normal mucosa, which was 114.8 +/- 106.6 x 10(3)) and 46.1 +/- 35.7 x 10(3)), respectively (P < 0.01). According to the following order, well differentiated moderately differentiated poorly differentiated, the IOD of MRP2 increased in ESCC (P < 0.01). The IOD of beta-catenin was higher in poorly differentiated ESCC than in well or moderately differentiated ESCC (P < 0.01). The IOD of Bcl-2 was lower in well differentiated ESCC than in poorly and moderately differentiated ESCC (P < 0.01). The IOD of beta-catenin and Bcl-2 was higher in the ESCC of specimens with infiltration depths that were in membrane mucosa than those in the muscular layer or serous coat (P < 0.01). The IOD of Bcl-2 was significantly higher in cases with lymph node metastasis than in cases without (P < 0.01). Positive correlations which were respectively between the expressions of P-gp and MRP2, the expressions of P-gp and Bcl-2 were found (r = 0.288 and r = 0.253, respectively, P < 0.01).</p><p><b>CONCLUSIONS</b>MRP2, P-gp, and Bcl-2 may be taken as prognostic factors for MDR of ESCC. beta-catenin may play an important role in carcinogenesis and progression of ESCC.</p>

Establishment of a stable nasopharyngeal carcinoma cell line with lentivirus-mediated RNA interference for EIF4G1 gene silencing / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a nasopharyngeal carcinoma (NPC) cell line with stable EIF4G1 gene silencing induced by small interfering RNA (siRNA).</p><p><b>METHODS</b>The EIF4G1 mRNA levels in 8 NPC cell lines including 5-8F, 6-10B, C666-1, CNE1, CNE2, HNE1, HONE1, and SUNE1 were detected by fluorescence quantitative RT-PCR (QRT-PCR). The recombinant lentivirus shRNA expression plasmid targeting EIF4G1 gene was packaged into mature lentivirus by 293FT cells and used to infect 5-8F cells. After blasticidin selection of NPC cells with constant expression of the EIF4G1-siRNA, the efficiency of EIF4G1 mRNA expression interference was determined using QRT-PCR.</p><p><b>RESULTS</b>The 8 NPC cell lines showed differential expression of EIF4G1 mRNA, among which 5-8F cells had the highest EIF4G1 expression. The recombinant lentivirus plasmid pLenti6/BLOCK-iT-DEST/EIF4G1-shRNA was successfully constructed and verified by PCR and sequencing. The EIF4G1 mRNA level of 5-8F cells infected with shRNA-EIF4G1 lentivirus was significantly reduced as compared with the negative control and the blank control cells.</p><p><b>CONCLUSION</b>The recombinant lentivirus vector pLenti6/BLOCK- iT-DEST/EIF4G1-shRNA we constructed results in marked downregulation of EIF4G1 mRNA expression and constant expression of EIF4G1-siRNA after infection of 5-8F cells.</p>

Expressions of NF--kappaBp65, TRAF2, cyclinD1 and their association with cell apoptosis in oral lichen planus / 南方医科大学学报

<p><b>OBJECTIVE</b>To examine the expression and distribution of NF-kappaBp65, TRAF2, and cyclinD1 and their association with cell apoptosis in oral lichen planus (OLP).</p><p><b>METHODS</b>Sixty OLP patients were divided into erosion-atrophy group (n=30) and non-erosion group (n=30) according to their clinical features. Immunohistochemistry with SP method was used to detect the expressions of NF-kappaBp65, TRAF2, cyclinD1 in the 60 OLP and 40 normal oral mucosa (control) specimens. TUNEL assay of randomly selected specimens from 10 normal and 15 OLP cases was performed to detect the cell apoptotic index (AI).</p><p><b>RESULTS</b>Compared with the control group, OLP group showed significantly increased AI of the epithelial cells (67.32-/+18.99) and decreased AI of the lymphocytes (34.12-/+9.89) (P<0.05). In the OLP group, the positivity rates for NF-kappaBp65, TRAF2, and cyclin D1 in the epithelial cells (85.00%, 76.67% and 71.67%, respectively) and in the lymphocytes (91.67%, 86.67% and 70.00%, respectively) were all significantly higher than those in the control group (P<0.05). NF-kappaBp65 expression was significantly increased in the lamina propria in the non-erosion OLP group as compared to the erosion-atrophy group. A positive correlation was noted between lymphocyte NF-kappaBp65 expression and AI of the epithelial cells, but an inverse correlation found between lymphocyte NF-kappaBp65 expression and the AI of the lymphocytes. Lymphocyte TRAF2 and cyclin D1expressions were also inversely correlated to lymphocyte AI. There was a positive correlation between TRAF2 and cyclin D1 expressions and the expression NF-kappaBp65 in the epithelial cells and lymphocytes in OLP.</p><p><b>CONCLUSIONS</b>Accelerated apoptosis of the keratinocytes and inhibition of lymphocyte apoptosis may coexist to contribute to the formation and progression of OLP. NF-kappaBp65 expression, particularly its abnormal nuclear expression, may play a partial role in the pathogenesis of OLP.</p>

Effect of Tiam-l gene silencing on human colorectal carcinoma cell line SW480 metastasis in nude mice observed by real-time whole-body fluorescence imaging / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of Tiam-l gene silencing on the metastasis of human colorectal carcinoma cell line SW480 in nude mice by real-time whole-body fluorescence imaging.</p><p><b>METHODS</b>Enhanced green fluorescence protein (EGFP)-labeled human colorectal carcinoma cells, SW480/EGFP(+)/Tiam-1(-) and SW480/EGFP(+), were implanted into nude mice via tail vein injection or orthotopic colonal inoculation, and real-time whole-body fluorescence imaging was performed to compare the difference in tumor progression and metastasis between the two cells.</p><p><b>RESULTS</b>Both SW480/EGFP(+) and SW480/ EGFP(+)/Tiam-1(-) cells stably expressed EGFP, and Tiam1 gene expression was reduced in SW480/EGFP(+)Tiam-1(-) to 30% of the expression level in SW480/EGFP(+) cells. The growth rate of the two cell lines had no significant difference in vitro (P>0.05), but SW480/EGFP(+)/Tiam1(-) cell proliferation and metastasis were depressed obviously in comparison with SW480/EGFP(+) in vivo (P<0.05).</p><p><b>CONCLUSION</b>Tiam-1 gene may play an important role in invasion and metastasis of human colorectal cancer.</p>

Expression and cloning of mCD99L2 gene from mouse B lymphoma cell line A20 and construction of its eukaryotic expression vector / 南方医科大学学报

<p><b>OBJECTIVE</b>To detect and clone mCD99L2 gene from mouse B lymphoma cell line A20 and construct its eukaryotic expression vector pcDNA3.1-mCD99L2.</p><p><b>METHODS</b>The expression of mCD99L2 mRNA in A20 cell line was detected by in situ hybridization. The total RNA of A20 cells was extracted to obtain the full-length cDNA of the coding region of mCD99L2 gene by RT-PCR, the product of which was ligated into pMD18-T vector and the DNA sequence of the insert was detected. The coding regions of mCD99L2 gene was amplified from pMD-mCD99L2 by PCR using primers containing EcoR I and Xho I sites and cloned into the eukaryotic expression vector pcDNA3.1/MycHis(+).</p><p><b>RESULTS</b>In situ hybridization identified positive expression of mCD99L2 gene in the A20 cell line. The full-length cDNA of mCD99L2 coding region of A20 cell line was obtained by RT-PCR, which yielded a product of 712 bp as expected, and the DNA sequence was completely homologus to the mCD99L2 cDNA reported in GenBank. Restriction endonuclease digestion and DNA sequencing indicated that the eukaryotic expression vector pcDNA3.1(+)- mCD99L2 had been constructed successfully.</p><p><b>CONCLUSION</b>mCD99L2 cDNA has been cloned from mouse B lymphoma cell line A20 and its eukaryotic expression vector pcDNA3.1(+)- mCD99L2 successfully constructed, which facilitates further functional study of mCD99L2 gene in mouse B lymphoma cell line A20.</p>

Expression of tumor suppressor gene PTEN, PIP3 and cyclin D1 in oral squamous cell carcinoma and their correlations / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To detect the expression of PTEN, PIP3 and cyclin D1 in oral squamous cell carcinoma and precancerous lesions and analyze their correlation.</p><p><b>METHODS</b>Immunohistochemistry SP method was used to detect the expression of PTEN, PIP3 and cyclin D1 in 63 cases of oral squamous cell carcinoma, 29 cases of simple hyperplasia, 33 cases of dysplasia, and 25 cases of normal oral mucosa.</p><p><b>RESULTS</b>The negative or low expression of PTEN in oral squamous cell carcinoma was 25%, which was remarkably lower than that in other groups. The positive expression of PIP3 in simple hyperplasia, dysplasia and oral squamous cell carcinoma was 66%, 64%, and 76% respectively, which were much higher than those in normal oral mucosa. The positive expression of cyclin D1 in oral squamous cell carcinoma was 49%, which was significantly higher than that in other groups. The negative correlation between PTEN with PIP3, cyclin D1 and the positive correlation between PIP3 and cyclin D1 were observed.</p><p><b>CONCLUSIONS</b>PTEN may play a role in the oncogenesis of oral squamous cell carcinoma, and PTEN may down-regulate the expression of PIP3, and then down-regulate the expression of cyclin D1, which leads to the suppression of cell growth.</p>