Preliminary screening of downstream proteins of Sox2 and role of Sox2 in colonic cancer cell migration and invasion / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen the down-stream proteins of transcription factor Sox2 and explore the role of Sox2 in the proliferation and migration of colonic cancer cells in vitro.</p><p><b>METHODS</b>The cellular proteins were separated by SDS-PAGE electrophoresis and stained with Coomassie blue and amine plated silver. The differentially expressed proteins was identified by mass spectrometry and verified by QPCR and Western blotting. A cell counting kit-8 (CCK8) assay was performed to evaluate the cell proliferation, and the cell migration was assessed using Transwell assay.</p><p><b>RESULTS</b>S3a was identified by proteomics technology as a Sox2-downregulated protein while ENO1 and gama-actin the up-regulated proteins. QPCR and Western blotting analyses showed that overexpression of Sox2 significantly decreased the expression of S3a (P<0.005) and increased the expression of ENO1(P<0.05), but had no significant effect on gama-actin expression. Sox2 overexpression obviously promoted cell proliferation and migration (P<0.05), while inhibition of Sox2 produced contrary effects (P<0.05).</p><p><b>CONCLUSION</b>Sox2 negatively regulates S3a expression and positively regulates ENO1 expression to promot the proliferation and migration of colonic cancer cells.</p>

Construction of colorectal cancer cell line stably expressing mir-101 and identification of the target gene of mir-101 / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a colorectal cancer cell line stably expressing mir-101 and identify the target gene of mir-101.</p><p><b>METHODS</b>Quantitative real-time PCR was used to detect mir-101 expression in colorectal cancer cell lines. The recombined lentiviral vector GV209-mir101 or the empty lentiviral vector GV209 was transfected into human colorectal cancer cells SW620. The recombinant psiCHECK-2-Rac1 vector containing RAC1 3'UTR was constructed, and site-directed mutagenesis of RAC1 3'UTR was induced to construct the psiCHECK-2-Rac1-Mut vector. In HEK293A and SW480 cells co-transfected with mir-101 inhibitors or negative control (NC) and these recombined vectors, luciferase activities was examined with a dual-luciferase reporter assay.</p><p><b>RESULTS</b>SW620 cells transfected with GV209-mir101 lentivirus exhibited higher mir-101 expression level than cells transfected with GV209 lentivirus. Mir-101 inhibitors significantly increased the luciferase activities of RAC1 3'UTR. Overexpression of mir-101 increased the expression of RAC1 while inhibition of mir-101 suppressed RAC1 expression.</p><p><b>CONCLUSION</b>We have successfully constructed a SW620 cell line stably overexpressing mir-101. mir-101 can suppress RAC1 gene expression by targeting the specific sequence of RAC1 3'UTR.</p>

Relationship between glypican-3 and Notch1 expressions in hepatocellular carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the correlation between the expressions of glypican-3 (GPC3) and Notch1 and their roles in the tumorigenesis and progression of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Immunohistochemistry and computerized image analysis were utilized to quantitatively detect the expressions of GPC3 and Notch1 in 30 HCC tissue specimens.</p><p><b>RESULTS</b>In the 30 HCC specimens, GPC3 expression decreased significantly as the grade of tumor differentiation increased (P<0.05 or P<0.01), while Notch1 expression presented with a reverse pattern of changes (P<0.05 or P<0.01). An obvious negative correlation was found between the expressions of GPC3 and Notch1 in HCC tissues (rp=-0.607, P=0.000; r=-0.692, P=0.000).</p><p><b>CONCLUSION</b>The expressions of GPC3 and Notch1 show a negative correlation in HCC, suggesting a possible mechanism for mutual regulation between them to contribute to the tumorigenesis and progression of HCC.</p>

Expression of hsa-miR-186 and its role in human colon carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the expression of hsa-mir-186 in colorectal cancer and study its role in regulating the biological behaviors of human colorectal cancer SW620 cells in vitro.</p><p><b>METHODS</b>The expression of hsa-miR-186 in colon cancer tissue and the adjacent tissues as well as 5 colon carcinoma cells were analyzed using real-time quantitative RT-PCR. The precursor sequence of miR-186 gene was amplified from the genomic DNA by PCR and cloned into the lentiviral vector PLVTHM labeled with GFP. The colorectal cancer cell line SW620 was transfected with PLVTHM-miR186 vector and the lentivirus-infected cells were sorted with flow cytometry. Cell counting kit-8 (CCK-8) assay was used to detect the proliferation of the cells. The migration and invasion of SW620 cells were investigated using Transwell assay and scratch test. Western blotting was used to detect the expression of YY1 protein in SW620 cell lines.</p><p><b>RESULTS</b>The relative expression of miR-186 in the cancer tissues was 0.0024∓0.0027, significantly lower than that in the adjacent tissues (0.066∓0.068, P=0.008); the relative expression level of hsa-miR-186 in SW620 and LoVo cells with a high metastatic potential was 0.118∓0.138 and 0.157∓0.001, respectively, significantly lower than that in HT-29 cells with a low metastatic potential (1.000∓0.00, P<0.05). The recombinant lentiviral vector PLVTHM-miR186, verified by enzyme digestion, sequencing and qPCR, caused significant inhibition of cell proliferation, migration and invasion and suppressed the expression of YY1 protein in SW620 cells.</p><p><b>CONCLUSION</b>As a tumor suppressor gene, Hsa-miR-186 is down-regulated in colon carcinoma tissues and in highly metastatic SW620 and LoVo cells. Has-miR-186 can inhibit the cell proliferation, migration and invasion of colon carcinoma cells in vitro possibly by suppressing YY1 expression.</p>

Expression of Id1 and Id3 in endometrial carcinoma and their roles in regulating biological behaviors of endometrial carcinoma cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of inhibitor of DNA differentiation/DNA binding 1 (Id1) and Id3 in endometrial carcinoma and explore their roles in regulating the proliferation, invasion, migration and adhesion of endometrial carcinoma cells in vitro.</p><p><b>METHODS</b>Id1 and Id3 expression in 4 fresh endometrial cancer tissue specimens and matched adjacent tissues were detected using Western blotting. Two endometrial cancer cell lines, HEC-1-B and RL-952, were both divided into 4 groups, namely the untreated group, blank virus group, promoter group and Id1/Id3 double-knockdown group, and their expressions of MMP2, CXCR4 and P21 were detected by qRT-PCR and Western blotting. The proliferation, invasion, migration and adhesion of the cells were evaluated with MTT, Transwell, wound-healing, and adhesion assays.</p><p><b>RESULTS</b>Endometrial carcinoma tissues showed significantly higher Id1 and Id3 expression than the adjacent tissues (P<0.05). In the two endometrial carcinoma cell lines, Id1/Id3 double-knockdown significantly decreased MMP2 and CXCR4 expression and increased P21 expression at both mRNA and protein levels (P<0.05), and resulted in suppressed cell proliferation, invasion, migration and adhesion.</p><p><b>CONCLUSION</b>Id1 and Id3 expressions are up-regulated in endometrial carcinoma to promote the proliferation, invasion, migration and adhesion of the tumor cells by increasing MMP2 and CXCR4 expression and reducing P21 expression. Therapies targeting Id1/Id3 can be a novel strategy for treatment of endometrial carcinoma.</p>

Initial screening of binding-peptide of the cell surface marker CD133 of cancer stem cells / 北京大学学报(医学版)

Objective:To select the binding-peptide of the cell surface marker CD133 of cancer stem cells from phage peptide library,and to find a new tool for research on stem cells,tumor therapy and anti-metastasis of cancer.Methods:Biotined mouse CD133 extracellular fraction was used as a target to screen phage 7-peptide library by the high affinity of streptavidin and biotin,and the clones were identified by sandwich ELISA and competitive experiment.Single strand DNA was extracted from these positive clones and was analyzed by single-strand dideoxy-sequencing.Results:After three turn solution panning,five peptides with high affinity shared the same amino acid sequence:APSPMIW and three identical peptides with high affinity shared the same amino acid sequence:LQNAPRS.Conclusion:The peptides that bind with mouse CD133 extracellular fraction with high affinity and specificity were first screened from the phage peptide library for the first time,which initially indicates that the feasibility of screening from phage peptide library with small molecule polypeptide biotined as a target.

Clinicopathological analysis and genetic diagnosis of subcutaneous panniculitic T-cell lymphoma / 临床与实验病理学杂志

Purpose　To explore the clinicopathological features of subcutaneous panniculitic T-cell lymphoma(SPTCL) and significances of genetic analysis in the diagnosis. Methods　Histopathology, immunohistochemitry and detection of clonal gene rearrangement by PCR were used in 3 cases of subcutaneous panniculitic T-cell lymphoma (SPTCL), which were originally diagnosed as relapsing nodular nonsuppurative panniculitis. Results　Three misdiagnostic cases were correctly redefined as subcutaneous panniculitic T-cell lymphoma, with immunophenotype of CD45+,CD45RO+, Mac387－,and clonal TCR-β gene rearrangement. Conclusions　Subcutaneous panniculitic T-cell lymphoma has distinctive clinicopathological features. Genetic analysis is an effective method for the diagnosis of SPTCL.

Silencing effect of lentivirus-mediated RNA interference on metastasis-associated gene 1(MTA1) in human nasopharyngeal carcinoma cell line / 解放军医学杂志

Objective To investigate the difference between two substrains,5-8F and 6-10B,of nasopharyngeal carcinoma cell line SUNE1on expressing metastasis-associated gene 1 (MTA1),and evaluate the silencing effect of lentivirus-mediated RNA interference (RNAi) on MTA1 in human nasopharyngeal carcinoma cell line.Methods The differential expression of MTA1 mRNA and MTA1 protein in 5-8F and 6-10B cell lines were detected by real time PCR and Western blotting respectively.Short interference RNA (siRNA) fragment targeting MTA1 gene was designed using online databases and software.A specific RNAi lentiviral vector targeting human MTA1 gene was constructed and transfected into nasopharyngeal carcinoma cell line 5-8F.Real time PCR and Western blotting were performed to detect the expressive changes in MTA1 mRNA and MTA1 protein in the transfected 5-8F cells respectively.Results Compared with cell line 6-10B,the expressions of MTA1 mRNA and MTA1 protein were higher in 5-8F cell line.Sequence analysis validated the correct insert of siRNA targeting MTA1 gene into the lentiviral vector.Real time PCR and Western blotting results showed that the expressions of MTA1 mRNA and MTA1 protein in 5-8F cell lines were down-regulated significantly after siRNA transfection.Conclusions MTA1 may promote the malignant transformation and enhance the metastatic potential of nasopharyngeal carcinoma cell lines.The expression of MTA1 in 5-8F cells can be inhibited effectively by MTA1-specific siRNA expression lentiviral vector,which provides a valuable tool for investigating the role of MTA1 gene in the carcinogenesis and progression of nasopharyngeal carcinoma.

Effect of CD133~+ stem cells from cord blood on the proliferation,adhesion and migration in human colorectal neoplasm cell line SW480 / 中国癌症杂志

Background and purpose:The most serious aspect of cancer is the emergence of metastases in organs distant from the primary tumor, since most deaths from cancer are due to metastases. In recent years, a series of studies has been undertaken both in vitro and in vivo, and demonstrated that the CD133+ hematopoietic stem cell is effective in increasing mice tumor cell metastases, but the efficacy of CD133+ stem cells is not well studied for human cancer.We investigated the effects of CD133+ stem cells on the proliferation, adhesion and migration in human colorectal neoplasm cell line SW480.Methods:CD133+ cells were separated from fresh cord blood mononuclear cells(MNC) by Ficoll density gradient centrifugation and magnetic activated cell-sorting system(MACS).The treatment group was the co-culture of CD133+ cells and SW480, the control group used the same culture medium without CD133+ cells. The effect of CD133+ cells on proliferation in SW480 cell was measured by MTT assay, the migration abilities of SW480 cell were assayed in Transwell cell culture, Cell adhesion assay was carried out in a 96 microplate well precoated with fibronection.Results:CD133+ cells could significantly improve the growth ability of SW480 cell, not only the ability of proliferation, but also the morphology. The morphology of the cells was remarkably changed. Irregular nucleus, double nucleus and polymorphic nucleus appeared in the treatment group, and the cells were shaped as polygon-like and leptosomatic. In the cell adhesion assay, A 490 of the treatment group was 0.11?0.01, A 490 of the control group was 0.05?0.01(P﹤0.05).Conclusions:Our results indicate that the CD133+ cell is effective in increasing tumor cell proliferation and invasion. Hematopoietic stem cell may play an important role in the invasion and metastasis of colorectal neoplasm.