Clinical Significance of co-expression of RNA Binding Protein PTBP1 and p-AXL in Osteosarcoma / 中山大学学报(医学科学版)

ObjectiveTo explore the co-expression of PTBP1 and p-AXL in osteosarcoma and its clinicopathological significance for prognosis evaluation. MethodsThe expression of PTBP1 and AXL and their prognostic value in osteosarcoma were analyzed by GEO and Target data. Paraffin biopsy specimens and clinical information from 76 cases of osteosarcoma and 37 cases of non-malignant bone tissue （callus， osteofibrous dysplasia and osteoid ostema） were obtained from the First Affiliated Hospital of Sun Yat-sen University from March 2016 to October 2020. The expressions of PTBP1 and p-AXL proteins in osteosarcoma were detected by immunohistochemistry. ResultsGEO database showed that the expression levels of PTBP and AXL in osteosarcoma tumor group were higher than those in normal tissues， but did not reach statistical significance. Target database showed that the high expression of PTBP1 had shorter Overall survival（OS） and Progression-free survival（PFS） than low PTBP1 expression， but did not reach statistical significance （P=0.064； P=0.134）. Immunohistochemical staining included 76 cases of osteosarcoma and 37 cases of non-malignant bone tissue. The expression rate of PTBP1 and p-AXL protein in osteosarcoma tissues was higher than that in non-malignant bone tissue. The expression of p-AXL is correlated with lung metastasis （P=0.025）. Kaplan-Meier analysis showed that lung metastasis， recurrence， PTBP1 expression， co-expression of PTBP1/p-AXL influence the prognosis of patients in OS. Multivariate Cox regression analysis showed that lung metastasis （P<0.000 1） and positive expression of PTBP1 （P=0.041） were independent risk factors for osteosarcoma patients in OS. Co-expression of PTBP1 and p-AXL had shorter OS （P=0.017） and PFS （P=0.043） than non-coexpression osteosarcoma patients. ConclusionsPTBP1 and p-AXL were highly expressed in osteosarcoma tissues. The co-expression of PTBP1 and p-AXL was associated with poor prognosis of patients， and PTBP1 could be used as an independent prognostic indicator of patients with osteosarcoma.

IMP3 is a novel biomarker to predict metastasis and prognosis of gastric adenocarcinoma: a retrospective study / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is a newly identified mRNA-binding protein that is involved in embryogenesis and carcinogenesis of some malignant tumors. The aim of this study was to detect the expression of IMP3 protein in gastric adenocarcinoma (GAC) and the correlation with clinicopathological features.</p><p><b>METHODS</b>IMP3 protein in 92 samples of GAC was evaluated by immunohistochemical method. The Mann-Whitney U test and Kruskal-Wallis H test were used to compare IMP3 expression and clinicopathological parameters. Kaplan-Meier survival curve, log-rank test and Cox-regression model were used to evaluate the correlation of IMP3 protein expression to the prognosis of patients.</p><p><b>RESULTS</b>Out of 92 cases of adjacent normal mucosa (ANM), 10 with dysplasia demonstrated weak expression of IMP3 and 82 without dysplasia showed negative expression. Out of 92 cases of GAC, positive immunohistochemical stain for IMP3 was identified in 75 (82%) cases. A comparison of IMP3 expression in GAC and ANM showed stronger immunohistochemical reactivity in GAC (P < 0.05). High expression of IMP3 was found to be associated with lymphoid metastasis, high Ki-67 labelling index, and patient poor outcome (P < 0.05). There was a significant TNM stage difference between GAC with and without IMP3 expression (P < 0.05). Tumors with higher stage showed higher level of IMP3 expression. In multivariate analysis, IMP3 emerged as an independent predictor of survival.</p><p><b>CONCLUSIONS</b>Increase of IMP3 expression suggests that IMP3 may play an importent role in the carcinogenesis and tumor metastasis in GAC. It could be regarded as a novel proliferation and prognostic indicator for patients with GAC.</p>

Suppression of osteosarcoma in vitro by coexpression of antisense VEGF165 cDNA and thymidine kinase gene / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions.</p><p><b>METHODS</b>Eukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations.</p><p><b>RESULTS</b>The eukaryotic expression vector pBI-HRE-AsVEGF165 -HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentration-dependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the "bystander effect" was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at G0 approximately G1 phase, apoptosis and necrosis.</p><p><b>CONCLUSIONS</b>Antisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.</p>

Expression of syndecan-1 at different stages in the course of gastric carcinoma and its significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of syndecan-1 protein at different stages in the course of gastric carcinoma and its significance in carcinogenesis and metastasis.</p><p><b>METHODS</b>There were 56 cases of chronic gastritis, 50 cases of chronic atrophic gastritis, 59 cases of intestinal metaplasia, 61 cases of displasia, and 112 cases of gastric carcinoma. Among the carcinoma cases, 55 were without and 57 with lymph node metastases. All paraffin-embedded tissue samples were assessed by immunohistochemistry.</p><p><b>RESULTS</b>The syndecan-1 positive rate was 96.43% (54/56) in gastritis, 98.00% (49/50) in chronic atrophic gastritis, 100.00% (59/59) in intestinal metaplasia, 91.80% (56/61) in displasia, 45.45% (25/55) in gastric carcinoma without, and 24.56% (14/57) in gastric carcinoma with lymph node metastases. There was no significant difference among chronic gastritis, chronic atrophic gastritis and intestinal metaplasia (P > 0.05). There was a significant difference between displasia group and gastric carcinoma group (P <0.05), as well as between gastric carcinoma with and without lymph node metastases. There was a significant difference among well, moderately and poorly differentiated carcinoma groups.</p><p><b>CONCLUSION</b>A decreasing expression of syedecan-1 in the development of gastric carcinoma is related with gastric carcinogenesis, and it may further promote metastasis of gastric carcinoma.</p>

The role of CD44 in the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the influence of CD44 a cell-matrix adhesion molecule on the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines, in order to investigate the growth and invasion mechanism of osteosarcoma.</p><p><b>METHODS</b>Three osteosarcoma cell lines MG-63, HOS and U2-OS were routinely cultured. Flow cytometry and Western blot analysis were used for detecting the positive rates and relative amount of CD44 protein in the three cell lines. RT-PCR method was also used to compare the differences in the expression of CD44 mRNA among the 3 cell lines. Then, MTT method, adhesion detection, and Microcon-migration assay were used to detect the changes of the cells' proliferation rate, adhesive and invasive abilities after blocking the role of CD44 by using a special neutralizing antibody.</p><p><b>RESULTS</b>The results of flow cytometry showed that the percentage of CD44 positive cells were both over 99% in HOS and U2-OS, while that in MG-63 was only (2.10 +/- 0.46)%. The average fluorescence density of CD44 in HOS was significantly higher than in U2-OS. Western blot also showed that the relative content of CD44 protein in HOS was notably higher than that in U2-OS, while CD44 was negatively expressed in MG-63. The expression of CD44 mRNA was significantly lower in MG-63 than in both HOS and U2-OS, which were consistent with the expression of CD44 protein. The proliferation rates and adhesive abilities of MG-63 and HOS have no significant difference, but both were significantly higher than that of U2-OS. The invasive abilities of HOS was dramatically higher than MG-63 and U2-OS. After the role of CD44 was blocked by anti-CD44 neutralizing antibody, the proliferation rates of the 3 cell lines did not change significantly. While the HOS and MG-63 adhesion indices decreased dramatically (P < 0.05), the invasive abilities of HOS and U2-OS also decreased notably (P < 0.01).</p><p><b>CONCLUSIONS</b>CD44 could promote the adhesiveness and invasiveness of osteosarcoma cell line HOS. CD44 may take part in promoting the process of U2-OS invasion and the adhesion of MG-63. On the other hand, CD44 could not affect the osteosarcoma cell proliferation rates.</p>

Targeted inhibition of vascular endothelial growth factor (VEGF) expression in human osteosarcoma cell line by antisense VEGF165 cDNA promoted by hypoxia reaction element / 中华病理学杂志

<p><b>OBJECTIVE</b>Utilizing the hypoxia inducible factor 1/hypoxia reaction element (HIF-1/ HRE) gene regulation system to construct antisense vascular endothelial growth factor (VEGF165) cDNA eukaryotic expression vector promoted by HRE, and investigate its targeted inhibiting VEGF expression of osteosarcoma cells in hypoxia environment.</p><p><b>METHODS</b>Eukaryotic expression plasmid with HRE promoter was constructed containing luciferase reporter gene and antisense VEGF165 cDNA by using PCR and recombinant DNA techniques. The recombinant vectors were transfected into osteosarcoma cells with lipofectin method. Hypoxia-inducible reporter gene expression was determined by liquid scintillation analyzer and the expression of VEGF protein was detected by ELISA method.</p><p><b>RESULTS</b>The eukaryotic expression plasmid containing antisense VEGF165 and luciferase promoted by HRE was constructed successfully. After being transferred into MG63 cells, luciferase expression was increased 3.5 x 10(2) times and VEGF protein expression decreased 45% under hypoxia condition.</p><p><b>CONCLUSION</b>Antisense VEGF165 cDNA expression, efficiently realized by HRE promoter under hypoxia condition, provides an experimental basis for targeted antiangiogenesis of tumors.</p>