OBJECTIVE: To investigate the association between the expression of caveolin-1(CAV-1) and the invasion of choriocarcinoma, and to explore the effect of CAV-1 small interfering RNA(siRNA) on the invasion of choriocarcinoma cell line JEG-3. METHODS: (1) Matrigel invasion assay and 3-(4,4)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumormide (MTT) assay were used to examine the difference in invasion and proliferation ability between JEG-3 cells and JAR cells;(2) Expression of caveolin-1 gene in the human chorionic villi tissues and chorionicnoma cell lines (JEG-3 cells and JAR cells) were detected by semi-quantitative RT-PCR. (3) The effect of CAV-1 siRNA transfection on the expression of CAV-1 mRNA, and the invasion and proliferation ability of JEG-3 cells were measured by RT-PCR, Matrigel invasion assay and MTT assay. RESULTS: (1) The invasion ability of JEG-3 cell line was stronger than that of JAR cell line (P<0.05), but the difference in proliferation ability between JAR and JEG-3 was not obvious (P>0.05);(2) The expression of caveolin-1 gene in chorionicnoma cell lines was significantly stronger than that in the human normal chorion(P<0.05), and the expression of caveolin-1 gene in JEG-3 cells was stronger than that in the JAR cells (P<0.05). The data suggested that there was significantly positive correlation between caveolin-1 and the invasiveness of chorionicnoma cells (r=0.086,P<0.05);(3) CAV-1 siRNA could knock-out the expression of CAV-1 mRNA, and inhibit the invasion and proliferation ability of chorionicnoma cells. CONCLUSION CAV-1 can promote the invasion ability of chorionicnoma cells. CAV-1 siRNA can inhibit the invasion and proliferation ability of chorionicnoma cells.
Caveolin 1 ; biosynthesis ; genetics ; Choriocarcinoma ; metabolism ; pathology ; Female ; Humans ; Neoplasm Invasiveness ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Tumor Cells, Cultured ; Uterine Neoplasms ; metabolism ; pathology

To compare the expression level of metastasis associated-1 (MTA1) gene in high and low metastatic human osteosarcoma cell lines and examine the relationship of MTA1 expression and the metastasis potentiality of osteosarcoma cells, the expression of MTA1 in MG-63 osteosarcoma cell lines with high and low metastasis potential was detected by semiquantitative TR-PCR. Boyden chamber invasion assay was used to evaluate the invasive capacity in vitro in two osteosarcoma cell lines. The low metastasis MG-63 cells were transfected with MTA1 full-length cDNA expression plasmid by lipofectamine and the changes of MTA1 expression and in vitro invasion potential were examined after the transfection. Our results showed that MG63 cell line with high metastasis potential expressed significantly higher MTA1 than that of MG63 cells with low metastasis as reavealed by RT-PCR. The invasion potential of low metastasis MG63 cell line was increased after MTA1 gene transfection. It is concluded that there may be a relationship between MTA 1 and invasive potentiality of human osteosarcoma cells, and the mechanism of MTA1 in osteosarcoma metastasis and its possible role in associated gene therapy deserve further study.
Bone Neoplasms/*metabolism ; Bone Neoplasms/pathology ; Gene Expression Regulation, Neoplastic ; Histone Deacetylases/*biosynthesis ; Histone Deacetylases/genetics ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Osteosarcoma/*metabolism ; Osteosarcoma/pathology ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Repressor Proteins/*biosynthesis ; Repressor Proteins/genetics ; Tumor Cells, Cultured

The study was purposed to investigate the effect of small interference RNA (siRNA) targeting Polo-like kinase 1 (PLK1) gene on cell cycle progression, proliferation and drug resistance in K562/A02 cells. siRNA plasmid vector specifically targeting PLK1 gene with enhanced green fluorescence protein (EGFP) was transfected into K562/A02 cells. Expressions of PLK1 mRNA and protein were assayed by RT-PCR and Western-blot; cell proliferation was evaluated by direct cell counting after trypan blue staining. Cell cycle and intracellular adriamycin (ADM) accumulation was determined by flow cytometry; 50% inhibition concentration (IC50) of ADM on K562/A02 cells was determined by MTT method. The results showed that, as compared with control cells, siRNA plasmid reduced PLK1 mRNA expression by (34.7 +/- 2.1)% for 24 hours and by (56.6 +/- 1.5)% for 48 hours, PLK1 protein significantly decreased simultaneously by (49.9 +/- 3.2)% and by (62.1 +/- 1.7)%. After being transfected for 24 and 48 hours, the rate of survival cells decreased by 30% and 59% respectively. Forty-eight hours after transfection, the ratio of K562/A02 cells at G2/M increased by 2.77-fold, at the same time, intracellular ADM accumulation increased and the relative efficiency of K562/A02 cells to ADM was 73.8%. It is concluded that PLK1 gene silence can inhibit K562/A02 cell proliferation, induce cell cycle arrest at G2/M, and increase intracellular ADM accumulation, so that enhance cell sensitivity to ADM.
Cell Cycle ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Proliferation ; Daunorubicin ; pharmacokinetics ; Drug Resistance, Neoplasm ; genetics ; Gene Silencing ; Humans ; K562 Cells ; Protein-Serine-Threonine Kinases ; biosynthesis ; genetics ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; pharmacology

OBJECTIVESTo explore the significance of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the development of human primary hepatocellular carcinoma (HCC).

METHODSPTEN protein expression in cancerous liver tissues and paired para-carcinoma liver tissues from 60 HCC patients was detected by immunohistochemistry and PTEN mRNA expression was analyzed by northern blot. The significance of PTEN in the development of HCC was analyzed by investigating the relationship between the expression levels of PTEN protein and mRNA, and the clinicopathological parameters of HCC patients.

RESULTSPTEN protein was immunohistochemically stained in the cytoplasmic region of para-carcinoma liver tissues in all the 60 patients, while only 48.3% (29/60) of the patients were positive for PTEN protein in cancerous liver tissues. The positive rate of PTEN protein in HCC tissues were relative to the histological gading and the presence of tumor thrombus. In grade I - II, III, and IV, the positive rates were 84.0%, 23.8%, and 21.4% respectively, and in the group with tumor thrombus was 26.7%, while in the group without tumor thrombus was 55.6%. Northern blot showed that there existed four PTEN mRNA transcripts with the length of 5.5kb, 4.4kb, 2.4kb, and 1.8kb respectively. The level of PTEN mRNA expression in HCC tissues was much lower than that in the paired para-carcinoma liver tissues. The low expression level of the 5.5kb and 4.4kb transcripts was significantly associated with serum AFP value, presence of tumor thrombus, state of satellite lesion and histological grading. The low expression level of the 2.4kb transcript in HCC was significantly associated with the presence of tumor thrombus and satellite lesions in HCC patients. However, no evident relationship between the lowered expression level of the 1.8kb transcript and the clinicopathological parameters of HCC was observed in these 60 patients.

CONCLUSIONSDown-regulation of PTEN expression may play an important role in the development of HCC and the expression level of PTEN may be a potential adjuvant parameter in forecasting the progress and prognosis of HCC.

Adult ; Biomarkers, Tumor ; Carcinoma, Hepatocellular ; metabolism ; Female ; Humans ; Liver Neoplasms ; metabolism ; Male ; Middle Aged ; PTEN Phosphohydrolase ; Phosphoric Monoester Hydrolases ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Neoplasm ; biosynthesis ; Tumor Suppressor Proteins ; biosynthesis ; genetics

OBJECTIVETo study the expression of cyclooxygenase-2 (COX-2) gene in breast cancer and its clinicopathologic characteristics.

METHODSWith beta-actin gene as reference, the COX-2 mRNA was monitored in 30 specimens of breast cancer tissue and adjacent normal breast tissue by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe COX-2 mRNA expression was significantly upregulated in most breast cancer tissues with range of 0.05 - 0.91 (median 0.56), which was rare in normal breast tissue with range of 0 - 0.09 (median 0). The difference of COX-2 mRNA expression between cancer and normal breast tissue was significant (rank sum test, P < 0.05). COX-2 overexpression in breast cancer was related to its lymph node metastasis (P < 0.05) but not to age, tumor size, pathologic grade or pathologic type (P > 0.05).

CONCLUSIONThe level of COX-2 mRNA expression is obviously higher in the breast cancer tissue than that in normal breast tissue. COX-2 overexpression may play a crucial role in the carcinogenesis, development of cancer and lymph node metastasis in breast cancer patients.

Adult ; Biomarkers, Tumor ; biosynthesis ; genetics ; Breast Neoplasms ; enzymology ; metabolism ; Cyclooxygenase 2 ; Female ; Gene Expression ; Humans ; Isoenzymes ; biosynthesis ; genetics ; physiology ; Membrane Proteins ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; genetics ; physiology ; RNA, Messenger ; biosynthesis ; RNA, Neoplasm ; analysis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo transfect a short hairpin RNA (shRNA) against survivin gene into human T lymphoblastic leukemia cell line Jurkat, and to explore the effects on apoptosis and proliferation of transfected cells.

METHODSThe survivin-shRNA expression vector were constructed and transfected into Jurkat cells. Expression of survivin mRNA and protein were assessed by RT-PCR and Western blot analysis respectively. Apoptosis index of transfected Jurkat cells was quantified by flow cytometry. The potential of cell proliferation was described by cell growth curves.

RESULTSIn survivin-shRNA transfected Jurkat cells, survivin mRNA levels were significantly reduced by 66.67% ( transient transfection) and 60.69% ( stable transfection) respectively, compared with that in control-shRNA treated group and PBS treated group (P < 0.05); and the levels of survivin protein were significantly reduced by 63.41% (transient transfection) and 60.18% (stable transfection), compared with that in the two control groups (P < 0.05). Apoptosis index was significantly increased during both transient and stable transfection, respectively [(22. 41 +/- 2.83)% and (20.73 +/- 2.56)% (P < 0.05)]. Survivin-shRNA also inhibited the proliferation of Jurkat cells.

CONCLUSIONSVector-based survivin-shRNA can effectively reduce the expression of survivin gene, induce apoptosis

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Gene Expression ; Gene Silencing ; Humans ; Inhibitor of Apoptosis Proteins ; Jurkat Cells ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA Interference ; RNA, Messenger ; biosynthesis ; RNA, Small Interfering ; pharmacology

OBJECTIVETo investigate the effect of a sequence-specific small interfering RNA (siRNA) in suppressing survivin expression and cell proliferation and inducing apoptosis of PC-2 cells.

METHODSThe plasmid expression vector of siRNA targeted against survivin was constructed and transfected into PC-2 cells with Lipofectamine 2000. The changes of survivin expression were detected by semi-quantitative RT-PCR and immunohistochemical SP methods. The effect of siRNA in suppressing the proliferation of PC-2 cells was detected by MTT assay, and its role in inducing PC-2 cell apoptosis evaluated by flow cytometry.

RESULTSThe sequence-specific siRNA effectively suppressed survivin expression at both mRNA and protein levels with inhibition rate of 81.25% at mRNA level and 74.24% at protein level. Survivin expression suppression significantly inhibited the proliferation of PC-2 cells, and at 24 and 48 h after cell seeding, the proliferation inhibition rate was 28.00% and 33.38% respectively; 24, 48 h after the transfection, apoptosis occurred in 8.46% and 7.53% of the cells, respectively.

CONCLUSIONSThe plasmid expression vector for the siRNA against survivin constructed in the study can effectively and specifically suppress survivin expression in PC-2 cells, and blocking survivin expression suppresses PC-2 cell proliferation and induces cell apoptosis. siRNA targeted against survivin has a potential value in gene therapy for pancreatic cancer.

Apoptosis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Pancreatic Neoplasms ; genetics ; pathology ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo prepare small interfering RNA (siRNA) targeting survivin for inhibition of endogenous survivin gene expression in Hela cell line and evaluate its effect on promoting Hela cell apoptosis.

METHODSThe recombinant plasmid pshRNA-survivin-1 and pshRNA-survivin-2 were constructed and transfected into Hela cells, in which the expression level of survivin was determined by immunofluorescence staining and survivin gene transcription detected by semi-quantitative RT-PCR.

RESULTSIntroduction of the plasmids pshRNA-survivin-1 and pshRNA-survivin-2 into Hela cells resulted in efficient and specific inhibition of survivin expression as demonstrated by immunofluorescence staining. Semi-quantitative RT-PCR showed that mRNA transcription of survivin gene was reduced. In contrast, the control plasmid did not exhibit any inhibitory effect on the protein expression and mRNA transcription of survivin gene. PI-Annexin V staining indicated an apoptosis rate of the transfected Hela cells of (36.02-/+2.12)% (P<0.01) and (35.29-/+2.02)% (P<0.01), respectively.

CONCLUSIONThe prepared siRNA targeting survivin gene is capable of inducing marked inhibitions of survivin protein expression and RNA transcription and significant enhancement of apoptosis in Hela cells, which shed light on a new strategy in gene silence therapy targeting survivin.

Apoptosis ; Flow Cytometry ; Fluorescent Antibody Technique ; Genetic Vectors ; genetics ; HeLa Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo explore the association of FMNL2 expression with the metastatic potential of colorectal cancer cells.

METHODSFMNL2 mRNA and protein expressions in 6 human colorectal cancer cell lines were detected by real-time RT-PCR and immunohistochemical method, respectively, and analyzed for their correlations to the in vitro invasiveness of the cell lines evaluated by Boyden assay. In SW620 and SW480/M5 cell lines, the expression of FMNL2 was repressed by FMNL2 short hairpin RNA (shRNA), and the changes in the invasiveness of the cells were observed.

RESULTSFMNL2 was highly expressed in SW480/M5, LoVo and SW620 cells derived from metastatic colorectal cancers in comparison with that in LS174T, SW480 and HT29 cells, which were derived from primary colorectal cancers. In vitro analysis of the cell invasiveness demonstrated that SW480/M5, LoVo and SW620 cells had higher invasiveness than LS174T, SW480 and HT29 in vitro. In SW480/M5 and SW620 cells, transfection with FMNL2 shRNA resulted in significantly lowered cell invasiveness.

CONCLUSIONFMNL2 may play an important role in the invasion and metastasis of colorectal cancer.

Colorectal Neoplasms ; metabolism ; pathology ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Proteins ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Tumor Cells, Cultured

OBJECTIVETo investigate trichorhinophalangeal syndrome 1 gene (TRPS-1) expression patterns in different subtypes of breast cancer and its correlations with other genes and survival using microarray data sets.

METHODSThe transcripts of TRPS-1 and its role in survival in breast cancer were analyzed using published microarray data sets#x02014;Netherlands Cancer Institute (NKI) cohort and Wang cohort.

RESULTSTRPS-1 expression was lower in basal-like breast cancer. The mRNA levels of TRPS-1 negatively correlated with Slug (Pearson correlation coefficient=-0.1366, P=0.0189 in NKI data set and Pearson correlation coefficient=-0.1571, P=0.0078 in Wang data set), FOXC1 (Pearson correlation coefficient=-0.1211, P=0.0376 in NKI data set and Pearson correlation coefficient=-0.1709, P=0.0037 in Wang data set), and CXCL1 (Pearson correlation coefficient=-0.1197, P=0.0399 in NKI data set and Pearson correlation coefficient=-0.3436, P<0.0001 in Wang data set), but positively correlated with BRCA1 (Pearson correlation coefficient=0.1728, P=0.0029 in NKI data set and Pearson correlation coefficient=0.1805, P=0.0022 in Wang data set). Low TRPS-1 expression associated with poor overall survival (hazard ratio 1.79, 95% CI of ratio 0.9894 to 3.238, P=0.054) and relapse-free survival (hazard ratio 1.913, 95% CI of ratio 1.159 to 3.156, P<0.05). The low TRPS-1 mRNA levels predicted poor outcome in breast cancer patients by the 70-gene signature.

CONCLUSIONThe strong expression of TRPS-1 may serve as a good prognostic marker in breast cancer.

Adult ; Biomarkers, Tumor ; biosynthesis ; Breast Neoplasms ; metabolism ; mortality ; pathology ; Cell Line, Tumor ; Cohort Studies ; DNA-Binding Proteins ; biosynthesis ; Disease-Free Survival ; Epithelial-Mesenchymal Transition ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Middle Aged ; Neoplasm Proteins ; biosynthesis ; RNA, Messenger ; biosynthesis ; RNA, Neoplasm ; biosynthesis ; Survival Rate ; Transcription Factors ; biosynthesis