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ABSTRACT: To establish a gastric cancer cell line with stable expression of metastasis-associated in colon cancer 1 (MACC1) and detect the changes in tumor-related gene expression profiles for investigating the possible regulation mechanisms between MACC1 and the differentially expressed genes.
The full-length MACC1 cDNA was amplified from human embryonic kidney 293FT cells and cloned into the pBaBb-puro vector. The recombinant pBaBb-puro-MACC1 expression vector, after identification with restriction enzyme digestion, was transfected into 293FT cells, and the expression of fluorescent reporter gene was observed. pBaBb-puro-MACC1 vector was transfected into human gastric cancer BGC-823 cell line to establish BGC-823/pBaBb-puro-MACC1 cell line stably expressing MACC1. Quantitative RT-PCR and Western blotting were used to detect MACC1 expression in both BGC-823/pBaBb-puro-MACC1 and control BGC-823 cells. High-throughout cDNA microarray was used to screen the effects of MACC1 on the gene expression profiles of gastric cancer cells.
The recombinant pBaBb-puro-MACC1 plasmid was successfully constructed and verified by PCR and sequencing. BGC-823/pBaBb-puro-MACC1 cells showed significantly increased MACC1 mRNA expression as compared with the control cells. The results of cDNA microarray identified 33 up-regulated and 24 down-regulated genes in the cells after MACC1 transfection involved were in various cellular functions.
The established BGC-823/pBaBb-puro-MACC1 gastric cancer cell line show some important molecular changes caused by MACC1.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 03/2012; 32(3):312-6.
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ABSTRACT: To construct glypican-3 (GPC3)-green fluorescent protein eukaryotic expression vector pEGFP-c3-GPC3, and analyze the effect of GPC3 on the proliferation of human hepatoma cell line MHCC-97L.
The eukaryotic expression vector pEGFP-c3-GPC3 was constructed with recombinant DNA technique and transfected into MHCC-97L cells via Lipofectamine 2000. The cells stably expressing GPC3 were screened by flow cytometry and G418. The mRNA expression of GPC3 was detected by RT-QPCR method, and the protein expression by Western blotting and fluorescence microscope. The effect of GPC3 gene on the growth of the cells was examined by MTT assay.
Restriction endonuclease analysis and DNA sequencing verified correct construction of the recombinant plasmid. The green fluorescence was detected in the transfected MHCC-97L cells under fluorescence microscope. RT-QPCR and Western blotting both confirmed successful expression of GPC3 in MHCC-97L cells. The growth curve showed a significant acceleration of the proliferation of the transfected MHCC97-Lsol;GPC3 cells as compared with MHCC97-L and MHCC97-L/C3 cells (P<0.001).
We have successfully constructed the eukaryotic expression vector pEGFR-c3-GPC3, which allows stable GPC3 expression in MHCC97-L/GPC3 cells. The upregulation of GPC3 expression can stimulate the growth of hepatoma cell line MHCC97-L in vitro.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 03/2011; 31(3):448-52.
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ABSTRACT: Cholangiocarcinoma (CCA) is a rare but lethal malignancy arising from the biliary tract epithelium. It has a poor prognosis largely due to the difficulties of early diagnosis and the lack of effective therapies. It is thus imperative to develop new and effective treatments for CCA, which depends heavily on the mechanistic understanding of the disease. Previous studies have suggested that somatic mutations in KRAS, BRAF, and PIK3CA genes are frequently found in several types of human cancers including colon, breast, and lung carcinomas as well as CCA. Yet, the frequency and the involvement of these oncogenic mutations in CCA in Chinese population have not been investigated. In this study, we evaluated the hotspot mutations of KRAS, BRAF, and PIK3CA genes in 34 Chinese CCA patients. Sequencing analysis revealed 13 (38.2%) and 11 (32.4%) patients bearing KRAS and PIK3CA mutations, in which two (5.9%) of them harbored both KRAS and PIK3CA mutations. Surprisingly, no BRAF mutation was detected in all 34 CCA samples. Our findings indicate that somatic mutations in KRAS and PIK3CA but not BRAF oncogenes are closely associated with the development of CCA in Chinese population and provide new potential targets for future therapeutic treatments of the disease.
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 11/2010; 65(1):22-6. · 2.24 Impact Factor
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ABSTRACT: To compare the short-term curative effect of photodynamic therapy (PDT), PDT combined with chemotherapy and chemotherapy alone on the advanced esophageal cancer patients.
Retrospective analysis of 90 patients of esophageal cancer underwent PDT, PDT combined with chemotherapy and chemotherapy alone from 2004 to 2007 (stages III-IV), including 27 cases received PDT alone, 33 cases received PDT combined with chemotherapy and 30 cases chemotherapy alone. The enrolled patients were treated with intravenous administration of Photofrin as the photosensitizer at a dose of 2mg/kg. 630 nm laser irradiation was performed through optical fiber that passed through the biopsy channel of a flexible endoscope after 48 h. Two days later, the necrotic tissue was removed, and the primary sites and other newly identified lesions were subjected to a second irradiation and then the residual necrotic tissue was removed according to the patients' condition. Electronic endoscopy was performed to observe the effectiveness on tumor after 1 month. In PDT combined with chemotherapy group, chemotherapy regimen was 5-FU and DDP, administered 4 cycles after PDT and chemotherapy alone group only chemotherapy regimen 5-FU and DDP for four cycles. All the 90 patients were followed up for 2 years.
Symptomatic palliation rate of the PDT alone group, the complex treatment group and chemotherapy alone group was 85.2%, 93.9% and 60.0%, respectively, and effective rate under endoscopy was 85.2%, 90.9% and 63.3%, respectively, there is no statistically significant difference; the survival rate of 2 years was 29.6%, 54.5% and 16.7%, respectively, and medium survival time is longer (III stages 13 m, 22 m, 10 m; IV stages 7 m, 5m, 4m), there is statistically significant difference (p=0.046).
PDT combined with chemotherapy for the advanced esophageal cancer is superior to PDT alone and chemotherapy alone.
Photodiagnosis and photodynamic therapy 09/2010; 7(3):139-43.
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ABSTRACT: To observe SHP-1 protein expression in breast cancer cell line MDA-MB-231 before and after SHP-1 gene transfer and its effect on the proliferation of MDA-MB-231 cells.
The eukaryotic expression vector pEGFP-C3-SHP-1 was constructed and transfected into breast cancer cell line MDA-MB-231 via Lipofectamine 2000, and the positive clones were selected using G418. SHP-1 expression in MDA-MB-231 cells was detected with immunocytochemistry and Western blotting, and the cell growth curve was observed using MTT assay.
SHP-1 was highly expressed in transfected MDA-MB-231 cells, whose proliferation was significantly inhibited (P<0.05).
SHP-1 gene transfer into MDA-MB-231 cells results in inhibition of the cell proliferation.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 05/2010; 30(5):1024-7.
