OBJECTIVETo construct a lentiviral vector carrying the γ-synuclein(SNCG) gene and establish a human colorectal carcinoma cell line SW1116 stably expressing this gene, and then investigate the inhibition of the growth and invasion capacity of SW1116 cells.

METHODSRNA interference fragment was designed according to the SNCG sequence (GenBank: No.NM003087.2), and then SNCG RNAi effective target genes were screened. After the Oligo DNA of target sequences was synthesized, the lentiviral vectors carrying LV-SNCG-RNAi-EGFP (RNAi group) and LV-SNCG-NC-EGFP (NC group) were constructed and packaged to produce lentivirus venom. The supernatants of different virus-producing cells were used to transfect SW1116 cells respectively. Wild SW1116 cells were used as blank control (CON group) EGFP fluorescence was detected by fluorescent microscopy and the differential expression of SNCG mRNA and protein was detected by real-time PCR and Western blot. CCK-8, soft agar assay and Transwell chamber were employed to estimate the inhibiting effect on growth and invasion of SW1116 respectively.

RESULTSRecombinant lentiviral vectors respectively carrying the SNCG-RNAi-EGFP and SNCG-NC-EGFP were successfully constructed and the supernatants of lentivirus could effectively infect SW1116 cells. The titer of the virus carrying LV-SNCG-RNAi-EGFP or LV-SNCG-NC-EGFP was 8×10(8) TU/ml. Real-time PCR and Western blot confirmed that compared with the NC group, SNCG-RNAi group had lower SNCG expression (1.009±0.161 vs. 0.114±0.030, P=0.009), and showed tremendous silencing effect as 76.8%(P<0.05). SNCG protein expression was also significantly reduced (RNAi:12.001±2.884, NC:32.443±4.731, CON:34.308±6.920, P<0.05). After SNCG knockdown, the number of proliferation cells was obviously reduced at 48, 72, 96 and 120 hours respectively(P=0.036). In soft agar assay, clones in RNAi group were smaller[RNAi:(0.582±0.103) mm, NC:(1.863±0.316) mm, CON:(1.749±0.525) mm]. Colony formation rate of RNAi group was down to (17.1±3.5)%, which was significantly lower than (36.5±4.3)% in NC group and (33.8±3.9)% in CON group. In migration test, the number of invasion cell was 37.4±9.3 in RNAi group, which was significantly less than 112.3±8.6 in NC group and 100±0.0 in CON group.

CONCLUSIONExpression of SNCG mRNA and protein plays an important role in the growth and the invasion capacity of SW1116 cells.

Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; pathology ; Genetic Vectors ; Humans ; Lentivirus ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; genetics ; Real-Time Polymerase Chain Reaction ; Transfection ; gamma-Synuclein ; genetics

OBJECTIVETo construct &gamma;-synuclein gene eukaryotic expression vector, and to study its effect on the invasion of colon cancer cell line SW1116 and the adhesion between SW1116 and human umbilical vein endothelial cells(HUVECs) in vitro.

METHODSTotal RNA was extracted from colon cancer cell line HT29 and the cDNA of &gamma;-synuclein was amplified using RT-PCR. The digested fragment of cDNA coding sequence was linked to the eukaryotic expression vector pEGFP-C1 containing the GFP gene. After identification by sequence analysis, the recombinant plasmid was transfected into colon cancer cell line SW1116 via lipofectamine. The stable cell line was selected with G-418. The invasion in vitro was tested by Transwell invasion chamber assay. HUVECs were previously seeded onto 96-well plates before SW1116 cells seeded, and fluorescence intensity of GFP was detected to represent the amount of adhesion cells by ELISA.

RESULTSHuman &gamma;-synuclein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-&gamma;-synuclein protein in vitro. &gamma;-synuclein facilitated SW1116 cell passing through matrigel and filter membrane(198.4±20.7 vs. 98.8±13.2, P<0.05) and elevated the adherence of SW1116 cells to HUVECs(3.08±0.36 vs. 1.22±0.21, P<0.05).

CONCLUSIONExpression of &gamma;-synuclein can strengthen colon cancer cell SW1116 potentiality of invasion and metastasis in vitro.

Cell Adhesion ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Genetic Vectors ; biosynthesis ; Human Umbilical Vein Endothelial Cells ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; gamma-Synuclein ; genetics

OBJECTIVESTo evaluate the expression of synuclein-&gamma; (SNCG) and metalloproteinase 9 (MMP-9) both in the invasive ductal breast cancer samples and T47D and T47D(SNCG)- cell lines, to investigate the correlation between SNCG and MMP-9.

METHODSTotally 96 invasive ductal breast cancer samples (female, mean age of (56 ± 8) years) were collected between June 2009 and June 2012. The expressions of SNCG and MMP-9 were investigated by immunohistochemistry. T47D and SNCG knock down T47D(SNCG)- cell lines were established and SNCG and MMP-9 protein expression were investigated by Western blot and gene expression by real-time PCR.

RESULTSAmong 96 samples, 26 (27.1%) of them co-expressed SNCG and MMP-9, 30(31.2%) of them expressed neither SNCG nor MMP-9. The expression of SNCG was correlated with the expression of MMP-9 (r = 0.655, P = 0.000).SNCG mRNA level of T47D cell line was 13.5 fold of T47D(SNCG)- cell line and SNCG protein expression was 2.1 fold. While MMP-9 mRNA level of T47D cell line was 7.3 fold of T47D(SNCG)- cell line and MMP-9 protien expression was 1.6 fold.When SNCG was knocked down, the expression of MMP-9 decreased.

CONCLUSIONSSNCG and MMP-9 are significantly correlated with each other in breast cancer. SNCG may promote the invasion and metastasis of breast cancer mediated by up-regulating the expression of MMP-9.

Aged ; Breast Neoplasms ; metabolism ; Female ; Gene Expression ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; Neoplasm Proteins ; metabolism ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; gamma-Synuclein ; metabolism

OBJECTIVETo investigate the expression of SNCG in colorectal cancer with liver metastasis and its clinical significance.

METHODSSurgical specimens were collected from 217 colorectal cancer patients with complete clinical and follow up data between January 1999 and December 2003. There were 113 cases with liver metastasis and 104 without liver metastasis. SNCG expression was identified by immunohistochemistry. Association of SNCG expression with clinicopathologic factors and prognosis of colorectal cancer was accessed.

RESULTSThe positive rate of SNCG in colorectal cancer with and without liver metastasis was 68.1% and 27.9%, respectively, and the difference was statistically significant(P<0.05). Logistic regression analysis showed that SNCG expression was an independent factor associated with the presence of liver metastasis(OR=8.29, 95%CI: 3.37-20.37, P<0.01). In synchronous colorectal liver metastasis, the median survival time of SNCG-negative and SNCG-positive was 12.6 months and 8.2 months, respectively(Log Rank, P<0.05). Multivariate Cox analysis showed that SNCG expression was an independent prognostic factor for colorectal cancer with synchronous liver metastasis(RR=1.97, 95%CI:1.10-3.53, P<0.05).

CONCLUSIONSHigh expression of SNCG is present in the tumor tissue in patients with liver metastasis from colorectal cancer. SNCG may be used as a predictive biomarker for colorectal liver metastases and is an important prognostic factor in patients with liver metastasis from colorectal cancer.

Colorectal Neoplasms ; metabolism ; pathology ; Female ; Follow-Up Studies ; Humans ; Liver Neoplasms ; secondary ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Prognosis ; Retrospective Studies ; gamma-Synuclein ; metabolism

The synuclein family consists of three distinct genes, alpha-synuclein, beta-synuclein, and gamma-synuclein. The alpha-synuclein and beta-synuclein are predominately expressed in brain and especially alpha-synuclein is related with Parkinson's disease, Alzheimer's disease, and dementia with Lewy bodies. The gamma-synuclein was first identified as breast cancer specific gene 1. It is expressed in the peripheral nervous system and also detected in breast and ovarian cancers. The gamma-synuclein is also known to mediate metastasis of breast and ovarian cancer cells. Insulin-like growth factor 1 (IGF-I) is one of the growth factors that plays an important role in cell proliferation and migration in cancer cells, as well as in normal cells. In this study, we investigated the migrations of SKOV-3, MDAMB-231, and HeLa cells by the recombinant synuclein proteins (alpha-, beta-, and gamma-synucleins) and IGF-I and the molecular mechanism. Furthermore, we investigated the membrane ruffle formation of SKOV-3 cells by recombinant synuclein proteins and IGF-I. As a result, synucleins and IGF-I were found to induce cancer cell migrations. Simultaneous synucleins and IGF-I treatment on the cancer cells induced more migrations than the individual synuclein or IGF-I treatments. The synucleins or IGF-I treatments increased the expressions of membrane-type1 matrix metalloproteinase (MT1-MMP) and cluster of differentiation 44 (CD44). Moreover, simultaneous synucleins and IGF-I treatments further increased the expressions of MT1-MMP and CD44. The synucleins and IGF-I promoted the conformational change of actin filaments, and then this led to the membrane ruffle formation.
Actin Cytoskeleton ; alpha-Synuclein ; Alzheimer Disease ; beta-Synuclein ; Brain ; Breast ; Breast Neoplasms ; Cell Movement ; Cell Proliferation ; Dementia ; gamma-Synuclein ; HeLa Cells ; Humans ; Insulin-Like Growth Factor I ; Intercellular Signaling Peptides and Proteins ; Lewy Bodies ; Matrix Metalloproteinase 14 ; Membranes ; Neoplasm Metastasis ; Ovarian Neoplasms ; Parkinson Disease ; Peripheral Nervous System ; Proteins ; Synucleins

BACKGROUNDOverexpression of breast cancer-specific gene 1 (SNCG) is associated with poor prognosis in advanced breast cancer patients. This study aimed to determine the effects of SNCG knockdown in breast cancer cells by using small hairpin RNA (shRNA).

METHODSFour different SNCG shRNA oligonucleotides were designed and chemically synthesized to construct mammalian expression vectors. These vectors were then stably transfected into a breast cancer MCF-7 cell line to knockdown SNCG expression. After SNCG knockdown was confirmed, the stable cell lines were inoculated into nude mice. SNCG mRNA and protein expressions were analyzed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively in both the stable cell lines and xenografts.

RESULTSAll four SNCG shRNA constructs significantly reduced SNCG mRNA and protein levels in MCF-7 cells, as compared to the unrelated sequence control shRNA and the liposome control mice (P < 0.05). SNCG-knockdown MCF-7 cells formed significantly smaller tumor masses than cells expressing the unrelated sequence control or the liposome control mice (P < 0.05).

CONCLUSIONSNCG shRNA effectively suppressed breast cancer cell formation in vivo and may be a useful clinical strategy to control breast cancer.

Animals ; Breast Neoplasms ; genetics ; therapy ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; Immunohistochemistry ; Mice ; Mice, Nude ; RNA, Small Interfering ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Xenograft Model Antitumor Assays ; gamma-Synuclein ; genetics ; metabolism

Liver cancer is one of the most common neoplastic diseases with high mortality in China. Currently, antimicrotubule drugs such as paclitaxel (PTX) and vincristine (VCR), are used as the common agents in the clinical chemotherapy for liver cancer. However, the responses of patients to these drugs vary markedly. Successful identification of intracellular factors influencing liver cancer's sensitivity to antimicrotubule drugs would be of great clinical importance. In this study, by engineering human hepatoma cell HepG2 to overexpress synuclein-gamma (SNCG), we investigated if SNCG is a molecular factor associated with the sensitivity to antimicrotubule drug treatment. Real-time RT-PCR and Western blotting assays showed SNCG was successfully overexpressed in HepG2/ SNCG cells compared with HepG2/Neo cells. The overexpressed SNCG altered the proliferation activity in HepG2 cells, which was 66% higher than that of HepG2/Neo cells through MTT method. The overexpressed SNCG also reduced sensitivity of HepG2 cells to antimicrotubule drugs: after PTX or VCR treatment, the proportion of HepG2/SNCG cells in G2/M arrest was significantly lower than that in HepG2/Neo cells. Correspondingly, HepG2/SNCG cells showed significantly lower mitotic index than HepG2/Neo cells. Meanwhile, HepG2/SNCG cells showed higher resistance to PTX and VCR than HepG2/Neo cells, with resistance index 21 and 15 respectively. Our studies suggested that the overexpression of SNCG could confer resistance to antimicrotubule drugs in hepatoma cells; and it indicated that SNCG may be as a potential response marker for antimicrotubule drugs in liver cancer chemotherapy.
Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Cycle ; Cell Proliferation ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Hep G2 Cells ; drug effects ; metabolism ; Humans ; Microtubules ; drug effects ; Mitosis ; drug effects ; Mitotic Index ; Paclitaxel ; pharmacology ; Plasmids ; RNA, Messenger ; metabolism ; Transfection ; Vincristine ; pharmacology ; gamma-Synuclein ; biosynthesis ; genetics ; physiology

OBJECTIVETo investigate the inhibitory effects of siRNA targeting BCSG1 gene expression in tumor transplants of human breast cancer cell line in nude mice.

METHODSFour-pairs of small interfering RNA sequences of BCSG1 were chemically synthesized and inserted into the plasmid expression vectors, and were then transfected into human breast carcinoma cell line MCF7 by liposome method. Plasmid vector with unrelated sequence was used as the vector control. Cells transfected with 4 siRNA sequences, control vector and naive FCF7 cells were transplanted into the nude mice. The tumor inhibition was analysised. Immunohistochemical SP method and semi-quantitative RT-PCR were adopted to detect the BCSG1 mRNA and protein expression, respectively. Breast tissue samples of human infiltrating ductal carcinoma, ductal hyperplasia and fibroadenoma were also used as the controls.

RESULTSThe inhibition rates of tumor growth in four BCSG1-siRNA transfected groups were remarkably higher than those of the vector control group and naive MCF7 cells (P<0.01). Compared with that of the vector control and naïve MCF7 cell group, there was a significant decrease of BCSG-1 protein expression in the four experimental groups by immuno-histochemistry staining (P<0.01). In addition, BCSG1 mRNA expression in the four groups transfected with BCSG1-siRNA were significantly less than that of the control vector group, naive MCF7 cell control group and human breast IDC (P<0.01).

CONCLUSIONBCSG1-siRNA down-regulates the expression of BCSG1 and inhibits effectively growth of the transplaned human breast cancer cell line in nude mice.

Animals ; Breast Neoplasms ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; metabolism ; Cell Line, Tumor ; Female ; Fibroadenoma ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Mice, Nude ; Neoplasm Proteins ; biosynthesis ; genetics ; Neoplasm Transplantation ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Random Allocation ; Transfection ; Tumor Burden ; gamma-Synuclein ; biosynthesis ; genetics

Hsc70-SNCG fusion protein cDNA fragment containing signal peptide sequence of Igkappa, MMP9, and P37 was inserted into the vector pVAX1 to construct recombinant plasmid pVAX-Igkappa-Hsc70-SNCG, pVAX-MMP9-Hsc70-SNCG, and pVAX-P37-Hsc70-SNCG. Three eukaryotic vectors were constructed and verified by restriction enzyme digestion and sequencing. After transfection with recombination plasmids in QM-7 cells, the transient expression and secretion of three fusion proteins were detected by ELISA and Western Blot. The results suggested that Hsc70-SNCG carrying three different signal peptides could be expressed and secreted by transfected cells, and three signal peptides effectively directed secretion of fusion protein by QM-7 cells. BALB/c mice were immunized by three plasmids using gene gun system. The serum levels of anti-SNCG antibodies in mice were measured by ELISA. The results showed that three secreted plasmids could stimulate humoral immune responses to SNCG in mice, which depended on the secreted expression levels induced by signal peptides.
Animals ; Genetic Vectors ; Humans ; Immunoglobulin G ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Neoplasm Proteins ; biosynthesis ; genetics ; immunology ; Plasmids ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection ; gamma-Synuclein ; biosynthesis ; genetics ; immunology

OBJECTIVETo explore the relationship between gamma-synuclein gene expression and CpG island demethylation in colorectal cancer(CRC), and the relationship between the demethylation and clinicopathological factors of CRC.

METHODSThe expression of gamma-synuclein mRNA was examined in 30 pairs of tumor tissues and tumor-matched non-neoplastic adjacent tissues(NNAT) by RT-PCR. CRC cell lines including COLO205, LoVo, and SW480 were used and treated with a demethylating agent, 5-aza-2'-deoxycytidine(5-aza-C). Before and after the treatment, the expression of gamma-synuclein mRNA in the cells was determined by RT-PCR, and bisulfite sequencing PCR was also used to analyze methylation status of CpG island. The methylation status of gamma-synuclein was then examined in 67 CRC samples and 30 NNAT samples by nested methylation-specific PCR (NMSP) and real time methylation-specific PCR(real-time MSP). The relationship between the demethylation of gamma-synuclein in CRC and clinicopathological factors was analyzed.

RESULTSThe mean gamma-synuclein mRNA expression was 0.66+/-0.34 in CRC samples, which was much higher than 0.45+/-0.26 in NNAT samples(P=0.011). 5-aza-C could induce expression and demethylation of gamma-synuclein in COLO205, LoVo and SW480 cells. gamma-Synuclein gene was demethylated in 80.0%(24/30) of the CRC samples and 50.0%(15/30) of the NNAT samples. The demethylated status of gamma-synuclein was much higher in CRC samples than that in NNAT samples(P=0.030), and was significantly correlated with clinical stage, lymph node involvement, and distant metastasis of CRC(P<0.05).

CONCLUSIONThe upregulation of gamma-synuclein expression in CRC is primarily attributed to the demethylation of CpG island, which may be used as a marker for prognosis.

Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; CpG Islands ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Humans ; Prognosis ; RNA, Messenger ; genetics ; gamma-Synuclein ; genetics ; metabolism