Construction of lentiviral vector of siRNA specific for γ-synuclein and its inhibition effect on colorectal carcinoma cell line SW1116 / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>RNA 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.</p><p><b>RESULTS</b>Recombinant 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.</p><p><b>CONCLUSION</b>Expression of SNCG mRNA and protein plays an important role in the growth and the invasion capacity of SW1116 cells.</p>

Construction of γ-synuclein eukaryotic expression vector and its effect on invasion and metastasis of colon cancer cell line SW1116 in vitro / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To construct γ-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.</p><p><b>METHODS</b>Total RNA was extracted from colon cancer cell line HT29 and the cDNA of γ-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.</p><p><b>RESULTS</b>Human γ-synuclein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-γ-synuclein protein in vitro. γ-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).</p><p><b>CONCLUSION</b>Expression of γ-synuclein can strengthen colon cancer cell SW1116 potentiality of invasion and metastasis in vitro.</p>

The correlation of synuclein-γ and matrix metalloproteinase 9 in breast cancer / 中华外科杂志

<p><b>OBJECTIVES</b>To evaluate the expression of synuclein-γ (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.</p><p><b>METHODS</b>Totally 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.</p><p><b>RESULTS</b>Among 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.</p><p><b>CONCLUSIONS</b>SNCG 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.</p>

Correlation between periostin and SNCG and esophageal cancer invasion, infiltration and apoptosis

OBJECTIVE@#To investigate the correlation between periostin and SNCG and esophageal cancer invasion, infiltration and apoptosis.@*METHODS@#A total of 78 cases esophageal surgical resection specimens were collected, expression of periostin and SNCG in esophageal cancer were detected. Effect of periostin and SNCG in esophageal carcinoma invasion and infiltration was analyzed.@*RESULTS@#The upregulated rate of periostin had significant difference in esophageal cancer tissues (39.74%), adjacent tissues (17.86%) and normal tissues (0.00%); The positive expression rates of SNCG had significant difference in esophageal cancer tissues (61.54%), adjacent tissues (32.14%) and normal tissues (1.96%); The upregulated rate of periostin had a significant correlation with lymph node metastasis, adventitia invasion, TNM stage; The positive expression rates of SNCG had a significant correlation with differentiation degree, lymph node metastasis, adventitia invasion, TNM stage; Apoptosis index of the positive of expression of SNCG of esophageal cancer tissue (4.541±2.267) was significantly lower than that of the negative expression (7.316±2.582) (P<0.05).@*CONCLUSIONS@#SNCG may play an important role in invasion, infiltration and apoptosis of esophageal cancer and serve as target spots in the targeted therapy of esophageal cancer.

SNCG expression and clinical significance in colorectal cancer liver metastasis / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of SNCG in colorectal cancer with liver metastasis and its clinical significance.</p><p><b>METHODS</b>Surgical 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>High 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.</p>

The Effects of Recombinant Synucleins and Insulin-like Growth Factor 1 on Cancer Cell Migration

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.

SNCG shRNA suppressed breast cancer cell xenograft formation and growth in nude mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Overexpression 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).</p><p><b>METHODS</b>Four 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.</p><p><b>RESULTS</b>All 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).</p><p><b>CONCLUSION</b>SNCG shRNA effectively suppressed breast cancer cell formation in vivo and may be a useful clinical strategy to control breast cancer.</p>

Overexpression of synuclein-gamma confers resistance to antimicrotubule drugs against human hepatoma cells / 药学学报

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.

Expression of BCSG1-siRNA in tumor transplants of human breast cancer cell line in nude mice / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effects of siRNA targeting BCSG1 gene expression in tumor transplants of human breast cancer cell line in nude mice.</p><p><b>METHODS</b>Four-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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>BCSG1-siRNA down-regulates the expression of BCSG1 and inhibits effectively growth of the transplaned human breast cancer cell line in nude mice.</p>

Construction, expression and immune response of eukaryotic plasmid secreting SNCG / 生物医学工程学杂志

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.

Demethylation of the gamma-synuclein gene CpG island in colorectal cancer and its clinical significance / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>The 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.</p>

Influence of SNCG transfection on the effectiveness of anti-tumor drugs in PC-3 cell lines / 中华男科学杂志

<p><b>OBJECTIVE</b>To observe the sensitivity of the PC-3 cell lines transfected with the PCI-NEO-SNCG plasmid to Cisplatin (DDP), 5-Fluorouracil (5-FU), Adriamycin (ADM), Vincristine (VCR) and Paclitaxel (TAX), and to explore the influence of the SNCG expression on the effectiveness of anti-tumor drugs.</p><p><b>METHODS</b>The plasmids PCI-NEO and PCI-NEO-SNCG were transfected into the hormone-independent prostate cancer cell lines PC-3. RT-PCR was adopted to examine the expression of SNCG in the PC-3 cell lines. The MTT method was employed to detect the suppressive effects of different anti-tumor drugs (DDP, ADM, 5-FU, VCR and TAX) on the cell lines transfected with PCI-NEO and PCI-NEO-SNCG. Flow cytometry was used to analyze the cell cycles and apoptosis of the transfected cells treated with TAX.</p><p><b>RESULTS</b>The 5 anti-tumor drugs suppressed the growth of the cell lines transfected with the plasmids PCI-NEO and PCI-NEO-SNCG in a time-dependant manner. The comparison between the growth-suppressing effects of different anti-tumor drugs on the PC-3 cell lines showed no significant differences between the group transfected with PCI-NEO and that with PCI-NEO-SNCG in DDP, 5-FU, ADM and VCR (P > 0.05), while the rate of suppression of TAX on the latter cell lines was significantly lower than that on the former (P < 0.01). Compared with the PCI-NEO-SNCG plasmid transfected cell lines, after treated with TAX for 48 hours, those transfected with the PCI-NEO plasmid exhibited a significantly larger proportion of cells remaining in the G2-M stage (P < 0.01), a smaller proportion in the G0-G1 and S stages (P < 0.01) and a significantly higher expression of Caspase-3 (P < 0.01).</p><p><b>CONCLUSION</b>The significant reduction of the growth-suppressing effect of TAX in the SNCG-transfected PC-3 cell lines suggests that the expression of SNCG may restrain the effect of TAX. These findings have provided evidence and guide to the individual chemotherapy of prostate cancer.</p>

The gamma-Synuclein Expression in Breast Cancer and its Correlation with the Expression of the HER-2/neu Gene / 한국유방암학회지

PURPOSE: Synuclein has been identified as an important neuroprotein for developing pathologic deposits in Alzheimer's and Parkinson's disease patients. gamma-synuclein is also known as a breast cancer-specific gene 1 thats's not found in normal breast tissues but it has been reported to be overexpressed in breast cancer, ovarian cancer and other tumors. To evaluate the availability of gamma-synuclein expression as a prognostic factor for infiltrative breast cancer, we analyzed its correlation with the clinical parameters and the HER-2/neu gene expression. METHODS: Two hundred fiffty samples of breast cancer tissues embedded in paraffin and that were obtained from the infiltrative breast cancer patients who were operated in our institution from January 1995 to December 2000 were analyzed with employing the tissue microarray technique. The expression of gamma-synuclein was studied with immunohistochemistry and with using gamma-synulcein antibodies. One hundred thirty one cases that showed favorable staining were selected and studied retrospectively. RESULTS: Fiffty five% (71/131) of the patients showed gamma-synuclein overexpression. The histopathological findings that significantly correlated with gamma-synuclein overexpression were the number of metastatic lymph nodes (p<0.01) and the cancer stage (p<0.01). Using the same tissue mircoarray, the HER-2/neu gene expression and gamma-synuclein expression also showed statistically significant correlation (p=0.04). CONCLUSION: gamma-synuclein overexpression showed significant correaltion with lymph node metastasis and cancer stage. It also showed significant relevance with the HER-2/neu gene expression, and that is already known to be a prognostic factor for breast cancer. Therefore gamma-synuclein may be a useful prognostic factor for infiltrative breast cancer and further studies on the its correlation with survival, local recurrence, and distant metastasis should be conducted

The effect of breast cancer specific gene 1 in the prognosis of breast cancer / 中华外科杂志

<p><b>OBJECTIVE</b>To detect breast cancer specific gene 1 (BCSG1) expression in different breast tissue, analysis its correlation with clinical parameters and evaluate the prognosis of breast cancer.</p><p><b>METHODS</b>The expression of BCSG1 was detected by reverse transcription-polymerase chain reaction (RT-PCR) in surgical specimens from 84 cases of breast disease patients selected randomly at XinHua Hospital affiliated with Shanghai Second Medical University from September 1999 to December 2002. Of 84 cases, 72 case were breast cancer. Statistic analysis BCSG1 gene expression correlation with clinical parameters of breast cancer. 72 breast cancers were followed up (4 - 43 months) to set up independent prognosis factor by survival analysis.</p><p><b>RESULTS</b>BCSG1 was undetectable in all benign breast lesions, while was detectable in 36.1% of all breast cancer samples (26/72), in which 79.2% of stage III/IV cases were positive (19/24). The expression of BCSG1 was tightly correlated with the stage (P = 0.000) and the size of tumor (P = 0.007). Both ER (P = 0.027) and BCSG1 (P = 0.001) were the independent prognosis factor of breast cancer.</p><p><b>CONCLUSION</b>BCSG1 is one of independent tumor marker of breast cancer, the expression of BCSG1 is closely correlated to the stage of breast cancer and the tumor size. Maybe, BCSG1 is a new prognosis factor of breast cancer.</p>

Study on gamma-synuclein gene in patients with idiopathic Parkinson's disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the relationship between idiopathic Parkinson's disease (PD) and two polymorphisms (C243G and A377T) of the gamma-synuclein gene in a Chinese Han population of Shanghai area.</p><p><b>METHODS</b>Polymorphic genotyping was performed with PCR-RPLP technique. Association analysis was carried out in 145 unrelated idiopathic PD patients and 184 age-matched healthy controls.</p><p><b>RESULTS</b>The authors failed to detect any distributional difference of the C243G and A377T polymorphisms of the gamma-synuclein gene between PD cases and control subjects, nor did they find any association.</p><p><b>CONCLUSION</b>These data do not support that gamma-synuclein gene C243G and A377T polymorphisms are involved in idiopathic PD onset in the Han population of Shanghai area.</p>