[Construction of lentiviral vector of siRNA specific for γ-synuclein and its inhibition effect on colorectal carcinoma cell line SW1116].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
Expression of SNCG mRNA and protein plays an important role in the growth and the invasion capacity of SW1116 cells.
Feng
F
Huang
Shaohua
S
Xu
Department of Gastrointestinal Surgery, Fujian Provincial Tumor Hospital, Fujian Medical University, Fujian Key Laboratory of Tumor Biological Treatment, Fuzhou 350014, China. yqbobo@163.com.
Qing
Q
Ye
Qiuhong
Q
Zheng
Yangmei
Y
Xu
Qinying
Q
Liu
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000473:pathology*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus; D034622:RNA Interference; D012333:RNA, Messenger; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D051845:gamma-Synuclein / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051845:gamma-Synuclein