Constructs and Lentivirus production refers to a method described previously 1617. MAT2A gene was cloned into the Xho I and EcoR I sites of vector pEGFP-C1 (Genechem) which was driven by CMV to yield plasmid pEGFP-C1-MAT2A. MAT2B gene was cloned into pEGFP-C1 at EcoR II and XhoI sites to generate pEGFP-C1-MAT2β. Two-pair of primers 5'-CCGCTC GAGCTATG AAC GGACAGCTCAACG-3' (sense), 5'-CCGGAATTCGAATATTTAAGCTTTTT GGGCAC-3' (antisense) or 5'-CCGCTCGAGCTATGAACGGACAGCTCAACG-3' (sense); 5'-CCGGAATTCG AATATTTAAGCTTTTTGGGCAC-3', (antisense) were used to amplify the MAT2A and MAT2β gene, respectively. The PCR products were then cloned into AgeI and EcoRII sites of pGCL-GFP to generate plasmid pGCL-GFP-MAT2A and pGCL-GFP-MAT2β, in which the MAT2A or MAT2β were fused in frame with the GFP gene and the expression of the fusion gene was driven by the CMV promoter. Four regions of the MAT2A gene and four regions of the MAT2β gene were selected as the targeted sequences of siRNA in this study. To construct single siRNA expression vector, two 64 nt primers, each containing a 19 nt or 21 nt target sequence in the sense and antisense forms from different regions of the MAT2A gene or MAT2β gene as indicated below, were systhesized:

5'-GAGAGCTATTAGAGATTGT-3' (MAT2A 1siRNA);

5'-GGATACAATCTACCACCTA-3' (MAT2A 2siRNA);

5'-GTGAGAGAGAGCTATTAGA-3' (MAT2A 3siRNA);

5'-AATATCTGGTGACTGTTGC-3' (MAT2A 4siRNA);

5'-GCAGTTCATCACATCATTCAT-3' (MAT2β1siRNA);

5'-CCTTACAGAGAGGAAGACATA-3' (MAT2β2siRNA);

5'-GCTGTGACTGTTATGTTTGAT-3' (MAT2β3siRNA);

5'-GCCTCTCAACTTAATGTGGA-3' (MAT2β4siRNA).

Sense and antisense primers were then cloned into Psc siRNA plasmid (Genechem) at EcoRI and XhoI sites after annealing according to the manufacturer's instructions. To generate the dual siRNA expression plasmid, two primers 5'-CCACGAGGCGTTCATCGAGG-3'(sense), and 5'-AAGTCTTGTAGTCAAAACCT-3'(antisense) were designed to amplify a DNA fragment containing U6 promoter and MAT2A 2siRNA expression cassette from recombinant plasmid Psc-U6-MAT2A. The PCR product was then cloned into AatII and EcoRI sites of plasmid Psc-U6-MAT2β1 to generate recombinant plasmid Psc-U6-MAT2A/β, which carries two independent siRNA expression cassettes. Correct insertions of shRNA cassettes were confirmed by restriction mapping and direct DNA sequencing. Recombinant lentiviruses vectors were produced by co-transfecting 293T cells with the lentivirus expression plasmid and packaging plasmids (pHelper 1.0 including gag/pol and pHelper 2.0 including VSVG) using Lipofectamine 2000 or calcium phosphate method 181920. pGCL-GFP plasmids and Psc siRNA plasmid were co-transfected in 293T cells, fluorescence-activated cell sorting analysis of GFP positive in 293T cells, then MAT2A 2siRNA, MAT2β1 siRNA plasmid was chosen as effective siRNA plasmid Infectious lentiviruses were harvested at 48 and 72 h post-transfection, centrifuged to get rid of cell debris, and then filtered through 0.22-lm cellulose acetate filters21. The infectious titer was determined by fluorescence-activated cell sorting analysis of GFP positive in 293T cells. The virus titers were at the range of 10⁹ transducing units/ml medium. lentiviruses vectors containing MAT2A 2siRNA, MAT2β1 siRNA and both of them simultaneous was constructed respectively, which was designated LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β.

Human hepatocelluar cancer HepG2 cell lines (purchased from Classic Specimen Culture and Storage Centre in Wuhan University) The cell lines were grown in 5% CO₂ saturated humidity at 37°C and cultured as monolayers in RPMI 1640 supplemented with penicillin/streptomycin, 2 mM glutamine and 10%FBS. Cells were always detached using Trypsin-EDTA. for 4–5 days and then subcultured at 1×10⁵ cells per well into six-well tissue culture plates. After 72 h of culture, the cells were transfected with siMAT2A and2β formulated into liposomes according to the manufacturer's instructions (TransMessengerTM, Qiagen, Valencia, CA). lentiviruses concentration was 0.5μl per well, and the final volume of culture medium was 2 mL per well. Growth curve was drafted. Untreated cell was used as control.

HepG2 cells were transfected with LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β respectively. At 72 h after transfection, total RNA was extracted with TRIzol reagent according to the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA, USA). Sequences of sense primer and antisense primer were seen as Table 1. Reverse transcription PCR (RT-PCR) reactions were performed as follows: cDNA was amplified for 25 cycles at 95°C for 5 min, 94°C for 1 min, 60°C for 45 s, 72°C for 1 min and 72°C for 10 min. MAT2β gene cDNA was amplified for 45 cycles at 95°C for 15 s, 94°C for 5 s, 60°C for 30 s, 72°C for 30 s and 72°C for 10 min. The PCR reaction for β-actin cDNAs was performed with 30 cycles and the reaction conditions were: denaturation at 94°C for 1 min, annealing at 53°C for 2 min, and extension at 72°C for 3 min. Semi-quantitative PCR for Bax: denaturation at 94°C for 45 s, annealing at 58°C for1 min, and extension at 72°C for 1 min. p21:95°C for30 s, 55°C for 90 s and 72°C for 120 s using 30 cycles, p27:94°C 30 s, 55°C 30 s, or 57°C 30 s, 72°C 60 s. PCR products were analyzed through 1% agarose gel electrophoresis and following ethidium bromide staining.

Cells were collected after different treatments, rinsed in ice-cold PBS, 15 mins, and lysed in lysis buffer containing 50 mmol/L HEPES (pH 7.9), 0.4 mol/L NaCl, 1 mmol/L EDTA, 2 Ag/mL leupeptin, 2 Ag/mL aprotinin, 5 Ag/mL benzamidine, 0.5 mmol/L phenylmethylsulfonylfluoride, and 1% NP40. The lysates were centrifuged at 14,000 rpm to remove any cellular debris. Protein concentrations of the lysates were determined by the Bio-Rad Dc protein assay system which was 2 μg/μl. Equal amount of protein was separated by12% SDS-PAGE, transferred to PVDF membrane, and blocked with 5% nonfat dry milk in TBS/Tween 20 (0.05%, v/v) for 1 hour at room temperature. The membrane was incubated with primary antibody overnight. anti-GAPDH anti-p21, anti-p27 and anti-Bax antibodies were obtained from Santa Cruz Biotech. Anti-MAT2A antibody were obtained from Genway company. Anti-MAT2β was purchased from Abnova corporation (Tai wan, China). After washing, the membrane was incubated with secondary antibody (diluted 1:4,000; Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 hour. Following several washes, the blots were developed by enhanced chemiluminescence. (Amersham, Arlington Heights, IL). Each experiment was repeated at least twice with similar results.

The inducing apoptosis effects of LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β on human hepatocelluar cancer cells and were analyzed by Flow cytometry. Briefly, Cells were incubated with LV-siMAT2A, LV-siMAT2β, LV-siMAT2A/2β and vehicle alone for 72 h, fixed with 70% ethanol treated with RNase (100 lg/ml, Sigma Chemical Co., St. Louis, MO), and stained with propidiumiodide (100 lg/ml, Sigma) for 30 min on ice. DNA content in the cells was analyzed by a flow cytometer (Epics XL; Beckman Coulter, Miami, FL).

Hepatic SAMe levels were measured using a method described previously25 with slight modifications. Liver specimens were homogenized in phosphate-buffered saline, and an aliquot was saved for protein assay. The rest was treated with 100 μl of 1 M perchloric acid (PCA) onice for 5 min and centrifuged at 1000 g for 15 min at 4°C. The aqueous layer was quantitatively removed, neutralized with 3 M KOH, and centrifuged at 3000 g for 10 min at 4°C. SAMe levels were deter mined in the neutralized PCA extracts by HPLC (LC-10ATVP pump, SCL-10AVP system control) with a SPD-10AVP UV detector and a SIL-10ADVPautosampler (Shimadzu) using a Partisil SCX 10 μm column (25 × 0.44 cm i.d.; Whatman Chem. Sep. Maidstone, Cleveland, OH). SAMe was eluted isocratically at 1 ml/min with 0.19 MNH4H2PO4 adjusted to pH 2.6 with 2 M H3PO4. SAMe levels were calculated using standard curve of SAMe prepared at the same time as the samples and are reported as nmol/mg protein.

All results are expressed as mean ± SD, and statistical analyses of the data is performed using ANOVA Post Hoc Dunnett t test. All p values are based on two-sided hypothesis testing, P < 0.05 is considered significant.

To assess the potential effects of Lentivirus vectors RNAi-mediated MAT2A and MAT2β silencing on cell growth, LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β were transfected into respectively, 72 hours after infection at multiplicity of infection of 25 through estimating GFP expression under a fluorescent microscope (Figure 1). Cells were counted every day for 5 days after transfections, growth curve was drafted. As a result, growth-inhibition effects were produced by LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β, in which caused by LV-siMAT2A/2β was most evident (p < 0.01) and LV-siMAT2A was most slight(p < 0.05) (Figure 2)

To investigate the effects of LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β on cell apoptosis. The distribution of cell cycle was determined by flow cytometry analysis. After HepG2 cells were transfected with LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β for 72 h, the percentage of both S and G2/M phase were decreased 72 h after LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β treatment, and the percentage of G0/G1 phase was increased, (Figure. 3). The expressions of p21 and Bax were up-regulated by LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β. p27 which was up-regulated by LV-siMAT2β and LV-siMAT2A/2β but not by LV-siMAT2A. the mRNA levels of p21 and Bax were highest induced by LV-siMAT2A/2β, but that there was no difference between that caused by caused LV-siMAT2A LV-siMAT2β (Figure 4). Western-blot was applied to assay the expressions of Bax, p27 and p21, After HepG2 cells were transfected with LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β for 72 h, GAPDH was used as control, It was showed in figure 5 that protein of Bax was increased by LV-siMAT2β, LV-siMAT2Aand LV-siMAT2A/2β, which was highest after cells was deal with LV-siMAT2A/2β, protein of p27 was increased by LV-siMAT2β and LV-siMAT2A/2β but not by LV-siMAT2A, The protein of p21 does not change compared with control.

After HepG2 cells were transfected with LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β for 72 h, RT-PCR was performed to determined the mRNA of MAT2A and MAT2β. As it showed in figure 6 mRNA of MAT2A and MAT2β were knocked down by LV-siMAT2A, LV-siMAT2β by 85.5% and 94.0% respectively, and both of them were suppressed LV-siMAT2A/2β by 89.5% and 97.8% simultaneously compared with control which was untreated. We found that LV-siMAT2A had no effect on mRNA of MAT2β, on the other hand, LV-siMAT2β had no impact on mRNA of MAT2A.

In order to figure out the suppression efficiencies caused by LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β, Western-blot was applied to detect protein of MAT2A and MAT2β after HepG2 cells were transfected with LV-siMAT2A, LV-siMAT2β and LV-siMAT2A/2β for 72 h. It was indicated in figure 7 that the expressions of MAT2A and MAT2β were inhibited by LV-siMAT2A and LV-siMAT2β respectively at protein level by 99.4% and 60.2%, both of them were suppressed by LV-siMAT2A/2β by 98.6% and 90.8%. We discovered that LV-siMAT2A did not have impact on protein expression of MAT2β, and LV-siMAT2β also did not have effect on impact on protein expression of MAT2A too.

The changes in MAT genes expression are likely to cause changes in the production of SAMe. We analyzed the effects of silencing MAT2A and MAT2β by different Lentivirus-Mediated RNAi on SAMe production by measuring levels of SAMe a in HCC cell. As a result, Intracellular SAMe content was decreased by LV-siMAT2A and increased by LV-siMAT2β as well as LV-siMAT2A/2β, compared with control. (Figure 8)