Inhibition Of Vascular Endothelial Growth Factor-C Expression By SiRNA And The Molecular Mechanism Study Of Lymphangiogenesis In Gastric Cancer

Gastric cancer is one of the most common malignancies in the world, and is thesecond leading cause of death after lung cancer. Transport of tumor cells via thelymphatics is the most common rounte and an important early event in gastric cancer,and lymph node metastasis is the strongest prognostic factor. As a whole, lymph nodemetastasis is observed in 2-4% of mucosal carcinoma and in 10-15% of submucosalcarcinoma; Actually,. 80% of patients with advanced gastric cancer have lymph nodemetastasis, and the remote lymph nodes, such as the para-aortic nodes, are involved in20% of gastric cancers. Accordingly, control of lymph node metastasis is the mostimportant strategy for the treatment of gastric cancer.Clinical and pathological data suggest that many people die principally as a resultof the metastastic spread of cancer (haematogenous metastasis, lymphatic metastasisand directly metastasis), Cells must first invade either blood or lymphatic vessels toenter the circulation. Over the past 10 years, little has been learned about themolecular regulation of lymphangiogenesis and the role of tumor lymphangiogenesisin the metastasis. Recently, specific markers for lymphatic vessels such as LYVE-1and VEGFR-3 have been found,,the growth of lymphatic vessels therefore hasimportant medical implications.VEGF-C is known to have a major role in lymphangiogenesis in cancer tissue,which belongs to the VEGF family of growth factors. The levels of VEGF-C inprimry tumors have significantly correlated with lymph node metastasis in a variety of cancers, including gastric, breast, prostate and thyroid cancer,, Many experimentalstudies indicated that lymphangiogenic factors (VEGF-C) could stimulatelymphangiogenesis in tumors by binding to their receptors (VEGFR-3) on thelymphatic endothelial cells and inducing proliferation and growth of new lymphaticcapillaries, then enhance the incidence of lymph node metastasis. VEGF-C canfacilitate metastasis by increasing the surface area of tumor cells in contact with LECs,by increasing vascular permeability, and/or by changing LEC adhesive properties orcytokine/chemokine expression. Recent work from many laboratories hasdemonstrated that the vascular endothelial growth factor-C/VEGFR-3 signalingpathway is crucial for lymphangiogenesis, this signaling system is therefore apromising target for inhibitors of lymphangiogenesis designed to restrict metastasis.inhibiting tumor lymphangiogenesis and lymph node metastasis include monoclonalantibody and a soluble VEGFR-3-IgG fusion protein, small moleculeinhibitors, Peptide drugs and antisense techniques.Recently a novel strategy of gene silencing using RNA interference(RNAi) hasbeen discovered, RNAi is the most potent AS technology discov-ered thus far. It isestimated that the half-maximal inhibition levels (IC50) of the siRNAs are some 100-to 1,000-fold lower than an optimal phosphorothioate oligodeoxynucleotide directedagainst the same target. RNA interference (RNAil), as commonly defined, is aphenomenon leading to post -transcriptional gene silencing (PTGS) after endogenousproduction or artificial intro-duction into a cell of small interfering double strandRNA (siRNA) with sequences complementary to the targeted gene. RNAi hasrecently become the method of choice for mammalian cell genetic analysis and hasthe potential to serve as a therapeutic treatment for a variety of acquired andhereditar-y diseases because of its potency and specificity, versatility. The potentialutility of RNAi in inhibiting cancer cell proliferation has recently been demonstratedin many in vitro as well as in vivo studies.The short interfering RNAs (siRNAs) are generated by chemical synthesis, in vitrotranscription, siRNAs expression cassettes, Construction of Plasmids and Viral Vectors for RNA Interference, an RNaseâ…¢-like processing reaction from longdsRNA. vectors produce shRNAs, which are transcripted intracellularly into shortduplex RNAs having siRNA-like properties, The process is economy and easilymanipulation. Such vectors which have antibiotics mark can mediate persistent genesilencing after stable integration of the vector into the host-cell genome. The ability tocreate permanent cell lines with a desired loss-of-function phenotype extends theutility of RNAi as method for probing gene function in mammalian cells. Thiscapability enables the production of large numbers of silenced cells for biochemicalanalysis and permits the evaluation of phenotypes over long time spans.Our research is to explore the role of VEGF-C on lymph node metastasis of gastriccancer. Two nucleotide sequences of siRNA corresponding to two various sites ofVEGF-C gene were designed. Expression vector of small interference RNA wasconstructed and transfencted into gastric cancer cells SGC-7901,RT-PCR, immunohistochemistry assay, western blot were used to detect the inhibitoryeffects of siRNA on the expression of VEGF-C at the level of protein and mRNA, thebest sequence inhibited was selected in order to go on exploring. Count assay wasused to determine the growth of transfected cells and SGC-7901 cells to observe ifinhibited expression of VEGF-C may influence the growth of cancer cells.Recently, nude mice transplanted tumor have become an important tool for researchof human tumor. In our study, nude mice transplanted tumor with the suppression ofVEGF-C gene were built to explore the relationship between VEGF-C gene andlymph node metastasis of gastric cancer, lymphangiogenesis, lymph node density. Ourresearch provides theoretical and experimental basis for anti-lymphangiogenesis andlymph node metastasis of gastric cancer.Part One: Expression of vascular endothelial growth factor-C indifferent human gastric adenocarcinoma cell linesMethods1. Two human gastric adenocarcinoma cell lines(BGC-823, SGC-7901) wereemployed during logarithmic growth phase and routinely cultured. Cell reproductive activity and growth curve were used by counting method. Expression of vascularendothelial growth factor-C were detected by RT-PCR and Western blot in thedifferent human gastric adenocarcinoma cell lines.2. Statistical analysis: All the data were analyzed by SPSS 13.0 program. Themeasurement data were expressed as standard deviation ((?)±S) and tested by t-test. Thelevel of significant difference wasα=0.05.Results1. Two human gastric adenocarcinoma cell lines(BGC-823, SGC-7901) were adherentto wall, cell densities were 5×10⁴/ml and exponential growth at the second or fouth dayafter changing culture fluid.2. VEGF-C mRNA Expression in Human Gastric Adenocarcinoma Cell LinesSGC-7901, BGC-823 expressed VEGF-C mRNA, Semi-quantitation RT-PCR(reversetriscription-ploymerase chain reaction) results showed that the VEGF-C mRNAexpression level in SGC-7901 cells with semi -quantitation value of 0.64±0.04,which is sadistically higher than that of BGC-823,0.45±0.02(p＜0.05).3. Western Blot AnalysisSGC-7901, BGC-823 expressed VEGF-C protein, Western Blot results showed thatthe VEGF-C protein expression level in SGC-7901 cells with density value of 0.79±0.06, which is sadistically higher than that of BGC-823,0.68±0.02(p＜0.05).4. Immunohistochemical Study of VEGF-C ExpressionAfter cell immobilization, Immunohistochemical Study of VEGF-C Expressionshowed that there were light brown particle diffusedly in the cytoplasm, whichindicated VEGF-C expression in two cell lines. Moreover, color in SGC-7901 isdeeper that that in BGC-823, which is according to Western blot results.Part Two: Construction and identification of siRNA expressionvector targeting Vascular Endothelial Growth Factor-CMethodsAccording to the computer aided design, oligonucleotide fragments containing VEGF-C specific sequences were synthesized, The complement form was obtained byannealing and being cloned into vector pSilencer 3.1-H₁ and the recombinant plasmidwas transformed into strain DH5a, plasmid were amplificated and depurated, thenidentified by sequencing and agarose electrophoresis.ResultsThe plasmid identified by restriction enzyme was used for sequencing and threerecombinant vectors were respectively pSienlcer3.1-VEGF-C₁,pSilencer3.1-VEGF-C₂, negative control Psilencer3.1-VEGF-C_N.Part Three:Detected Suppression Effect of Eukaryotic Expression Plasmid ofSmall Hairpin RNA(shRNA) of VEGF-C on SGC-7901 Cell LinesMethods1. SGC-7901 cell lines were transfected by Eukaryotic Expression Plasmid of SmallHairpin RNA(shRNA) of VEGF-C with liposome2000. Stabilized transfectedSGC-7901 cells were constructed by selection with 600μg/ml G418-sulfate, Stabletransfected cells were obtained and routinely maintained in selection media cantaining200ug/ml of G418-sulfate to avoid overgrowth of nontransfected cells, inhibitoryeffect of VEGF-C mRNA and protein were detected by semi-quantitative RT-PCRand Western-blot. Observed the effect of knockdown of VEGF-C on proliferationability of. SGC-7901 cells by cell counting method.2. Statistical analysis: All the data were analyzed by SPSS 13.0 program. Themeasurement data were expressed as standard deviation ((?)±S) and tested by One-WayANOVA. The level of significant difference wasα=0.05.Results1. After selected with media cantaining 600μg/ml of G418-sulfate, cells becameconstrcting, rounding and floating at the third day. A lot of cells decease excepttransfected cells at the eighth day, Stable transfected cells were obtained and formed clone at the fourteenth day and routinely maintained in selection media cantaining200ug/ml of G418-sulfate to avoid overgrowth of nontransfected cells.2. We examined mRNA expression of VEGF-C and VEGF-A, VEGF-B, VEGF-D, aclosed related member of the VEGF family, in these transfected cells to show thespecificity of VEGF-C gene silencing. We observed significant inhibition of VEGF-CmRNA expression in pSienlcer3.1-VEGF-C₁ cells, pSilencer3.1-VEGF-C₂ cellscompared with negative control Psilencer3.1-VEGF-C_N cells and non-transfected cells(p＜0.05), VEGF-C mRNA expression could be inhibited 82%, 65% respectively byvector pSienlcer3.1-VEGF-C₁, pSilencer3.1-VEGF-C₂.3. Western blot analyses showed that pSienlcer3.1-VEGF-C₁ cells andpSilencer3.1-VEGF-C₂ cells had a significant decrease in VEGF-C protein levelcompared with negative control Psilencer3.1-VEGF-C_N cells and non-transfected cells,VEGF-C protein expression could be inhibited 81.2% respectively by vectorpSienlcer3.1-VEGF-C₁(p＜0.05).4. There was no significant difference in the rate of cell proliferation amongpSienlcer3.1-VEGF-C₁ cells, negative control Psilencer3.1-VEGF-C_N cells andnon-transfected SGC-7901 cells.Part Four: Down-Regulation of Vascular Eendothelial Cell GrowthFactor-C Expression Using Small Interfering RNA Vectors in vivoMethods1. Transfected cells were transplanted subcutaneously in nude mice to observe thetumorigenesis. Tumor volume and the weight of node mice were measured in thevarious time. The level of protein and mRNA of VEGF-C were explored in thedifferencer gastric cancer tissues by IHC, western blot. Lymph node density wascounted by IHC, It was evaluated whether small interference RNA expression vectorcould inhibit the VEGF-C and lymph node metastasis by in vivo.2. Statistical analysis: All the data were analyzed by SPSS 13.0 program. Themeasurement data were expressed as standard deviation ((?)±S) and tested by One-Way ANOVA. The level of significant difference wasα=0.05.Results:1. Tumor value in vivoTumor grows at the fourth day after tumor cells injection, and we measured tumorvolume at the ninth day. Tumors volume and murine weight were measured withcalipers twice a week. Tumor volume was calculated by the following formula:volume=W²Ã—L/2, where W= short diameter and L=long diameter. Mice weresacrificed 4 weeks following tumor cells injection and tumors were harvested. Weexamined the impact of siRNA-mediated VEGF-C gene silencing on murine gastrictumor growth. We did not observe a statistically significant difference in tumorgrowth and murine weight among pSienlcer3.1-VEGF-C₁ cells group,Psilencer3.1-VEGF-C_N cells groups and non-transfected SGC-7901 cellsgroup(p＞0.05).2. The value of tumor specimen2.1 Tumor weight was calculated after Mice were sacrificed 4 weeks following tumorcells injection and tumors were harvested. There were not a statistically significantdifference in tumor weight among pSienlcer3.1-VEGF-C₁ cells group,Psilencer3.1-VEGF-C_N cells groups and non-transfected SGC-7901 cellsgroup(p＞0.05).2.2 Immunohistochemical analysisVEGF-C protein expression showed that there were light brown particle diffusedlyin the cytoplasm. Immunohistochemical analysis showed that pSienlcer3.1-VEGF-C₁cells group had a significant decrease in VEGF-C protein levels compared with othergroups.We examined the effect of VEGF-C gene silencing on lymphangiogenesis amongpSienlcer3.1-VEGF-C₁ cells group, Psilencer3.1-VEGF-C_N cells groups andnon-transfected SGC-7901 cells group produced by Immunohistochemical analysisusing an anti-VEGFR-3 antibody. Lymphatic vessels are mostly present in themargins of tumor Data showed fewer lymphatics in pSienlcer3.1-VEGF-C₁ cells group compared with other groups. There were not a statistically significantdifference in lymphangigenesis among pSienlcer3.1-VEGF-C₁ cells group,Psilencer3.1-VEGF-C_N cells groups and non-transfected SGC-7901 cellsgroup(p＜0.05).3. Western blot analysisWestern blot analyses showed that pSienlcer3.1-VEGF-C₁ cells group had asignificant decrease in VEGF-C protein level compared with negative controlPsilencer3.1-VEGF-C_N cells and non-transfected cells, VEGF-C protein expressioncould be inhibited 81.2% respectively by vector pSienlcer3.1-VEGF-C₁ However, Itsinhibited rate was lower than stable transfected cells.Conclusions1. Two human gastric adenocarcinoma cell lines (BGC-823, SGC-7901) expressVEGF-C mRNA and protein, but the expression level of SGC-7901 cell linesaccompanied with lymph node metastasis were higher than that of BGC-823 celllines.2. We Successfully construct and identify siRNA expression vector targetingVascular Endothelial Growth Factor-C3. Stable transfected cells are obtained by selection media cantaining 600μg/ml ofG418-sulfate and routinely maintained in selection media cantaining 200μg/ml ofG418-sulfate. The expression level of VEGF-C mRNA and protein in stabletransfected cells could be inhibited by small interfering RNA in vitro and in vivo.4. We successfully build nude mice subcutaneouly transplanted tumor model, and datasuggested that inhibited of VEGF-C could inhibit lymphangiogenesis and themicrolymph node density were decreased.5. Inhibition of VEGF-C have no effect on tumor growth.6. The expression of VEEGF-C can be specially and effectively inhibited by smallinterfering RNA, and the effect of inhibitory could maintain over four weeks.Down -regulation of VEGF-C might provide a therapeutic strategy forinhibiting lymph node metastasis in gastric cancer.