Reversal Of Multidrug Resistance Of Gastric Carcinoma Cells By Small Interfering RNA Targeting Glucosylceramide Synthase Gene

Gastric carcinoma is one of the malignant tumors most frequently seen in our country and threatens seriously to the health and life of humans. Chemotherapy plays an important role in its combined therapies, but the chemotherapeutic effect may be affected by many factors in which multidrug resistance(MDR) is one of the major causes for the failure of chemotherapy-based treatment modalities of malignant tumors. Thus, the study on generation mechanisms of MDR and reversing methods has become an important issue in tumor research. MDR is characterized by a cross-resistant phenotype against several unrelated drugs that differ widely with respect to molecular structure and target specificity. Many mechanisms related with MDR have been known, such as high expression P-gp (P-glycoprotein), MRP (multidrug resistance-associated protein), heat shock protein and LRP(lung resistance-related protein), increase of glutathione S-transferase and glutathione,decrease of topology isomerase activity,inhibitory apoptosis and alterations of biochemical characteristics in tumor cells. Recently overexpressed GCS (glucosylceramide synthase) has been found to be closely related with MDR of rumors.GCS is one kind of glucose transfering enzyme which participates in the metabolism of ceramide. It can convert ceramide to glucosylceramide (GlcCer), prevent cells from apoptosis induced by ceramide and generate MDR. Ceramide, the main molecule of sphingolipid metabolism, plays a critical role in MDR as a second messenger of cellular apoptotic signaling. GlcCer, a simple glycosylated form of ceramide, is prosoma substance for synthesizing other glycosphingolipids. It is correlative with biological behaviour of tumor cells, maintaining the normal structure and functions of cell as well as participating in cell proliferation, differentiation and MDR of tumor cell. Therefore, inhibiting the expression of GCS can decrease the potential ceramide glycosylation, increase the content of ceramide in cells, and even reverse MDR of tumor cells efficiently. Some medicine inhibiting GCS has been discovered in recent years but was limited in clinical application due to low specificity, grave adverse effects, difficulty in getting effective plasma concentration to reverse MDR. And although ASODN technology can also reverse MDR of tumor, the defects such as low specificity and efficacy are existing. Therefore, it is urgently needed to develop an efficient therapeutic strategy to reverse MDR.RNA interference(RNAi)is a novel technology of post-transcriptional gene silencing induced by double stranded RNA(dsRNA). Because of high efficacy and specificity, RNAi has been widely applied in the research of genomic function and disease therapy. In the study of the mechanisms of MDR in gastric carcinoma treament, a great lot of research had been focused on mdr1 and P-gp in the past. As so far, the relation between GCS and gastric carcinoma drug resistance has not been reported domestically and abroad.To explore the mechanism of GCS mediated MDR in gastric carcinoma and the effect of siRNA targeting GCS, the expression of GCS in several gastric carcinoma cell lines were detected by RT-PCR, immunohistochemistry and western blot in the experiment, revealing the relations between GCS and the occurrence, development of gastric cancer and MDR.Three siRNA eukaryotic expression vectors(Rl-pSUPER EGFP, R2-pSUPER EGFP, R3-pSUPER EGFP)targeting GCS gene were constructed, then transiently transfected into human gastric carcinoma cells SGC7901/VCR with MDR.The interferences to GCS were compared and the recombinant plasmids with high inhibitition were selected for further trials. The selected R1-pSUPER EGFP was stably transfected into SGC7901/VCR cells, then the expression of the related gene of GCS and mdr1, apoptosis related factors bcl-2, bax, caspase-3 and apoptosis rate were detected. At same time, the growth of transfected cells and the sensitivity to chemotherapeutics were observed. Finally, the stable transfection cells were inoculated subcutaneously into the nude mice. The inhibitory effect of siRNA on transplantation tumor growth, cells apoptosis and the expression of GCS were observed. According to the experiments both in vitro and in vivo, the relation between GCS and MDR in gastric carcinoma, even molecular mechanism of GCS mediated MDR, were studied from gene, protein level and tumor cytobiology behaviour. Reversing MDR by the decrease of expression of GCS were extremely explored as well. The reliable theoretic bases are provided for siRNA vector targeting GCS gene to increase gastric carcinoma chemotherapeutic sensitivity and be applied in clinics.Methods:1. The cells GES-1, BGC823, SGC7901 and SGC7901 /VCR were cultured in medium RPMI-1640 routinely.The cells SGC7901/VCR were cocultured with 0.5～1.0μg/ml of vincristine(VCR)to maintain MDR phenotype. It was followed by cultivation with no VCR for two weeks before detection.2. The expression of GCS in cells GES-1, BGC823, SGC7901 and SGC7901/VCR were examined by RT-PCR, immunohistochemistry and western blot on the level of mRNA and protein.3. MTT method was used to detect the sensitivity of cells SGC7901 and SGC7901 /VCR to chemotherapeutic drugs such as ADR, VCR, 5-FU and DDR4. Three pairs of reverse repeated motifs of sequence targeting GCS gene were designed and synthesized in vitro, then they were cloned into the expression vectors pSUPER EGFP respectively to construct GCS siRNA eukaryotic expression vectors R1-pSUPER EGFP, R2-pSUPER EGFP and R3-pSUPER EGFP.5. Three GCS siRNA expression vectors were transiently transfected into human drug-resistant gastric carcinoma cells SGC7901/VCR respectively. The effects on GCS mRNA and expression of protein were assayed by RT-PCR and western blot after transfection. Then their interference effects were compared and the one with the highest efficacy was selected for further trials.6. The recombinant plasmid R1 -pSUPER EGFP with high interference was transfected into the cells SGC7901/VCR. Then the positive cell was selected and amplified to construct a stable transfection cell line.7. MTT and growth curve were used to detect the growth of cells SGC7901/VCR transfected with recombinant plasmid R1-pSUPER EGFP.8. The expressions of GCS, mdr1 mRNA and P-gp in cells SGC7901/VCR with stable transfection of recombinant plasmid were examined by RT-PCR and western blot.9. RT-PCR and immunohistochemistry were used to examine the expression levels of the related apoptosis factors bcl-2, bax and caspase-3 in the cells SGC7901/VCR transfected with recombinant plasmid. Flow cytometry was applied to detect the cell apoptosis of the cells SGC7901/VCR transfected with recombinant plasmid.10. MTT method was applied to detect the sensitivity of the cell SGC7901/VCR transfected with recombinant plasmid to chemotherapeutics such as ADR, VCR, 5-FU and DDP.11. The cells SGC7901/VCR transfected with GCS recombinant plasmid, blank vector and the blank contrast without transfection were inoculated subcutaneously into the nude mice respectively to construct the transplantation tumor models of gastric carcinoma in nude mice. The size of tumor was measured regularly every six days, and the growth curve was drawn. The tumor weight and volume were measured and the tumor growth inhibition rate was calculated. The growth of different groups of transplantation tumor in nude mice was observed.12. The structure of transplantation tumor was examined with HE staining method. The expression of GCS was detected with RT-PCR and immunohistochemistry. The cell apoptosis of transplantation tumor was detected by TUNEL.13. The SPSS10.0 statistical package program was used to make data statistics. Fornumerical variable information, the data were expressed with x±s, the t-test andANOVA were used to compare these data. The positive rate was calculated with count information. The Chi-square test was used in comparison of positive rates. The level of significant difference isα=0.05. Results:1. It is shown with the results of RT-PCR, immunohistochemistry and western blot that GCS were expressed in cells GES-1, BGC823, SGC7901 and SGC7901/VCR. With the RT-PCR, the relative expression amount of GCS mRNA were 0.23±0.01 in GES-1,0.36±0.02 in BGC823, 0.49±0.04 in SGC7901, 0.98±0.01in SGC7901/ VCR, lowest in GES-1 and highest in SGC7901/VCR, significantly higher than in parent cells SGC7901(P<0.05).The GCS immunoreactivity in buffy-coloured granules were located in the intracytoplasm. The positive rate were the highest in cells SGC7901/VCR, significantly higher than in other cells(P<0.05). Based on the western blot, the expression levels of GCS in accordance of increased order were the GES-1, the BGC823, the SGC7901 and the SGC7901/VCR(highest) under the same amount of samples.2. The values IC₅₀ of the cells SGC7901/VCR to ADR,VCR, 5-FU and DDP was higher than those of the cells SGC7901 through extraorgan drug sensitivity analysis(P<0.05).3. Through double enzyme digestion, three recombinant plasmids, R1-pSUPER EGFP, R2-pSUPER EGFP and R3-pSUPER EGFP released about 285bp fragments, conforming to the plasmid atlas numerical data. The DNA sequence confirmed that base composition of insertion fragment into recombinant plasmids was the same as the designed DNA sequence. It was manifested that siRNA expression vectors were successfully constructed.4. The results of RT-PCR and western blot showed that the expression level variation of GCS was different with transient transfection of three recombinant plasmids into the cells SGC7901/VCR, the expression of GCS in the cells with Rl-pSUPER EGFP declined most significantly, while not evidently in the cells transfected with the other two. It indicated that siRNA targeting GCS could be expressed in eucaryotic cells and had the best inhibitory effect on GCS with the expression of R1-pSUPER EGFP.5. The cell growth curve ascended slowly and the growth speed is reduced in cells SGC7901/VCR with transfection of recombinant plasmids comparing to the blank vector group and the control group.6. By RT-PCR, the relative contents of GCS mRNA and mdr1 mRNA in cells thansfected with recombinant plasmid were evidently lower than those in the other two groups (P<0.05). By western blot, the transfected recombinant plasmid R1-pSUPER EGFP can evidently inhibited expression of GCS and P-gp in cells SGC7901/VCR, with inhibition rates at 83.5% and 74% respectively.7. After stable transfection, the expression levels of promoting apoptosis factors such as bax and caspase-3 increased, while inhibitory apoptosis factor bcl-2 decreased in cells transfected with recombinant plasmids. Compared to blank vector group and the control group, the difference was signiflcant(P<0.05).8. Flow cytometry showed that the apoptosis rate of the cell with recombinant plasmids is 33.35±0.11%, dramatically higher than that of the blank vector group and the control group(5.09±0.02% and 3.98±0.04% respectively). The difference had statistical significance(P<0.05).9. MTT method demonstrated that the values IC50 of the cells transfected with recombinant plasmids to ADR, VCR, 5-FU and DDP decreased, while the sensitivity increased comparing to those of the other two groups. The difference had statistical significance(P<0.05).10. For the nude mice transplanted with recombinant plasmid, the tumor growth in vivo is delayed. The volume and weight were smaller than those of the blank contrast group and blank vector group(P<0.05).The inhibition rate of tumor weight and volume were 64.13% and 63.81% respectively. By histopathology, much tumor tissue has necrosis in the group with recombinant plasmids. By RT-PCR and immunohistochemistry, the expression level of GCS mRNA and protein in the tumor tissue of nude mice transfected with recombinant plasmid declined obviously comparing to the other two groups(P<0.05). By TUNEL assay, the positive rate of cell apoptosis in the tumor tissue of nude mice with recombinant plasmid was significantly higher than that in the control group and blank vectors group(P<0.05). Conclusions:1. GCS gene is expressed in all cells of gastric carcinoma, but expression level different in cell sublines. In cells with MDR, the expression of GCS mRNA and protein show the highest level, significantly higher than its parent sensitivity cells. It reveals that GCS participates in the course of occurrence and development of gastric carcinoma and is related with MDR of gastric carcinoma cells.2. Three GCS siRNA eukaryotic expression vectors are successfully constructed. Their different interference effects to GCS in gastric carcinoma cells of MDR reveal that the inhibitory effect of recombinant plasmids on target gene is closely related to the structure and site of target sequence. The recombinant plasmids R1-pSUPER EGFP with optimal interference effect was selected through experiment to ensure the specificity and high efficacy of gene silencing, providing the basis for further exploring the effect on gastric carcinoma cells MDR of targeted GCS gene.3. The gastric carcinoma cell line with stable transfection of GCS siRNA expression vector is successfully constructed. GCS siRNA can increase the cells sensitivity to chemotherapeutics through inhibiting GCS expression, decreasing mdr1 expression, increasing factors caspase-3 and bax expression, inducing drug resistance cell apoptosis.4. The GCS siRNA vector can inhibit specially the expression of GCS in nude mice, induce the xenografts cell apoptosis, inhibit the growth of xenografts. It indicates that the expression vector of GCS siRNA may reverse MDR and be a novel gene-therapy method in gastric carcinoma, also providing theoretic base for clinical gene-therapy in gastric carcinoma.