| 【Introduction】Multidrug resistance(MDR) of gastric cancer is the major reason for causing the fail of chemotherapy. Therefore, exploring the underlying mechanisms and reversing the taget of MDR are the most important direction in cancer research. The mechanisms of MDR in gastric cancer are complicated, but ATP-binding cassette transporter and micro RNA play an important role in MDR.Our research team study the MDR of gastric cancer for a long time. 1. We established the MDR cell lines of gastric carcinoma. e.g SGC7901/ADR, SGC7901/VCR. 2. By using the differential expression analysis, we compared gene, protein and micro RNA between MDR cell lines of gastric carcinoma and wild-type cell. We found different expression in these two cell lines. 3. Using a variety of molecule and bioinformatics methods, we certified the role of mi R-15 b, mi R-16, let-7, Pr PC, HIFI in GC MDR. 4. We also proved that mi R-19a/b which target PTEN lead to GC MDR.We found MDR of GC mainly relate to the regulation of gene expression, especially epigenetics. Special epigenetics include DNA methylation and histone modification. General epigenetics also include the regulation of micro RNA. The most studies are methylation regulation. Many studies have reported epigenetics play an important role in cancer, but epigenetics is not studied in GC MDR.Previously we used 5-aza-dc to stimulate GC cell SGC7901, and found IC50 changed. Then we used micro RNA microarray to analysis the change of micro RNA after treatment with 5-aza-dc. The most difference is mi R-19a/b. Then by Bioinformatics software analysi-s, we predictd Me CP2 may be the target of mi R-19a/b. Me CP2 is a member of MBDs, and plays an important role in gene methylation. Thus we hypothesis that mi R-19a/b may be an important regulator in MDR of gastric cancer through DNA methylation. And we will discuss this hypothesis next step.【Aims】1. To explore the role of mi R-19a/b in MDR of gastric cancer induced by 5-aza-dc; 2. To predict and prove Me CP2 is the target of mi R-19a/b; 3. To explore the role of Me CP2 in MDR of gastric cancer induced by 5-aza-dc; 4. To verify the relation between mi R-19a/b and Me CP2 in gastric cancer tissues.【Methods】1. To explore the role of mi R-19a/b, we upregulated the expression of mi R-19a/b by transfecting pre-mi R-19a/b into SGC7901,Then we used flow cytometry to analyze cell apoptosis and used MTT assay to measure the half inhibition concentration(IC50) value; 2. To predict and prove the target of mi R-19a/b, we used multiple mi RNA target database, luciferase reporter gene and Western blot; 3. We downregulated and upregulated expression of Me CP2 by RNA interference and overexpression. Then we used MTT assay to analyze IC50 value; 4. To predict Cp G island in promoter region of mi R-19a/b by Cp G Island Searcher; 5. To investigate the role of methylation in MDR, the expression of mi R-19a/b and target molecule by MTT assay, q RT-PCR, Western blot after 5-aza-dc stimulated; 6. To test the role of mi R-19a/b, target molecule in methylation process by transfect inhibitor of mi R-19a/b or Me CP2 si RNA then stimulate by 5-aza-dc; 7. To verify the relation between mi R-19a/b and target molecule by hybrifization in situ and Immunohistochemistry in Gactric cancer tissues.【Results】1. 5-aza-dc can regulate GC MDR through mi R-19a/bWe used 5-aza-dc to stimulate SGC7901, and examined IC50 value by MTT. The IC50 value with 5-aza-dc was increased. Micro RNA microarray and q RT-PCR showed that the expression of mi R-19a/b were upregulated, We transfected pre-mi R-19a/b into SGC7901. Then MTT assay and flow cytometry results showed that compared with negative control,the IC50 value of SGC7901 transfected with pre-mi R-19a/b was siginificantly increased,cell apoptosis of SGC7901 transfected with pre-mi R-19a/b was siginificiantly decreased. We stimulated SGC7901 with or without treatment by mi R-19a/b inhibitor by 5-aza-dc. MTT showed that mi R-19a/b inhibitor downregulated the IC50 value of SGC7901 stimulated by 5-aza-dc. These indicated that 5-aza-dc can regulate GC MDR through mi R-19a/b.2. Me CP2 is the target of mi R-19/bMany multiple mi RNA target database predicted Me CP2 was the target of mi R-19/b. Luciferase reporter gene, and Western blot verified mi R-19a/b could target and inhibit Me CP2. The expression of Me CP2 in SGC7901 were higher than in SGC7901/ADR, SGC7901/VCR.3. 5-aza-dc can regulate GC MDR through MeCP2We used 5-aza-dc to stimulate SGC7901. Western blot showed that the expression of Me CP2 was inhibited by 5-aza-dc, then stimulated by 5-aza-dc after treating SGC7901 with mi R-19a/b inhibitor, 5-aza-dc didn’t downregulate the expression of Me CP2. Over-expression vector of Me CP2 upregulated the IC50 value in SGC7901, Me CP2 si RNA downregulated the IC50 value was upregulatedin SGC7901. These results indicated that Me CP2 reverse GC MDR. Me CP2 si RNA upregulated expression of mi R-19a/b. We stimulate SGC7901 with or without treatment by Me CP2 si RNA then stimulated by 5-aza-dc; Me CP2 si RNA upregulated expression of mi R-19a/b and the IC50 value. These data indicated that 5-aza-dc can regulate GC MDR through mi R-19a/b and Me CP2.4. mi R-19a/b and MeCP2 were negative correlation in Gastric cancer tissuesWe used hybrifization in situ to examine expression of mi R-19a/b in gastric cancer tissues. We found expression of mi R-19a/b in Gastric cancer tissues were higher than in adjacent tissues. We test the expressin of Me CP2 by immunohistochemistry, the results showed that the expression of Me CP2 in gastric cancer tissues were lower than in adjancent tissues. And there were negative correlation between mi R-19a/b and Me CP2.【Conclusion】Demethylation treatment can promote the MDR phenotype of GC, mi R19a/b-Me CP2 can regulate GC MDR through methyalation. Our study provide a useful supplement for the MDR of GC, and these results provide theory evidences to explore the new targets of gastric cancer MDR for us. |
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