The Expression And Function Of Ribosomal Protein L6 In Gastric Cancer

【Background】The gene of human ribosomal protein L6(RPL6) was located in chromosome 12.241, gene length was 989bp, molecule weight was 3340kD, encoding 288 amino acid and 6 intron and 6 extron. RPL6 is located in 50S large subunit of ribosome, in the combination site of t-RNA, center of transfer enzyme, linking with the known 23sRNA slightly, playing an catalyzing role in the activies of transfer enzyme. On the surface of protein, there may be some combination site with ribosome material, such as N site may have some relationships with protein-protein reaction, while C site may have link site with RNA. RPL6 expressed in all organs and tissues of human, playing an unsteaded role in ribosome synthesis, transcription and translation.Our previous study found that RPL6 expression was significantly higher in multidrug resistant gastric cancer cell lines than that in gastric cancer cells, further research demonstrated that RPL6 may up-regulate Bcl-2 and down-regulate Bax to inhibit cell apoptosis, and which enhances gastric cancer multidrug-resistant. Then RT-PCR results demonstrated that RPL6 expression in gastric cancer tissues were higher than that in adjacent nonneoplastic tissues, which suggested that RPL6 may play an important role in the tumorigenesis and development of gastric cancer.Previous study demonstrated that RPs played an important role in the tumorigenesis and development of cancer. More and more RPs, such as RPL14, RPS1, RPS7 were up-regulated in tumor, and may have some relationships with tumorigenesis and development of cancer. Otherwise, how is RPL6 expressed in gastric cancer, and whether it involves in the tumorigenesis and development of gastric cancer or not, whether it promotes the tumorigenesis and development of gastric cancer or not, we are not clear, so we designed the present study to explore RPL6 expression and its function in gastric cancer.【Objective】1. To explore the expression of RPL6 in gastric cancer tissues and cell lines.2. To construct and identify RPL6 sense eukaryotic expression vector pcDNA3.1and small interference siRNA vector.3. To further investigate the role of RPL6 in tumorigenesis and development of gastric cancer and its molecule mechanism.【Methods】1. Observation of RPL6 expression in gastric cancer tissues and adjacent nonneoplastic gastric mucosa by immunohistochemistry and analyze clinicopathological association with RPL6 expression statistically.2. Observation of RPL6 expression in gastric cancer cell line AGS, MKN45, SGC7901, MKN28 and the immortalized human gastric mucosa epithelial cell line GES. 3. Construction and identification of RPL6 sense eukarya expression vector pcDNA3.1 and small interference siRNA vector, transfecting RPL6 sense eukarya expression vector pcDNA3.1 and RPL6 small interference siRNA vector into GES cell and RPL6 small interference siRNA vector into SGC7901 cell by Lipofectamine2000, respectively, and to obtain stable clones by G-418 selection.4. Observation of RPL6 expression in transfected cells and the effect of different vectors on transfected cells.5. By MTT assay to draw cell growth curves and observe the effect of different vectors on the proliferation of transfected cells.6. By flow cytometer experiment to analyze cell cycle progression and distribution of transfected cells.7. Plate colony formation assay to observe the colony formation ability of different transfected cells in vivo.8. Nude mouse transplantation experiment to observe the effect of RPL6 on tumor formation of gastric cancer cells in vitro. 9. Western blot experiment to observe cell cycle related molecule P16, P21, P27, cyclinE, cyclinD expression in transfected cells.【Results】1. To explore the expression of RPL6 in gastric cancer tissues and cell lines.(1) RPL6 localized almost in cytoplasm and only occasionally in nuclei in gastric cancer cells, the positive ratio of RPL6 was 90% (54 of 60 patients) in gastric cancer tissues and was significantly higher than 25% (5 of 20 patients) in adjacent non-neoplastic tissues, which demonstrated that RPL6 was over-expressed in gastric cancer tissues (P<0.05).(2) Further statistically analysis of the 60 gastric cancer specimens showed a positive association of RPL6 expression with tumor differentiation, TNMstage and metastasis but not with patient's age and gender (P<0.05).(3) RPL6 expression level in gastric cancer cell lines AGS, MKN45, SGC7901, MKN28 was obviously higher than that in immortalized gastric epithelial cell GES. Interestingly, poorly-differentiated AGS and MKN45 cells exhibited stronger expression of RPL6 than well-differentiated MKN28 and moderately-differentiated SGC7901 cells, which was consistent with the findings that RPL6 in gastric cancer tissues negatively correlated to tumor differentiation.2. To construct and identify RPL6 sense eukaryotic expression vector pcDNA3.1 and small interference siRNA vector.(1) We successfully constructed RPL6 sense eukaryotic expression vector and identified its sequencing.(2) We successfully constructed RPL6 small interference siRNA vector and identified its sequencing.3. To further investigate the role of RPL6 in tumorigenesis and development of gastric cancer and its molecule mechanism.(1) We successfully constructed RPL6 sense eukaryotic expression vector and small interference siRNA vector and transfected them into GES cell by Lipofectamine2000, respectively, while transfected RPL6 small interference siRNA vector into SGC7901 cell by Lipofectamine2000 alone. To attain RPL6 stable expression mix-clones by G-418 selection and western blott results demonstrated that we successfully got RPL6 down-regulated and up-regulated stable cell lines.(2) MTT assay detected growth of variant transfected cell lines, and the results showed that RPL6 up-regulated GES cell, its growth rate was significantly fast, while RPL6 down-regulated GES cell, its growth rate was significantly low; RPL6 down-regulated SGC7901 cell, its growth rate was significantly low.(3) Flow cytometer analyzed cell cycle distribution and cell cycle progression in variant transfected cells, and the results showed that RPL6 up-regulated GES cell, the proliferation index increased, cell cycle G1-S phase transition accelerated, while RPL6 down-regulated GES cell by siRNA, the results was definitely opposite; at the same time, RPL6 down-regulated SGC7901 cell by siRNA, the proliferation index decreased, cell cycle arrested in G1 phase.(4) Plate colony formation assay showed that GES cell transfected with sense eukaryotic expression vector, the growth rate accelerated and colony formation ability in vitro strengthened, which was significantly higher than control cells; while GES and SGC7901 cells transfected with siRNA , the growth rate decelerated and colony formation ability in vitro weaken, which was significantly lower than control cells.(5) Nude mouse transplantation experiments showed that SGC7901 cell transfected with siRNA, the tumor formation ability in vivo was weaken, which was significantly lower than control cells.(6) Western blot experiments detected the expression of cell cycle related proteins, and the results demonstrated that except for cyclinE expression in variant transfected cell lines exhibited differences, all other cell cycle related proteins P16, P21, P27, CDK2 exhibited no differences, otherwise, expression of cyclinE was in accordance with RPL6 expression.【Conclusion】1. RPL6 was over-expressed in gastric cancer tissues and cell lines, and was negatively correlated with differentiation of gastric cancer, which suggested that RPL6 might be involved in the tumorigenesis and development of gastric cancer.2. Up-regulating RPL6 expression could promote GES cell growth and cell cycle progression, ability of colony formation in vitro; while down-regulating RPL6 expression could suppress gastric cancer cell growth and cell cycle progression, ability of colony formation in vitro and tumor formation in vivo weaken, which demonstrated that RPL6 could served as a new target for gastric cancer.3. RPL6 affected cell cycle progression might by regulating cyclinE expression, further affected cell biological behavior, and the molecule mechanism need further exploration.