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[Background] human ribosomal protein L6 (RPL6) whose gene is located on chromosome 12, two District 4 with a sub-band gene is 989bp, molecular weight 33kD, encoding 288 amino acids, there are six introns and six foreign exons. RPL6 in 50S ribosomal large subunit, the positioning in the aminoacyl-tRNA binding site, peptidyl transferase center, with known 23sRNA closely, the peptidyl transferase activity has an important catalyst. On its surface proteins, may exist with the other components of the ribosome binding sites of interaction. Such as N-terminal proteins and protein interaction, whereas the C-terminal may be a RNA binding site. RPL6 widely distributed in various tissues of the human body, in the formation of ribosomes, transcription and translation process plays an irreplaceable role. Our previous studies have found, RPL6 resistant cells in gastric cancer was significantly higher than that in gastric cancer cells, further research showed that RPL6 by upregulating Bcl-2 and Bax decreased inhibition of apoptosis of gastric cancer cells to promote multi- drug resistance. Further RT-PCR results showed that, RPL6 in gastric cancer tissues was significantly higher than that in adjacent tissues, which suggests that, RPL6 in the occurrence and development of gastric cancer may play an important role. Past studies have shown that ribosomal protein in tumor occurrence and development plays an important role. Many ribosomal proteins, such as RPL14, RPS1, RPS7 were all elevated expression in tumors, and tumor occurrence and development are closely related. Well, RPL6 in gastric cancer is like, which is not with the occurrence and development of gastric cancer is closely related to the incidence of gastric cancer or promote and develop it, we are still unclear, so the design of the experiment, it will RPL6 in gastric carcinoma and its functional studies were discussed. [Objective] 1. Explore RPL6 in gastric cancer tissues and cells. 2.RPL6 justice eukaryotic expression vector pcDNA3.1 and small interfering siRNA vector and identification. 3 further explore RPL6 in gastric carcinogenesis and its further role in the development of the molecular mechanism. [Methods] 1. Immunohistochemical observation RPL6 in gastric carcinoma and non-tumor tissues in normal gastric mucosa, and RPL6 expression and the relationship between gastric cancer clinical and pathological data were statistically analyzed. 2.Western blot RPL6 detected in gastric cancer cell lines AGS, MKN45, SGC7901, MKN28 and immortalized normal gastric mucosa cells GES expression. 3.RPL6 justice eukaryotic expression vector pcDNA3.1 and small interfering siRNA vector and identification, and through liposome respectively RPL6 justice eukaryotic expression vector pcDNA3.1 and small interfering GES siRNA vector transfected into cells, RPL6 small interfering siRNA transfected into SGC7901 cells by G-418 screening to obtain stable clones. 4.Western blot RPL6 were detected in the above expression in transfected cell lines to observe the effect of different vector transfection. 5.MTT France cell growth curve, the difference observed RPL6 vector transfection on cell proliferation. 6 Flow cytometry analysis of transfected cell cycle distribution and periodic progress. 7 colony forming assay the effects of different vector transfected cells in vitro colony formation. 8 RPL6 nude mice experimental observation on gastric cancer cells in vivo tumorigenicity impact. 9.Western blot RPL6 detected in transfected cells in the above cycle related molecules (P16, P21, P27, CDK2 and cyclinE) in the expression. [Results] 1. Explore RPL6 in gastric cancer and cells. 1) RPL6 in 60 cases of primary gastric carcinoma cells in the cytoplasm of the positive rate can reach 90% (50/60), significantly higher than the non-neoplastic mucosa 25% (5/20), suggesting RPL6 in high expression in gastric cancer (P lt; 0.05). 2) Further statistical analysis revealed that overexpression RPL6 with the patient's age and gender were not significantly different, but not with the degree of tumor differentiation, TNM stage and metastasis were significantly different (P lt; 0.05). 3) RPL6 in gastric cancer cell lines AGS, MKN45, SGC7901, MKN28 was significantly higher than that of normal gastric GES immortalized cells. RPL6 in low differentiated gastric cancer cell lines AGS and MKN45 Expression of SGC7901 were higher than moderately differentiated and higher differentiation MKN28 cells, suggesting RPL6 expression is closely related to the degree of tumor differentiation, and with immunohistochemical results. 2.RPL6 justice eukaryotic expression vector pcDNA3.1 and small interfering siRNA vector and identification. 1) RPL6 successfully constructed the eukaryotic expression vector justice and confirmed its sequenced correctly. 2) successfully constructed RPL6 small interfering siRNA vector and confirmed its sequenced correctly. 3 further explore RPL6 in gastric carcinogenesis and its further role in the development of the molecular mechanism. 1) constructed RPL6 justice eukaryotic expression vector pcDNA3.1 and small interfering siRNA vectors, and through liposome Lipofectamine2000 respectively RPL6 justice eukaryotic expression vector pcDNA3.1 and small interfering siRNA vector transfected into GES cells, RPL6 small interfering siRNA vector transfected into SGC7901 cells were screened through a G-418 hybrid clones stably expressing, Western blot results confirmed RPL6 upregulation successfully obtained stable cell lines and down. 2) MTT method for detection of different cell lines transfected cells, the results show up RPL6 expression GES cells, which grow significantly faster, down RPL6 expression GES cells, the growth rate slowed down; while the expression of SGC7901 down RPL6 cells, the growth rate slowed down. 3) Flow-cycle analysis of transfected cell lines of different cell cycle distribution and cell cycle progression, the results show increased expression GES RPL6 cells, increased cell proliferation index, cell cycle G1-S phase transition speed, down through siRNA RPL6 GES expressed in cells, which results contrary; expression through siRNA down RPL6 SGC7901 cells, the cell proliferation index decreases, cell cycle arrest in G1 phase. 4) colony formation assay showed that justice GES transfected cell growth accelerated, and its ability to enhance colony formation in vitro, was significantly higher; while siRNA transfection GES and SGC7901 cell growth slowed, in vitro clonogenic diminished capacity, significantly lower than that of the control group. 5) nude mice experiments showed, siRNA transfected SGC7901 cells, in vivo tumorigenicity diminished capacity, significantly lower than the control group. 6) Western blot assay of cell cycle-related protein expression changes in circumstances, the results show in addition to cyclin cyclinE in different cell lines transfected with the obvious differences, the rest of the cell cycle related protein, P16, P21, P27, CDK2 in different cell lines transfected with no significant differences and changes cyclinE consistent with RPL6. [Conclusion] 1.RPL6 in gastric cancer tissues and cells showed high expression, and negatively correlated with tumor differentiation, suggesting their involvement in the occurrence and development of gastric cancer. (2) increase RPL6 GES expression can promote cell growth and cell cycle progression in vitro clonogenic capacity enhancement, down RPL6 expression inhibits cancer cell growth and cell cycle progression, reduced colony formation in vitro and in vivo tumorigenicity diminished capacity, Tip RPL6 can be used as a new target for cancer therapy. 3.RPL6 may regulate the expression of cyclinE affect cell cycle progression, thereby affecting the biological behavior of cells, but the molecular mechanism needs further study.
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