| Objective: The study displays that long non-coding RNA(Long non-coding RNA,Lnc RNA)is closely related to the tumor's occurrence and development.The study first determines that Lnc RNA RP1-163G9.1 is aberrant expression in gastric carcinoma patients.We carry out the research about the relationgship between abnormal expression and clinicopathological parameters in cell and animal level.Methods: 1.According to the data of Lnc RNA expression profiles by our subjects' group,analysis of the expression data and review the literature,the Lnc RNA is first studied by us and the differentiated expression was statistically significant.2.The detection of expression levels about the Lnc RNA RP1-163G9.1 in tissues and cells: GADPH as reference gene,detecting the expression levels in the gastric carcinoma and corresponding adjacent tissues with using real-time quantitative PCR,analysis of the relationship between the expression levels and clinicopathological data.3.The detection of expression about the RP1-163G9.1 in situ: designing the fluorescence hybridization probe of Lnc RNA RP1-163G9.1 and detecting cellular localization and expression levels of RP1-163G9.1 by using fluorescence in situ hybridization(FISH)in 30 cases of gastric carcinoma paraffin-embedded tissue.4.The functional research at a cellular level: Constructing of eukaryotic expression vector of pc DNA3.1-Lnc RNA RP1-163G9.1,screening the cell line which was expressed stably Lnc RNA RP1-163G9.1 and corresponding empty vector by G418 in gastric cancer cells BGC 823 and SGC7901.We study the proliferation and invasion with CCK-8 proliferation assay,Transwell experiments,colony formation assay and detect the apoptosis and analysis cell cycle by flow cytometry5.Animal experiments: BALB/c-nu nude mice,4-6W,were selectedand inoculated the gastric cells which stably expressed Lnc RNA RP1-163G9.1 and corresponding empty vector in outer arm subcutaneous,8*106/0.1ml/per.And then tumor volume was measured and growth curve was drawed every seventh day.The mice were killed at the end of the experiment and then isolating tumor tissue,measuring the volume and weight,analysis the tumor growth between Lnc RNA RP1-163G9.1 group and corresponding empty vector group by t test statistical.The tumor RNA was extracted for detecting the expression of Lnc RNA RP1-163G9.1 by qRT-PCR.At the same time,the tumor tissue HE staining was carried to determine the nature of the tumor.Results: 1.Compared with adjacent tissues,Lnc RNA RP1-163G9.1 is downregulated andthe ratio reaches to 140.67 in gastric adenocarcinoma.2.qRT-PCR results: Detecting 112 gastric adenocarcinoma and adjacent tissues,we found that the expression of Lnc RNA RP1-163G9.1 in gastric cancer tissues was significantly decreased(p <0.001),the rate of downrehulation was 75.89% and the times was 23.94.The expression of the Lnc RNA RP1-163G9.1 in gastric cancer cell BGC823,SGC7901 and AGS also showed downregulation(P <0.001),compared with normal gastric epithelial cells GES-1.3.The correlation between clinicopathological parametersand immunocytochemistry markers: Chi-square statistical analysis showed that the expression of Lnc RNA RP1-163G9.1 was related to the depth of invasion(p =0.001),lymph node metastasis(p = 0.001),tumor size(p = 0.028),and immunocytochemistry markers TS(p=0.040)and Ki-67(p=0.017).Multivariate Logic Regression statistical analysis showed that high expression of Lnc RNA RP1-163G9.1 possible inhibit of tumor metastasis,invasion and tumor size.4.In situ hybridization: The results of fluorescence in situ hybridization experiments which come from 30 pairs of gastric carcinoma paraffin-embedded sections show that Lnc RNA RP1-163G9.1 is mainly located in the cytoplasm and the expression in the gastric carcinoma is lower than normal gastric tissues,which also further validates qRT-PCR results.5.The experiment of cell function: The results which come from CCK-8 proliferation assay,transwell experiments,colony formation assay by the use of gastric cancer cell line SGC7901 and BGC 823 that stablly expressed the RP1-163G9.1 and the corresponding empty vector showthe overexpression of Lnc RNA RP1-163G9.1can significantly inhibit cell proliferation.Flow cytometric analysis of cell cycle showed that cell lines stably expressing cycle Lnc RNA RP1-163G9.1change ratio of S stage,which is lower in the experiment group Lnc RNA RP1-163G9.1,compared with the corresponding empty vector group.Apoptosis detection displays that ratio of early and late apoptosis has no difference in the experiment group and control group.6.Animal experiments:T test statistics shows that Lnc RNA RP1-163G9.1 tumor growth curve and final tumor weight are significantly lower than the experimental group(p<0.05),and the difference is statistically significant.Conclusion: 1.The low expression of Lnc RNA RP1-163G9.1 in gastric adenocarcinoma and gastric cancer cells are related with gastric cancer invasion,lymph node metastasis and tumor size 2.Lnc RNA RP1-163G9.1 mainly expressed in the cytoplasm and is lower expressed in gastric carcinoma,compared with normal gastric tissues.3.Cell and animal experiments show that Lnc RNA RP1-163G9.1 mainly effect the proliferation and invasion of gastric cancer.4.Lnc RNA RP1-163G9.1 was related with proliferation of gastric cancer which may be used as a potential prognostic indicator. |
PDF Full Text Request