| BackgroundChemotherapy resistance is a major cause of cancer-related death during the treatment,but the molecular mechanism of tumor chemoresistance is not clear. Long non-coding RNA(lncRNA) is a kind of non-coding RNA,which length is more than 200nt. At present,more and more studies showed that lncRNA plays an important role in the process of tumor progression. The mechanism of lncRNA is complex, recent studies showed that lncRNA can absorb microRNA to regulate gene expression indirectly ; IncRNA could also absorb protein,to regulate protein levels by protein modification; lncRNA, enriched in the nucleus, could regulate the gene expression by recruiting or hindering some nuclear transcription factor;IncRNA can also realizethe nucleosome space construct to change gene expression. The regulatory mechanism of lncRNA, makes IncRNA play various functions in the process of tumor development. It has been reported that some lncRNA are involved in the tumor chemotherapy resistance. Therefore, study the key role of IncRNA in tumor chemotherapy resistance, will be able to clarify new mechanism of tumor chemotherapy resistance, tumor progression, to provide new strategies and drug targets for clinical treatment.Objective:This study was to screen the high expression of IncRNA in gastric cancer,and to analyze its role in the gastric cancer chemotherapy resistance.Methods(1) lncRNA chip was used to screen the high expression of lncRNA in gastric cancer tissue; (2)The expression level of lncRNA in 60 patients with gastric cancer was detected by qRT-PCR technique, and the clinical significance of the lncRNA was analyzed by combining with clinical pathological data;(3)The expression of lncRNA in different gastric cancer cells was detected, and interfering lentivirus was used to decrease lncRNA in gastric cancer cells;(4)CCK8 assay, Transwell assay, scratch assay, flow cytometry assay, subcutaneous tumor model, peritoneal metastasis model, lung metastasis model were used to detect the function of lncRNA;(5) The localization of lncRNA in gastric cancer cells and gastric cancer tissues was detected by in situ hybridization;(6)The specific functions of the lncRNA were analyzed by the expression profile chip;(7) RNA pull-down technology was used to detect the proteins that bind to the lncRNA;(8) The effect of lncRNA expression on gastric cancer cell sensitivity to oxaliplatin was detected.Result:(1)Screening thehigh expression of lncRNA in gastric cancer tissues: lncRNA chip was used to screen high expressed lncRNA in gastric cancer tissues,and 15 highly expressed in gastric cancer tissues the lncRNA were picked. At the same time,qRT-PCR was used to detect expression of 15 lncRNA in 10 cases of gastric cancer tissue and adjacent tissue.Finally,IncRNA AK095203 was chose according to the experiment.(2)The expression and clinical significance oflncRNA AK095203: qRT-PCR was used to detection expression of IncRNA AK095203in 60 cases of gastric cancer tissues and adjacent tissues, we found that lncRNA AK095203 was high expressed in gastric cancer tissue,P=0.00662.Expression of lncRNA and tumor size was positively related, while the differentiation of tumor and the expression of IncRNAwasnegative correlated.(3)The effect of lncRNA AK095203 on gastric cancer cell behavior: Gastric cancer cell lines SGC7901 and BGC823 were used as the cell model, CCK8 showed low expression of lncRNA AK095203 and gastric cancer cell proliferation was obviously inhibited; flow cytometry showed that interfering expression of lncRNA AK095203 could preventgastric cancer cells enter the G2 period; Transwell assay and scratch assay demonstrated that interference of IncRNA AK095203 can significantly inhibit the migration and invasion of gastric cancer cells; subcutaneous tumor formation experiments, peritoneal metastasis experiment, lung metastasis experiments proved evidence that interfering expression of IncRNA AK095203 could inhibit the proliferation and invasion of gastric cancer cells.(4)Localization of lncRNA AK095203 in gastric cancer cells and tissues: we foundthat IncRNA AK095203 mainly in nuclear enrichment by in situ hybridization.(5)Specific function of lncRNA AK095203 was analyzed by microarray: Microarray showed that cell division related genes and DNA damage repair related geneswere significantly inhibited, when the expression of IncRNA AK095203 in gastric cancer was decreased, indicating that the lncRNA AK095203 was closely related with cell division and DNA damage repair.(6) RNA pull-down experiments demonstrated that lncRNA AK095203 could bindSSRP1 and H2A / H2B.SSRP1 is closely related with gastric cancer oxaliplatin induced DNA damage repair, indicating that IncRNA AK095203 may cause oxaliplatin resistance of gastric cancer by binding with SSRP1.(7)Sensitivity of SGC7901 and BGC823to oxaliplatin was significantly increased after interfering expression of lncRNA AK095203.Conclusion:(1) Through lncRNA chips and qRT-PCR test, we found lncRNA AK095203 was highly expressed in gastric cancer,and the expression of lncRNA AK095203 is closely related to the size and differentiation degree of gastric tumor, by clinical pathological data analysis.(2) By transfecting lentivirus, we obtained two cell lines in which the expression of IncRNA AK095203 was stablely inhibited. Combined in vitro and in vivo experiments, we found that the ability of proliferation and invasion of gastric cancer cells were significantly inhibited after the expression of lncRNA AK095203 was decreased.(3) According to the analysis of expression profile chip, we found that lncRNA AK095203 level is significantly related to cell division and DNA damage repair process.(4) Through RNA pull-down experiment and in situ hybridization combine IHC experiment, we demonstrated that lncRNA AK095203 can integrate with SSRP1 and H2A/H2B, Reducing the expression of lncRNA AK095203 can significantly increase GC cells’ susceptibility to oxaliplatin.BackgroundIn the first part of study, we found that the expression of lncRNA AK095203 was the most significant lncRNA, according to the results of lncRNA chip. We also found that the tumor size and the differentiation degree of gastric cancer are closely associated with the expression of lncRNA AK095203, through 60 clinical specimens by using qRT-PCR detection.What’s more,we initially found that lncRNA AK095203 may be involved in the function of DNA damage repair by binding SSRP1 and H2A / H2B, according to RNA pull-down experiments. IncRNA AK095203 may promote the gastric cancer oxaliplatin resistance in this manner.Oxaliplatin is a kind of third generation platinum chemotherapy drugs,which has been widely used in clinical cancer therapy. But the treatment of some patients is not perfect due to the drug resistance, leading to the result of malignant proliferation, invasion and metastasis of gastric cancer, which is the main cause of gastric cancer death in patients. To solve this clinical problem, researchers try to find out the specific molecular mechanisms of oxaliplatin-resistant in different kinds of cancer. In order to achieve the purpose of treating cancer, new anticancer drugs are also being synthesized constantly. In these new drugs,nanomedicine has been widespread concerned by researchers, due to its own unique properties such as their size, surface charge, surface area, etc.Cerium oxide nanoparticles (CNPs) is a kind of metal compound nanomaterials. Cerium ions can be converted in the +3 and +4, which makes CNPs owns the unique ability of binding / release reactive oxygen species (ROS). ROS is a kind of important intracellular second messengers, which involved in a variety of life activities in vivo. CNPs have got a great concern in the medical field because of its unique ability of absorb / release ROS. Pesic et al found that cerium oxide nanoparticles has a significant cytotoxic effect on the colon cancer cell line HT-29 and melanoma cell line 518A2, but CNPs showed no cytotoxic effect on normal cells,suggesting that CNPs may serve as an effective potential treatment of melanoma and colon cancer. In addition, in the study of lung cancer, it also found that CNPs could increase the cytotoxic effect and reactive oxygen species level to lung cancer cells, and thus play a role in. What’s more, this cytotoxic effect of CNPs was positively correlated with the concentration and co-culture time. Recently, researchers developed a drug delivery system,by taking the advantage of sensitive nature for the ROS of CNPs. This system can carry a different drug into the cell to effect on tumor. Above documents show that cerium oxide nanoparticles have the potential properties of treating cancer. However,whether CNPs can play a role in the treatment of gastric remains unclear.Objective:This study aims to find out that whether cerium oxide nanoparticles can play an efficient treatment to gastric cancer, and the possible mechanism of how CNPs functioned.Methods and Materials:(1) Transwell invasion assay and CCK 8 proliferation assay were used to detect the invasion and proliferation ability of gastric cancer cell lines BGC823 and MKN28, after co-cultured with different concentrations of cerium oxide nanoparticles (0ug / ml, 0.01ug / ml,0.1ug/ml, 0.5ug/ml,1ug/ml,10ug/ml).(2) Microarray, qRT-PCR, Western blot experiments were used to find out the most obvious expression of genes in MKN28, which were co-cultured with different concentrations of cerium oxide nanoparticles.(3) Western blot and Immunofluorescence were used to detect the possible mechanism of the gene.(4) Two nude mice models, subcutaneous tumor model and peritoneal metastasis model,were used to detect the effect of cerium oxide nanoparticles on the proliferation and invasion ability of gastric cancer cells in vivo.Results:(1) Every concentration of cerium oxide nanoparticles can suppress the ability to migrate of gastric cancer cell lines MKN28 and BGC823, but only high concentration (10ug / ml) may inhibit proliferation of MKN28 and BGC823.(2) After the co-cultured with gastric cancer cells, cerium oxide nanoparticles mainly enriched in cell lysosomes.(3) Cerium oxide nanoparticles mainly upregulate DEAH-box helicase 15 (DEAH-box helicase 15, DHX15) of the gastric cancer cells.(4) DHX15 can increase p38 expression, but p38 was localized in the cytoplasm.(5) Subcutaneous tumor model showed that high concentrations of cerium oxide nanoparticles can inhibit the proliferation of gastric cancer cells, but low concentration of cerium oxide nanoparticles has no effect on the proliferation.(6) Peritoneal metastasis model in nude mice showed that cerium oxide nanoparticles could inhibit the peritoneal metastasis ability of gastric cancer cells in a dose-independent manner.Conclusions:(1) Low concentrations of cerium oxide nanoparticles can inhibit the invasion ability of gastric cancer cells and high concentrations of cerium oxide nanoparticles may inhibit the proliferation and invasion ability of gastric cancer cells.(2) Cerium oxide nanoparticles may achieve the above functions by promoting the expression of DHX15. |
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