| 【Background】Chemotherapy is an important means of treatment of gastric cancer, one of the most common malignant tumors in China. Multidrug resistance(MDR) is responsible for the most cases of failure of chemotherapy in patients with gastric cancer. The puzzle of MDR of gastic cancer can’t be solved from the perspective protein-coding genes, whose mechanisms need to be considered at the non-coding gene level. It is statisticed that non-coding RNAs which consist of long non-coding RNAs and small non-coding RNAs account for much greater proportion than that of protein-coding m RNAs in the human transcriptome. Long non-coding RNAs(lnc RNAs) that do not encode proteins are transcripts of more than 200 nucleotides in length. lnc RNAs are involved in the regulation of gene expression through a variety of mechanisms in a protein-coding independent measure. In recent years, lnc RNAs have been shown to play important roles in the regulation of growth, metastasis, and drug resistance of many types of cancers, however, little attention has been paid to the relationship between lnc RNAs and MDR in gastric cancer. In the previous study, high-throughput microarray profilings were applied to obtain a number of differentially expressed lnc RNAs and mi RNAs between SGC7901/ADR, SGC7901/VCR cells and parental SGC7901 cells. In this study, the expression profiling data was analyzed to identify MDR-associated lnc RNAs and mi RNAs, whose expressions and functions would be verified. Moreover, bioinformatic analysis and molecular biology experiments were employed to predict and confirm the lnc RNA/mi RNA interaction, further elucidating its effect on the expression of mi RNA downstream targets, finally revealing the mechanisms of MDR in gastric cancer.【Objectives】1. To screen and identify MDR-associated lnc RNAs in gastric cancer. 2. To study the role of lnc RNA UCA1 in the regulation of MDR of gastric cancer in vitro and in vivo. 3. To elucidate molecular mechanisms of lnc RNA UCA1 in promoting MDR of gastric cancer.【Methods】1. Differentially expressed lncRNAs was abtained by analysis of high-throughput microarray data, molecules. The expression levels of candidate molecules were verified by q RT-PCR. The expression of UCA1 was verified by q RT-PCR in different cell lines and gastric cancer tissues. 2. The role of UCA1 in MDR of gastric cancer was determined by MTT assay. The influence of UCA1 in cell apoptosis was studied by flow cytometry. Subcutaneous xenotransplanted tumor model in nude mice were used to study the role of UCA1 in promoting drug resistance of gastric cancer in vivo. 3. Fluorescence in situ hybridization(FISH) and the cell cytoplasm/nucleus separation experiment were used to determine the sub-cellular localization of UCA1. 4. Bioinformatics analysis was applied to analysis UCA1/mi RNA and UCA1/protein interaction. The UCA1/mi RNA interaction was determined by dual luciferase reporter gene assay and the UCA1/protein interaction by RNA binding protein immunoprecipitation. 5. The expression levels and the roles of let-7e and HMGA2 in MDR of gastric cancer were determined by q RT-PCR, MTT and cell apoptosis detection. western blot verify let-7e relation to the regulation of its target gene HMGA2 the negative. Western blot experimenst were used to explore the role of let-7e and UCA1 in regulating HMGA2 expression. The correlation between UCA1 and HMGA2 in expression leves was analyzed. The remedy experiments were used to prove that the regulatory role of UCA1 in HMGA2 was let-7e-dependent.【Results】1. Through high-throughput microarray, 23 lncRNAs were identified to be upregulated in SGC7901/ADR and SGC7901/VCR cell lines by more than 10 times. The q RT-PCR validation demonstrated UCA1 were significantly upregulated in two strains of MDR cell lines. Similarly, UCA1 was upregulated in the leukemia cell line of MDR. Compare to normal cells, the expression level of UCA1 was higher in gastrointestinal cancer cells such as gastric cancer, colorectal cancer and esophageal cancer. Moreover, the expression levels of UCA1 in gastric cancer tissues were significantly higher than in adjacent normal ones. 2. UCA1 could significantly promote gastric cancer cells to resist the damage caused by chemotherapeutic drugs such as ADR, 5-FU and CDDP, which was associated with reduced cell apoptosis. Likely, in vivo experiments also confirmed that UCA1 could promote MDR of gastric cancer cells. 3. UCA1 was mainly located in the cytoplasm and a small amount of one in the nucleus. 4. UCA1 was predicted to contain let-7a, let-7b, let-7c, let-7d, let-7e and let-7f binding sites by Reg RNA, PITA, FINDTAR3 and RNAhybrid database. By lnc RNAtor database, UCA1 was predicted to have binding potential of the AGO2 protein. Dual luciferase reporter gene assay demonstrated that let-7e could combine with the full-length sequence of UCA1, whose effect was compromised by let-7e binding sites mutation. Relative to a control Ig G antibody, anti-AGO2 antibody could significantly enrich the AGO2-UCA1-let-7e complex.5. HMGA2 was a downstream target of let-7e. HMGA2 was upregulaed and let-7e downregulated in drug-resistant cells of gastric cancer. The overexpression of let-7e or silencing of HMGA2 coule reduce IC50 of ADR, 5-FU and CDDP and increase apoptosis rates induced by chemotherapeutic drugs in SGC7901/ADR cells. Let-7e upregulated while UCA1 downregulated the protein expression of HMGA2. The upregulation of HMGA2 by UCA1 depended on its let-7 binding sites, whose mutation attenuated this effect. Correlation analysis showed that the expression of UCA1 was positively correlated with that of HMGA2.【Conclusion】UCA1 was upregulated in MDR cells of gastric cancer or leukemia, a variety of gastrointestinal cancer cells and gastric cancer tissues. The upregulation of UCA1 played important roles in promoting MDR of gastric cancer cells both in vitro and in vivo. As a ce RNA, UCA1 could combine with let-7 especially let-7e, thereby modulating the derepression of let-7 target HMGA2. The derepression of HMGA2 led to its upregulation, which inhibited apoptosis caused by chemotherapeutic drugs and promoted MDR of gastric cancer cells. In summary, the elucidation of the UCA1-let-7-HMGA2 pathway was expected to provide new molecular targets for reversing MDR of gastric cancer. |
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