| Background:Gastric cancer is molecular disease caused by activation of different oncogene and/orinactivation of anti-oncogene in different periods. The mechanisms may involve mutationof multiple genes on different chromosomes, among which the activation of telomeraseplays a significant role in the development of gastric cancer. Telomerase expressed in morethan85%of cancer tissues, whereas not expressed or low expressed in normal somatic cells.Human telomerase reverse transcriptase,(hTERT), as the rate-limiting subunit oftelomerase, directly determines the activity of telomerase. Therefore, the activation oftelomerase caused by hTERT’s activation is an important part in tumorigenesis. Ourprevious studies mainly focused on miRNA-related gastric hTERT regulation, whilenon-coding RNA has increasingly got much focus on post-translational modifications ingene by its abundant regulating methods. Now, a significant portion of the noncodingtranscriptome, including long noncoding RNAs and pseudogenes, harbors microRNA(miRNA)-response elements (MRE). A totally new regulation pattern of gene expressionwas put forward recently, namely, competing endogenous RNAs. That is to say, lncRNA,false gene transcripts and mRNA transcripts can competitively combine with miRNAresponse element (MRE) to influence the regulation of gene after transcription. This studywas aimed to explore lncRNA’s regulation of gastric cancer hTERT and its function.Methods：1. lncRNA gene chip technology was applied to screen lncRNA which may beinvolved in regulation of hTERT;2. Bioinformatics technique was used to predict the possible lncRNA which might playa role in regulating hTERT; 3. Real-time quantitative PCR (real-time PCR) analysis was performed to validatelncRNA may be regulated by selected hTERT;4. The specific lncRNA siRNA oligo was transiently transfected into gastric cancercells and the lentivirus loading shRNA-lncRNA was used to infect the cells todown-regulate the lncRNA expression level;5. MTT assay, flow cytometry, colony formation assay, wound healing assay and theinvasion assay and tumorigenicity in nude mice experiments were applied to analyze thefunction of lncRNA regulating hTERT;6. Bioinformatics technique was used to predict the possible miRNA which mightinteract with lncRNA;7. The probable lncRNA and miRNA targeting hTERT was verified by dual luciferasereporter assay;8. Western blot was applied to validate the regulating role of lncRNA and miRNA ontarget genes of hTERT protein content;9. TRAP was used to detect the regulating role of lncRNA and miRNA on target genesof hTERT protein activity.10. Real-timePCR was applied to validate the content of miR-1207-5p after overexpression of lncRNABC032469.11. RNA dot blot test showed that lncRNA BC032469and hTERT3’UTR which bothcan interact with miR-1207-5p directly.Results:1. Prognosis and certification of lncRNA regulating hTERT.There are4620lncRNAs varied a lot in comparing the hTERT-positive gastric cancertissues and hTERT-negative normal pericarcinous tissues in gene chip screening, and1134of which were up-regulated more than5-fold or more in hTERT-positive tissues.Bioinformatics predicted that twelve lncRNAs near the fifth genome hTERT, andreal-time PCR was used to verify five pairs of gastric cancer and corresponding adjacenttissues, and8lncRNAs were screened out which expressed significantly higher inhTERT-positive gastric cancer tissues than in hTERT-negative adjacent mucosa. Comparethose8lncRNAs mentioned above with lncRNA gene chip results, and we found that there were3of them overlapped each other, and the most obvious difference is lncRNABC032469with differential expression of20.566.2. Expression and clinical significance of lncRNA BC032469in gastric cancer tissue.Real-time PCR results showed lncRNA BC032469in cancer tissues was significantlyhigher than in adjacent gastric tissue and the ratio of lncRNA BC032469’s relative amountin gastric cancer and adjacent tissues(C/P) had a relation with hTERT protein’s relativeamount in corresponding gastric cancer and adjacent tissues (C/P), p <0.01Further analysis was made of the ratio of lncRNA’s relative amount in58cases ofgastric cancer and adjacent tissues(C/P) was related the size of tumor (p <0.05); and it’salso associated with the degree of tumor’s differentiation (p <0.05).Survival analysis showed survival time of patients after surgery from lncRNABC032469C/P> C/P group decreased comparing with the patients from lncRNABC032469(C/P <C/P group, but there’s no significant statistical difference, maybebecause of short follow-up period and higher5-year survival rate of gastric cancer.3. Functional study of lncRNA BC032469in gastric cancer cell.By loading lentiviral shRNA-BC032469to infect MKN28and SGC-7901cellssuccessfully, MTT experiments show that decreased lncRNA BC03249can inhibit MKN28and SGC-7901cell proliferation (p <0.01), and flow cytometry showed down-regulatinglncRNA BC032469can block MKN28and SGC-7901cell cycle in G1phase; colonyformation assay showed that down-regulating lncRNA BC032469can inhibit SGC-7901cells’ coloning ability; scratch experiments showed down-regulating lncRNA BC032469can inhibit MKN28and SGC-7901cells in vitro migration; Transwell invasion assayshowed down-regulating lncRNA BC032469had no influence on MKN28and SGC-7901cells in vitro invasion. Tumor formation in nude mice of interference group of lncRNABC032469was lower than the negative controlled group.4. Molecular mechanism of lncRNA BC032469regulating hTERT expression ofgastric cancerIn our previous study, we found five miRNAs（miR-138, miR-491-5p, miR-1182,miR-1207-5p, miR-1266）related to regulation of gastric cancer hTERT. It was furtherverified in PCR experiments that the differences of these five miRNAs in58pairs of gastriccarcinoma and adjacent tissue expression, among these miRNAs, the abundance of miR-1207-5p, miR-1266in hTERT-negative cancer adjacent normal mucosa weresignificantly higher than that in hTERT-positive gastric cancer.（p<0.05）Bioinformatics predicted that in lncRNA BC032469sequence, there were multiplemiR-1266and miR-1207-5p seed region binding sites with high score. Western blot resultsshowed in gastric cancer cells interfering lncRNA BC032469could down-regulateexpression of hTERT, on this basis, forced expression of hTERT ORF region (lackinghTERT3’UTR) can recover the downregulation of hTERT protein. Dual-Luciferase foundthat miR-1266, miR-1207-5p can down-regulate the expression of hTERT3’UTR luciferase.Combining the results from western blot experiments, interferring lncRNA work byaffecting hTERT3’UTR. It’s speculated that perhaps lncRNA BC032469interact withmiR-1266and miR-1207-5p to regulate the expression of hTERT.After lncRNA BC032469was disturbed, the activity of hTERT3’UTR luciferasedecreased, meanwhile, transfecting anti-miR-1207-5p on hTERT3’UTR luciferase wasweakened; and transfected with anti-miR-1266did not significantly down-regulate theexpression of hTERT3’UTR fluorescence luciferase. Based on the disturbance of lncRNABC032469, transfection of mutated miR-1207-5p binding sites in hTERT3’UTR luciferasereporter gene, luciferase activty significantly increased; transfected with mutated miR-1266binding sites of hTERT3’UTR luciferase reporter gene, down-regulating of luciferase wasnot weakened.Western blot results showed when interfering lncRNA BC032469, antagonizingmiR-1207-5p can partially recover the expression of hTERT protein. However,antagonizing miR-1266can not recover the expression of hTERT protein. TRAP resultsshowed that the interference of lncRNA BC032469could decrease the activity ofSGC-7901cell hTERT, on the basis of interfering lncRNA BC032469, exogenouslyantagonize the miR-1207-5p, may be partially recover the activity of hTERT protein.Real-timePCR found that over expression of lncRNABC032469, decreased the contentof miR-1207-5p, suggesting that lncRNA BC032469may promote the degradation ofmiR-1207-5p.The RNA dot blot test showed that lncRNA BC032469and hTERT3’UTR can bedirectly combined with miR-1207-5p, prove that lncRNABC032469can compete withmiR-1207-5p, which part of the lifting of the inhibition of miR-1207-5p on hTERT. Conclusion:1. By biochip and bioinformatics, it was confirmed for the first time that lncRNABC032469was involved in the regulation of gastric cancer cell hTERT and it was closelyrelated to poor prognosis for patients with gastric cancer.2. Functional study showed that lowering lncRNA BC032469can obviously inhibitgastric cancer cell proliferation, cell cycle, cloning and migration, and restrain cancer cellproliferation in vivo, and play a carcinogenic role in gastric cancer.3. lncRNA BC032469could possibly remove the negative regulation of hTERTthrough blocking miR-1207-5p, to promote the increase of hTERT protein content andactivity, and then promote gastric cancer cell proliferation and migration. Illustrate newmechanism in the development of gastric cancer from the angle of lncRNA.Above mentioned showed that lncRNA BC032469was involved regulating theexpression of gastric cancer cell hTERT, and it was closely related to prognosis for gastriccancer. It provided a new target for treatment of gastric cancer. |
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