MiR-1182Inhibits HTERT Through Binding To Its Open Reading Frame In Gastric Cancer Cells

Objective：Telomerase， a ribonucleoprotein enzyme that consists of telomerase reversetranscriptase (TERT) and a template-containing RNA component (TR), is responsible formaintaining the integrity of chromosomal ends. Telomerase activity is characterized by theexpression of the TERT, indicating TERT acts as the major limiting agent for telomeraseactivity. Most tumors acquire the immortal ability by increasing the expression of TERT.Nowadays, hTERT has become a crucial target of tumor treatment. So it is of greatsignificance to understand the regulatory mechanisms of hTERT. Many researches havefocused on hTERT regulation in transcriptional level, while the regulation ofpost-transcriptional level, such as the modulation of miRNAs, has not been exploredenough. microRNAs (miRNAs) are small endogenous nucleotide RNAs that play animportant role in most biological processes via regulating their target genes. To the best ofour knowledge, miR-138is the only miRNA that has been reported to directly target hTERT3’UTR and down-regulate its expression. As a gene could be targeted by different miRNAs,we hypothesize there maybe some other miRNAs target hTERT. In our previous study,through the combination of miRNA expression profling with bioinformatics analyses andqRT-PCR, we identifed nine miRNAs target hTERT potentially. Western blot suggested thedown-regulation of hTERT by miR-1266, miR-1207-5p and miR-1182. Dual-luciferaseassays revealed that only miR-1266and miR-1207-5p had functional binding sites inhTERT3’UTR, while miR-1182could not target hTERT3’UTR. In recent years, a growingnumber of evidences demonstrated the existence of MREs (miRNA response elements) inthe open reading frame (ORF). Thus we wonder whether miR-1182modulates hTERTthrough binding to some other regions, such as ORF. In the present study, we focus on themechanism of how miR-1182regulates hTERT, and discuss the dysregulation of miR-1182in gastric cancer cells and its clinical relevance. Methods：Part I:1. Quantitative real-time PCR was performed to investigate the relative expression ofmiR-1182in gastric cancer cell lines.2. Western blot was used to detect the protein level of hTERT after treatment ofmiR-1182mimics.3. Dual-luciferase assay was applied to assay the effect of miR-1182on the activity ofhTERT ORF.4. Bioinformatics (miRanda v3.3a software) was employed to predict the potentialbinding sites of miR-1182in hTERT ORF, and the conversation of the binding sites wasalso testified among several species.5. Dual luciferase reporter assay was carried out to further identify the effectivebinding site of miR-1182in hTERT ORF.6. ORF1synonymous mutation and western blot were conducted to further confirm therecognition sites for miR-1182located in hTERT ORF1.Part II:1. Quantitative real-time PCR was employed to detect the expression level ofmiR-1182in cancer tissues and corresponding adjacent tissues of40gastric cancer patients.2. The relationship between miR-1182and hTERT was testified in cancer tissues andcorresponding adjacent tissues of40gastric cancer patients.3. CCK-8assay was used to analyze the effect of miR-1182on cell proliferation invitro.4. Transwell migration assay was performed to explore the influence of miR-1182onmigration of gastric caner cells in vitro.5. Gastric cancer xenografts were employed to testify the inhibition of tumorproliferation by miR-1182in vivo.6. Tail vein metastatic assay was conducted to detect the suppression of tumormetastasis by miR-1182in vivo.Results：Part I:1. Quantitative real-time PCR indicated the relatively low-expression of miR-1182inMKN28and HGC-27cells among gastric cancer cells. 2. The protein level of hTERT was decreased in the gastric cancer cells aftermiR-1182treatment.3. Dual-luciferase assay demonstrated that miR-1182effectively repressed the activityof hTERT ORF.4. miRanda v3.3a software predicted there were two putative binding sites ofmiR-1182within hTERT ORF (ORF1and ORF2). What’s more, the miR-1182targetsequences, especially the ‘seed region’, were found to be conserved among five species.5. Dual-luciferase assay demonstrated that the activity of ORF1(but not ORF2) wasinhibited by miR-1182.6. ORF1synonymous mutation and western blot revealed miR-1182repressed theprotein level of hTERT through recognizing the seed-matched sequences within its ORF1.Part II:1. Quantitative real-time PCR demonstrated the miR-1182was signifcantly decreasedin gastric cancer tissues campared with corresponding adjacent tissues (P<0.05).2. The relative expression of miR-1182in gastric cancer and corresponding adjacenttissues had negative-relation with hTERT’s expression.3. CCK-8assay indicated the inhibition of cell proliferation by miR-1182in vitro.4. Transwell assay demonstrated miR-1182suppressed the migration of cancer cells invitro.5. Gastric cancer xenografts suggested miR-1182reduced cell proliferation and tumorgrowth in vivo.6. Tail vein metastatic assay indicated miR-1182attenuated lung metastasis of gastriccancer cells in vivo.Conclusion：The expression of miR-1182in gastric cancer tissues is signifcantly lower thanadjacent tissues and has negative relationship with hTERT.miR-1182reduces hTERT expression through targeting its ORF1.miR-1182represses the proliferation and migration of gastric cancer cells.