Long noncoding RNA BC032469, a novel competing endogenous RNA, upregulates hTERT expression by sponging miR-1207-5p and promotes proliferation in gastric cancer AbstractLong noncoding RNAs (lncRNAs) are emerging as new players in gene regulation and are associated with the development of cancers. To investigate the important role and mechanism of lncRNAs in the progression of gastric cancer, we screened lncRNAs in gastric cancer tissues and corresponding adjacent tissues, and assessed the effects on gastric cancer. Here, we report that BC032469, a novel lncRNA, expressed highly in gastric cancer tissues, and the upregulation was clinically associated with larger tumor size, poor differentiation and shorter survival of gastric cancer patients. Downregulation of BC032469 resulted in a significant inhibition of proliferation in vitro and in vivo. Mechanistically, BC032469 could directly bind to miR-1207-5p and effectively functioned as a sponge for miR-1207-5p to modulate the derepression of hTERT. Thus, BC032469 may function as a ceRNA to impair miR-1207-5p-dependent hTERT downregulation, suggesting that it may be clinically valuable as a poor prognostic biomarker of gastric cancer. IntroductionGastric cancer (GC) has a high incidence worldwide, and there are nearly one million new cases each year. Similar to other tumors, GC is a molecular disease caused by the activation of different oncogenes and/or inactivation of tumor suppressor genes.1 Activation of telomerase plays a significant role in maintaining the immortality of tumor cells. Telomerase is highly expressed in more than 85% of cancers. However, telomerase is hardly expressed by normal somatic cells.2 Human telomerase reverse transcriptase (hTERT) is the rate-limiting subunit of telomerase and directly determines telomerase activity.3 Many studies have clearly demonstrated that the activation of telomerase and elevated hTERT are implicated in the initiation and progression of malignancies.4, 5 hTERT expression in GC is positively correlated with tumor size, depth of tumor invasion, lymph node metastasis, histological grade and clinical stage.6 As a result, hTERT is considered to be a promising therapeutic target for cancer treatment, and understanding the regulation of hTERT is extremely important.Many studies suggest that the aberrant expression of noncoding RNA plays an important role in gene control, and may contribute to cancer biology as oncogenes or tumor suppressor genes. Our previous studies examined miRNA-related gastric hTERT regulation. We found that mature miRNAs bound to argonaute, and were associated with the RNA-induced silencing complex by pairing with partial complementary sites in 3鈥?untranslated regions (UTRs).7, 8 We have confirmed that miR-1207-5p and miR-1266 mediate the post-transcriptional silencing of the hTERT gene by binding to the 3鈥?UTR of hTERT mRNA.9The development of new sequencing technologies have led to the identification of another subset of noncoding RNAs, which are termed as long noncoding RNAs (lncRNAs).10 The lncRNAs are crucial regulators of epigenetic modulation, transcription and translation in a particular spatiotemporal manner.11, 12 The regulatory function of lncRNAs is more extensive and complicated than that of miRNAs. A completely new model of gene regulation has been suggested recently, which proposes a role of competing endogenous RNAs (ceRNAs) in gene regulation. In other words, lncRNA, mRNA transcripts and false gene transcripts can affect each other by competitively combining with a miRNA response element (MRE) to influence post-transcriptional regulation.13, 14 Preliminary studies have shown that lncRNAs are present in GC. However, the overall pathophysiological functions and detailed signaling pathways of lncRNAs in GC remain largely unknown.15In this study, we first identify BC032469, a novel lncRNA related to hTERT that expressed higher in GC tissues than that in paired adjacent tissues. By downregulating BC032469, we found a significant inhibition of proliferation in vitro and in vivo. Our data indicate that BC032469 could directly bind to miR-1207-5p and effectively functioned as a sponge for miR-1207-5p to modulate the derepression of hTERT. Furthermore, the overexpression of BC032469 in GC was clinically associated with larger tumor size, poor differentiation and shorter survival of GC patients, which may be valuable as a poor prognostic biomarker of GC.ResultshTERT-related lncRNA BC032469 is frequently overexpressed in GCWe hypothesized that restricted expression would be a general pattern for the lncRNA regulators of hTERT in GC as compared to non-cancer samples. We isolated total RNA from three paired hTERT-positive GC tissues and hTERT-negative corresponding adjacent tissues that were confirmed by immunohistochemistry (Figure 1a). The samples were subjected to lncRNA microarray analysis (Kang Cheng Biotech, Shanghai). The array results are listed on the website http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58828 and the gene expression omnibus accession number is GSE58828. Hierarchical clustering showed there were thousands of lncRNAs with significantly different expression profile when the lncRNA expression threshold was set at greater than twofold with P 0.05 (Figures 1b and c). We then used the University of California Santa Cruz (UCSC) genome browser to identify 12 lncRNAs transcribed near ( 100鈥塳b) the hTERT gene coding region on the fifth chromosome (http://genome.ucsc.edu/; Figure 1d). These lncRNAs might represent the key candidates for exploring the transcriptional regulation of neighboring genes. We examined lncRNA AK126199 (fold change=5.21), NR_003713 (fold change=13.03) and BC032469 (fold change=20.566) from the 12 lncRNAs that were aberrantly expressed higher in the hTERT-positive gastric tumor tissue as compared to the hTERT-negative corresponding adjacent tissue. To validate microarray results, we analyzed the expression of 12 lncRNAs using qPCR in five pairs of GC and corresponding adjacent tissues, and found that the differential expression of lncRNA BC032469 was most significant in the GC tissues as compared to the corresponding adjacent tissues. Thus, BC032469 was chosen to be a potential candidate to further study its role in the progression of GC (Figure 1e).Figure 1Microarray-based lncRNA expression profiling and bioinformatics. (a) The expression of hTERT in gastric cancer tissues and corresponding adjacent tissues was measured by immunohistochemistry. (b) The volcano plots show thousands of lncRNAs are significantly different by using lncRNA expression thresholds of more than twofold change with P 0.05. (c) The heatmap shows samples in groups based on their expression levels. Each row represents one lncRNA and each column represents a sample (A, corresponding adjacent tissues; B, gastric cancer tissues). The color scale shown at the top illustrates the relative expression level of an lncRNA; red represents high expression and green represents low expression. (d) The UCSC genome browser identified 12 lncRNAs 100鈥塳b upstream or downstream of the hTERT gene coding region on the fifth chromosome. (e) Quantitative real-time PCR analysis of the expression of 12 lncRNAs in five pairs of GC and corresponding non-cancer gastric tissues. All values of lncRNA expression levels were normalized to 尾-actin. *P 0.05; #P 0.01.Full size imageExpression of lncRNA BC032469 is correlated with poor prognosis of GC and positively correlated to hTERT expressionWe examined the expression level of lncRNA BC032469 in a panel of 58 paired GC/adjacent tissue specimens to investigate its roles in GC. Our data showed that lncRNA BC032469 was overexpressed in GC tissue as compared with the adjacent tissue (P 0.05; Figure 2a), which linked elevated expression of lncRNA BC032469 with pathological gastric tissue. We further investigated the relationship between the expression level of BC032469 in the patients鈥?GC tissues with their survival time. Each C/P-value was determined as BC032469 expression level in the GC tissue as compared with that in paired corresponding adjacent tissue. The patients were classified into a high lncRNA expression group (relative C/P-value the average relative C/P of all patients), and a low lncRNA expression group (relative C/P-value the average relative C/P of all patients). We found that patients in the lower lncRNA expression group survived longer than those in the higher lncRNA expression group. In addition, there was a significant correlation (Log-rank (Mantel-Cox) test, P=0.0243) between lncRNA BC032469 expression and survival (Figure 2b), and also a significant correlation between lncRNA BC032469 expression and both tumor size (P 0.05) and tumor differentiation (P 0.05; Supplementary Table S1). Furthermore, the C/P-values of lncRNA BC032469 expression were associated with the C/P-values of hTERT expression in 68 pairs of tumor/adjacent tissue specimens (P 0.0001; Figure 2c).Figure 2The expression of lncRNA BC032469 is clinically relevant. (a) In total, 58 pairs of GC tissues and matched corresponding adjacent tissues were collected and analyzed by real-time PCR. *P 0.05; Wilcoxon matched-pair test). (b) Kaplan鈥揗eier analysis of overall survival time of patients with GC according to the expression of lncRNA BC032469. (c) The C/P-values for lncRNA BC032469 were associated with the C/P-values of hTERT protein in 68 pairs of GC tissues and matched corresponding adjacent tissues (P 0.0001). Each C stand for GC tissue; P stand for peficancerous tissues; C/P-value was determined as the lncRNA expression level in the GC tissue as compared with the expression level in paired corresponding adjacent tissue.Full size imageDownregulation of BC032469 leads to a significant inhibition of proliferation in vitro and in vivoSince, BC032469 was overexpressed in GC tissues, we attempted to downregulate its expression to investigate the effects on GC. First, we detected the expression level of lncRNA BC032469 in GC cell lines, SGC-7901, KATO-III, MKN28, AGS and MKN45 cells, and found that BC032469 had higher expression level in SGC-7901 and MKN28 cells (Supplementary Figure S1A). We then constructed lentiviruses containing shRNAs targeting BC032469 and transduced them into SGC-7901 cells and MKN28 cells (Supplementary Figure 1B), and found that the expression of BC032469 could be effectively reduced by shRNAs (Supplementary Figures S1C and D). Importantly, downregulation of lncRNA BC032469 could significantly inhibit the proliferation of GC cells (Figure 3a), while co-expressing hTERT could restore sh-lncRNA mediated inhibition of cell proliferation (Supplementary Figures S3C and D), suggesting the effects of BC032469 on proliferation are closely related to effects observed on hTERT expression. The decrease of lncRNA BC032469 significantly suppressed SGC-7901 cell clonality (Figure 3b), and induced cell cycle arrest at the G1-to-S transition as measured by flow cytometry (Figure 3c). However, downregulation of lncRNA BC032469 could hardly affect the migration of SGC-7901 and MKN28 cells (Supplementary Figure S1E). We next examined whether lncRNA BC032469 mediated tumorigenesis in vivo, and found that both the growth (Figures 4a and b) and the volume (Figure 4c) of tumor xenografts were significantly suppressed in shRNA-BC032469 group as compared with the control group. Expression of hTERT in the transplanted tumors was significantly downregulated in the interference groups as compared with the controls (Figure 4d). Moreover, immunochemistry analysis of both PCNA and Ki-67 revealed that knockdown of lncRNA BC032469 repressed tumor cell proliferation (Figure 4d).Figure 3Biological effects of lncRNA BC032469 on gastric cancer cells. (a) Effects of lncRNA BC032469 on cell proliferation. MTT assays were performed after 7 days. (b) Colonies were stained with crystal violet and counted after 14 days. (c) Flow cytometry was performed to detect the effects of lncRNA BC032469 on cell cycle *P 0.05.Full size imageFigure 4Downregulation of lncRNA BC032469 inhibits the growth of transplanted SGC-7901 cells in nude mice. (a) The tumors in vivo were photographed with an in vivo imaging system. Left flank: shRNA-NC-LV groups; right flank: shRNA-lncRNA-LV groups. (b) The harvested transplanted tumors. (c) The volume of the harvested transplanted tumors. (d) The gastric cancer xenografts were H E stained. hTERT, PCNA and Ki-67 were detected by immunohistochemistry.Full size imageDownregulation of lncRNA BC032469 reduces the expression of hTERT via increasing miR-1207-5p expressionThe above studies demonstrated that the aberrant expression of lncRNA BC032469 may be involved in gene control and oncogenic function. Thus, we further investigated whether BC032469 could affect hTERT expression to exert its important role in the growth and progression of GC. We found that downregulation of lncRNA BC032469 could reduce hTERT protein abundance in GC cells (Figure 5a). However, lncRNA BC032469 could not affect the promoter activity of hTERT at the transcriptional level (Figure 5b). Therefore, we attempted to seek some other potential mechanisms to elucidate the effect of BC032469 on hTERT. Our previous study has demonstrated that miR-1207-5p and miR-1266 could inhibit hTERT expression at the post-transcriptional level by directly binding to the 3鈥?UTR of the hTERT mRNA. Interestingly, we also found that downregulation of BC032469 could significantly inhibit the activity of hTERT-3鈥?UTR in this study (Figures 6a and b). Most recently, a unifying hypothesis indicated that lncRNA could act as a ceRNA using MREs to exert its regulatory functions. We hypothesized that lncRNA BC032469 might have the same interaction with miR-1207-5p and miR-1266 to regulate expression of hTERT. We predicted MREs using miRanda 3.3 software (New York, NY, USA), and found that lncRNA BC032469 contained many elements complementary to the miRNA-1207-5p and miR-1266 seed regions (Figure 5c and Supplementary Figure S2). We then tested the effects of shRNA-mediated downregulation of lncRNA BC032469 on the expression of miR-1207-5p and miR-1266. Notably, miR-1207-5p was upregulated in the shRNA-BC032469-treated SGC-7901 cells as compared with scramble control-treated ones (Figure 5d), suggesting that lncRNA BC032469 could affect the expression of miRNAs. Furthermore, we also found that the expression of lncRNA BC032469 was negatively associated with the expression of miR-1207-5p in the same paired samples, but had no significant relationship with the expression of miR-1266 (Figures 6e and f). To verify that lncRNA BC032469 interacted with the miRNAs to regulate the activity of hTERT-3鈥?UTR, we then performed reporter assays and found that downregulation of lncRNA BC032469 could greatly inhibit the activity of hTERT-3鈥?UTR, which could be partially restored by anti-miR-1207-5p, but not anti-miR-1266 (Figures 6a and b). Furthermore, mutation on the predicted MREs for miR-1207-5p in the hTERT-3鈥?UTR led to the failure of lncRNA BC032469 reduction to inhibit the activity of hTERT-3鈥?UTR. Conversely, mutation of MREs for miR-1266 had no influence on the inhibitory effect of lncRNA BC032469 reduction on the activity of hTERT-3鈥?UTR (Figures 6a and b).Figure 5Downregulation of lncRNA BC032469 reduces the expression of hTERT and increases the expression of miR-1207-5p. (a) The effect of lncRNA BC032469 on hTERT protein abundance. Western blot analyses were performed to test hTERT protein abundance after SGC-7901 cells were infected with shRNA-NC-LV or shRNA-lncRNA-LV. *P 0.05 one-way analysis of variance. (b) Luciferase assays for hTERT promoter. The depletion of lncRNA BC032469 did not reduce luciferase activity of hTERT at the pretranscriptional level. (c) Partial sequence of lncRNA BC032469 and sequence of miR-1207-5p are shown. Nucleotides of the miRNA are in red and those of lncRNA are in blue. Numbers indicate the nucleotides relative to the transcriptional start site of lncRNA BC032469. The portions of the profile for four mir-1207-5p-binding sites with free energy of the RNA duplex score are shown on the right. (d) Expression profiling of miR-1207-5p and miR-1266 after downregulation of lncRNA BC032469 in SGC-7901 cells. *P 0.05 one-way analysis of variance).Full size imageFigure 6lncRNA BC032469 maintains the expression and activity of hTERT by acting as a ceRNA for miR-1207-5p to regulate the activity of hTERT-3鈥?UTR. (a and b) Luciferase assays show that inhibiting endogenous lncRNA BC032469 repressed Luc-hTERT-3鈥?UTR, and such repression could be partially restored by anti-miR-1207-5p but not anti-miR-1266. When the predicted MREs in the hTERT-3鈥?UTR were mutated, decreased lncRNA BC032469 was not able to effectively inhibit the luciferase activity. *P 0.05. hTERT-3鈥?UTR (1207鈥?鈥塸M): miR-1207-5p seed region on hTERT-3鈥?UTR was mutated; hTERT-3鈥?UTR (1266鈥塸M): miR-1266 seed region on hTERT-3鈥?UTR was mutated. (c) Western blot analyses were used to determine whether inhibiting lncRNA BC032469 repressed hTERT protein expression. (d) TRAP assay was performed to examine whether inhibiting lncRNA BC032469 repressed telomerase activity. (e) There was a significant negative correlation between the expression level of lncRNA BC032469 and the expression level of miR-1207-5p in the same paired gastric samples (P=0.0399, Pearson). (f) There was no significant correlation between the expression level of lncRNA BC032469 and the expression level of miR-1266 in the same paired gastric samples (P=0.4427, Pearson).Full size imageWe then examined the protein level and activity of hTERT to further confirm the interaction between BC032469 and the miRNAs in the regulation of hTERT using western blot analysis and telomeric repeat amplification protocol (TRAP). As shown in Figure 6c, downregulation of lncRNA BC032469 repressed hTERT protein abundance, which could be partly restored by anti-miR-1207-5p but not anti-miR-1266 (Figure 6c). Furthermore, we found that upregulation of BC032469 significantly promoted hTERT protein expression and cell proliferation, which was significantly attenuated by overexpression of miR-1207 (Supplementary Figures S3A and B), strongly suggesting that BC032469 regulated hTERT expression in a miR-1207-dependent manner. Furthermore, TRAP assay demonstrated that decreased lncRNA BC032469 inhibited telomerase activity, and such repression could be partly restored by anti-miR-1207-5p but not by anti-miR-1266 (Figure 6d). These results suggest that lncRNA BC032469 may act as an endogenous competing RNA to upregulate hTERT expression and activity in GC.lncRNA BC032469 acts as a ceRNA directly binding to miR-1207-5pTo verify that lncRNA BC032469 can directly bind to the miRNAs, we performed dot blot assay. Dot blot experiments were performed as shown in the schematic presentation in Figure 7a. As shown in Figure 7b, with the increase of concentrations of lncRNA BC032469 and wild hTERT-3鈥?UTR, the hybridization of miR-1207-5p was increased, whereas the hybridization activity of the hTERT-3鈥?UTR deletion mutant RNA and the hTERT-3鈥?UTR seed site mutation RNA with miR-1207-5p probe was as weak as the negative control probe. Similarly, miR-1266 showed a significant hybridization with wild-type hTERT-3鈥?UTR but not BC032469 (Figure 7b). To further confirm the interaction in vivo, we performed RNA fluorescence in situ hybridization (FISH) assay in GC samples. The data revealed that the co-localization of lncRNA and miR-1207-5p existed in GC tissues (Figure 7c). These results indicate that lncRNA BC032469 can directly bind to miR-1207-5p to function as a sponge to impair miR-1207-5p-dependent hTERT downregulation in GC (Figure 8).Figure 7lncRNA BC032469 directly binds to miR-1207-5p. (a) Workflow for the dot plot experiments. (b) Dot blot analysis was performed using specific miRNA probes for different transcripts. lnc, lncRNA BC032469; Tert, hTERT-3鈥?UTR; Tert d1, miR-1207-5p MREs in hTERT-3鈥?UTR were depleted; Tert m1, miR-1207-5p seed region in hTERT-3鈥?UTR were mutated; Tert d2, miR-1266 MREs in hTERT-3鈥?UTR were depletion; Tert m2, miR-1266 seed region in hTERT-3鈥?UTR were mutated. Six concentrations of RNA were used (the highest concentration is 100鈥塶g/渭l) and the concentrations were diluted using a threefold dilution from line 1 to 6. (c) RNA FISH showed that co-localization of lncRNA and miRNA-1207-5p existed in the gastric cancer sample. lncRNA oligonucleotides were labeled with rhodamine (red), and miRNA oligonucleotides were labeled with FAM (green).Full size imageFigure 8Schematic presentation of possible molecular mechanism underlying the interaction between lncRNA BC032469 and miR-1207-5p.Full size imageDiscussionIt is well accepted that lncRNAs are involved in regulating the complexity of biological processes with specific regulatory mechanisms. lncRNAs are attracting considerable research interest.14 Recent studies have suggested that lncRNAs potentially influence mRNA levels by competing for miRNAs through interactions. The ceRNA can inhibit miRNAs through MREs and protect the target RNAs from repression.13 For example, lncRNA IPS1 cleaves miR-399 by mimicking its binding site, leading to the accumulation of the miR-399 target PHO2 in Arabidopsis thaliana.16 In addition, lncRNA, highly up-regulated in liver cancer acts as a 鈥榮ponge鈥?to downregulate miR-372, resulting in the reduction of translational repression of its target gene PRKACB in liver cancer.17 Furthermore, Cesana et al.14 showed that lnc-MD1 was a decoy for miR-133 and miR-135, causing an increase in MAML1 and MEF2C accumulation during muscle differentiation. Cumulatively, these findings suggest that RNA transcripts may communicate through the ceRNA language.Many ncRNAs surround or overlap protein-coding genes and are predicted to function through a variety of regulatory mechanisms. In this study, we used UCSC genome browser to identify 12 lncRNAs transcribed near the hTERT gene coding region on the fifth chromosome. We preliminarily selected lncRNAs within 100鈥塳b of the hTERT gene based on the efficiency of genetic analysis and the previous studies.18, 19 The lncRNAs and hTERT-encoding genes are syntropic transcripts. Thus, lncRNAs may influence adjacent coding gene expression. Finally, our results revealed that lncRNA BC032469 differently expressed in the paired GC and corresponding non-cancer gastric tissues using qPCR and microarray analysis. lncRNA BC032469 is transcribed by enzyme ( 200鈥塶t) and contains five exons and the polyA tail structure. We compared the human genes with other species and found that lncRNA BC032469 reciprocal well with sequences, which are present in many vertebrates in UCSC online software. These local conserved segments are often distributed in fragile chromosome sites and tumor-related areas as that evolved to be targeted by miRNAs despite their full-scale low conservation.20, 21 We found that lncRNA BC032469 was overexpressed in GC tissue as compared with the corresponding adjacent tissue. In addition, there was a significant correlation between lncRNA BC032469 expression and both tumor size and tumor differentiation. Furthermore, lncRNA BC032469 expression was associated with the hTERT protein expression in 58 paired tissue samples. The downregulation or overexpression of lncRNA BC032469 was correlated with a reduction or an increase of the hTERT protein abundance, respectively. These results suggest that lncRNA BC032469 may be an oncogenic lncRNA to promote tumor progression by regulating hTERT in GC.To confirm whether lncRNA BC032469 affected hTERT at the pretranscriptional level, we applied a dual-luciferase reporter assay to test the potential interaction between the hTERT promoter and lncRNA BC032469. The reporter assays demonstrated that there was no significant effect on luciferase activity in the presence or absence of lncRNA BC032469 at the pretranscriptional level. In our previous studies, we found that miR-1207-5p and miR-1266 were significantly repressed in GC tissues. The reversal of the lncRNA BC032469 in the same paired samples revealed that miR-1207-5p and miR-1266 repressed hTERT by binding to hTERT-3鈥?UTR.9 Interestingly, we also found that downregulation of BC032469 could significantly inhibit the activity of hTERT-3鈥?UTR. We hypothesized whether lncRNA BC032469 had the same mechanism as miR-1207-5p and miR-1266 to regulate hTERT expression. We further predicted MREs using miRanda 3.3 software and found that lncRNA BC032469 contained many elements complementary to the miRNA-1207-5p and miR-1266 seed regions. Downregulation of lncRNA BC032469 enhanced miR-1207-5p in SGC-7901 cells. Therefore, we focused on targeting hTERT at the post-transcriptional level. Our reporter assay indicated that the knockdown of lncRNA BC032469 downregulated the activity of hTERT-3鈥?UTR in SGC-7901 cells by relieving miR-1207-5p but not miR-1266. Furthermore, the inhibition of lncRNA BC032469 reduced hTERT protein abundance and telomerase activity in SGC-7901 cells. The hTERT suppression could be partially rescued in the presence of anti-miR-1207-5p but not anti-miR-1266. We propose the following explanation for these abovementioned results. First, the MREs on ceRNA are not equal.13 Although two miRNAs are predicted to bind the same ceRNA, the nucleotide components of their MREs may be different. Analogously, miRNAs are predicted to work on thousands of RNAs. However, they do not induce equal inhibition for all targets. The primary targets of a given miRNA are preferentially affected, whereas the rest are finely tuned.22, 23 Second, the efficacy of a ceRNA would principally depend on the ability of miRNA molecules to act as a sponge.24, 25 The overall influences of ceRNAs also depend on the specific developmental and pathological context of the cells because not all miRNAs exist everywhere and at all times.26On the basis of the information stated above, we hypothesize that lncRNA BC032469 may act as a ceRNA to bind miR-1207-5p and regulate its function in GC cells. In principle, any RNA molecule that contains one MRE accessible to miRNA binding is described as a ceRNA. Therefore, it is imperative to identify the MREs within RNA molecules in ceRNA networks accurately.27, 28 We used miRanda, which is a popular computational algorithm to identify miRNA binding to their recognition elements.29 Among the four predicted miR-1207-5p sites in lncRNA BC032469, the site with its starting nucleotide at position 1467 is an 8-mer seed site with strong compensatory base pairing for the 3鈥?end of miR-1207-5p. This is the closest location to the target sequence bound by siRNA. The sites at positions 108 and 671 have strong base paring in the seed region despite involving several G:U pairs. The findings on the seed site at position 200 suggest noncanonical sites can function in target regulation.21, 30 Previous studies suggest that miRNA responsive elements in lncRNAs reveal a positional preference of the Ago-binding sites in mid regions and at the 3鈥?ends of the lncRNAs. These sites harbor a possible pattern of regulatory elements across transcripts.31 We decided to further examine the hypothesis that lncRNA BC032469 interacts with miRNAs directly, and found an interaction among lncRNA BC032469, miR-1207-5p and hTERT-3鈥?UTR using dot blot assay. This result demonstrates that the interaction of lncRNA BC032469 with miR-1207-5p is direct. Thus, the deletion mutant or seed site mutant forms of the hTERT-3鈥?UTR with reduced complement to miR-1207-5p affects the hybridization between miR-1207-5p and the hTERT-3鈥?UTR. Further studies are required to fully define the degradation of miRNA.16 A recent study has suggested that exoribonucleases found in Arabidopsis depress mature miRNAs.32 Moreover, repression of miRNAs likely occurs through the pathway involving the RNA-induced silencing complex or deadenylation, which causes miRNAs to be degraded sooner.33 However, we think that the repression of miR-1207-5p is not only the result but is also a driving factor for greater inhibition of lncRNA BC032469 and other miRNAs. However, these additional mechanisms require further studies.Cumulatively, our results show that the novel lncRNA BC032469 may be applicable as a poor prognostic biomarker and contribute to the growth and prognosis of GC. BC032469 functions as a ceRNA by acting as a sponge to impair miR-1207-5p-dependent hTERT downregulation. The ceRNA regulatory network involving BC032469, and the positive interaction between BC032469 and hTERT may contribute to better understanding the miRNA鈥搇ncRNA interaction and facilitate the development of lncRNA-directed diagnostics and therapeutics against GC.Materials and methodsEthics statementAll animal experimental procedures were performed following the 鈥楪uide for the Care and Use of Laboratory Animals鈥?published by the National Institutes of Health, and the study was approved by the Clinical Research Ethics Committee of the Third Military Medical University. Informed consent was obtained for all patients recruited or their guardians.Patients and GC cell linesGC tissues and corresponding adjacent tissues were collected from 68 patients who underwent curative surgery for GC (the study was approved by the Clinical Research Ethics Committee of the Third Military Medical University). KATO-III cells were obtained from the American Type Culture Collection (Manassas, VA, USA). SGC-7901, MKN28, AGS and MKN45 cells were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured routinely in Dulbecco鈥檚 modified Eagle鈥檚 medium (DMEM) supplemented with 10% fetal bovine serum at 37鈥壜癈 in a humidified atmosphere containing 5% CO2.9Microarray and computational analysislncRNA microarrays were used to profile lncRNA expression from three GC tissues and their corresponding adjacent tissues by KangChen Biotech (Shanghai, China). The labeled cRNAs were hybridized onto the Human LncRNA Array v2.0 (Arraystar, Rockville, MD, USA). Quantile normalization and subsequent data processing were performed using GeneSpring GX v11.5.1 software (Agilent Technologies, Santa Clara, CA, USA). The differentially expressed lncRNAs with statistical significance between the two groups were identified by volcano plot filtering. A P-value was calculated using the paired t-test. When the lncRNA expression level changed at least twofold with P 0.05, the lncRNA expression was then considered significantly different. The gene expression omnibus accession number of the microarray data is GSE58828. We used the UCSC genome browser to identify lncRNAs 100鈥塳b upstream or downstream of the hTERT gene coding region on the fifth chromosome (http://genome.ucsc.edu/). These databases were generated by the phastCons program through multiple sequence alignments of several mammalian genomes to the human genome (hg38). The miRNA-binding sites on lncRNA BC032469 were predicted using a miRNA target prediction algorithm miRanda 3.3. We used a threshold score 130 and free energy 鈭?5鈥塳cal/mol to identify the potential sites.Quantification of LncRNAs and miRNAs by quantitative real-time PCRTotal RNA was extracted by RNAiso Plus (Takara, Japan). The RNA was reverse transcribed to cDNA using PrimeScript RT polymerase (Takara, Japan). Quantitative PCR was performed using cDNA primers specific for lncRNAs and hTERT. The gene 尾-actin was used as an internal control. Stem-loop reverse transcriptase PCR (RT鈥揚CR) for mature miRNAs was performed as described previously. The gene U6 was used as an internal control. All the real-time RT鈥揚CR reactions were performed with SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Bulge-loop miRNA Primer Sets including one RT primer and a pair of qPCR primers were designed by RiboBio (Guangzhou, China). All of the reported results are the average ratios of three independent experiments.Cell proliferation and cell cycle analysisA modified 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to determine the relative cell growth and viability of each group as previously described.34 Cells at ~50% confluence were fixed by the addition of 70% ice-cold ethanol for at least 24鈥塰. The cell cycle distribution was analyzed by flow cytometry analyses (Beckman Coulter, Inc., Danvers, MA, USA), as described previously.9 Triplicate experiments with triplicate samples were performed.Clonogenic assayThe survival of wild-type cells and cells transfected with LV-shRNA-lncRNA or LV-shRNA-scramble were measured by the ability of one single cell to form a colony in a six-well plate after 14 days. The colonies were fixed and stained with 0.05% crystal violet (Beyotime, China) before counting.35 Triplicate experiments with triplicate samples were performed.Wound scratch assayCells from each group were seeded into 24-well tissue culture plates at a density that reached 80% confluence as a monolayer at 24鈥塰. The monolayer was gently scratched across the center of the well with a 10-渭l pipette tip. After scratching, the well was gently washed with the medium to remove the detached cells. Images were obtained at 0 and 24鈥塰 by using a microscope.36 Triplicate experiments with triplicate samples were performed.Animal workThe studies were conducted in accordance with the National Institutes of Health (NIH) Animal Use Guidelines. Male nude mice (~4 weeks old) were purchased from Hua Fu Kang (Beijing, China). Approximately, 1 脳 106 SGC-7901 cells from each group were injected into the bilateral axilla of nude mice. The volume of the GC xenografts was assessed each week. After 4 weeks, the tumors were visualized with an in vivo imaging system (Cambridge Research Instrumentation, Woburn, MA, USA), and were then harvested to assess the weights. Four mice were used for each group and the mice were randomly separated into each groups.19 All animal studies were followed by a blind randomized animal study protocol.ImmunohistochemistryThe tissues were harvested in 4% phosphate-buffered saline-buffered formaldehyde, embedded in paraffin and then sectioned. The antibodies against hTERT (R D Systems, Minneapolis, MN, USA), Ki-67 and PCNA (Abcam, Cambridge, MA, USA) were used for immunohistochemical analyses. Immunoreactivity was detected in the sections using a horseradish peroxidase (3,3鈥?diaminobenzidine) kit (BioGenex, Fremont, CA,USA). The slides were then counterstained with hematoxylin, dehydrated and mounted.Construction of the luciferase reporterThe hTERT-3鈥?UTR was cloned into the SpeI and HindIII restriction sites of the pMIR-REPORT vector (Applied Biosystems, Foster City, CA, USA). The putative miRNA-binding sites in the hTERT-3鈥?UTR were mutated using the QuikChange site-directed mutagenesis kit (Agilent Technologies). The mutated pMIR-REPORT reporter vectors are pMIR-1266 M and pMIR-1207-5p M. Wild-type or mutated versions of Luc-hTERT-3鈥?UTR and the pRL-TK vector plasmid (Promega, Madison, WI, USA) were co-transfected into SGC-7901 cells infected by LV-shRNA-lncRNA with either anti-miR-1266 or anti-miR-1207-5p by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24鈥塰, the luciferase activities were assayed using the Dual-Luciferase assay system (Promega). The hTERT-3鈥?UTR 1763-bp promoter was ligated and cloned into pGL3-basic vector and sequenced. All data represent three independent experiments.Western blotProtein was extracted and separated by SDS鈥揚AGE, and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The antibodies against hTERT and 尾-actin were from Abcam. The signals were visualized using enhanced chemiluminescence (Thermo Scientific, West Palm Beach, FL, USA), and recorded on a Gel Doc 2000 imaging scanner (Bio-Rad).TRAP assayTo analyze telomerase activity, 105 cells from each group were suspended in 200鈥壩糽 of 1 脳 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) lysis buffer. The cells were lysed on ice for 30鈥塵in. The sample was centrifuged at 12,000鈥?i>g for 20鈥塵in at 4鈥壜癈 and 160鈥壩糽 of the supernatant was collected. A 2-渭l sample of each protein extract was combined with 48鈥壩糽 of the 鈥楳aster Mix鈥?(Millipore) in RNase-free PCR tubes. The telomerase-positive control and telomerase-negative control were prepared in the same manner. The tubes were incubated at 30鈥壜癈 for 30鈥塵in, and the sample was amplified by PCR. The PCR protocol was described as follows: 30 cycles at 94鈥壜癈 for 30鈥塻, 59鈥壜癈 for 30鈥塻 and 72鈥壜癈 for 1鈥塵in. After the completion of PCR, 25鈥壩糽 of the PCR product was separated on a 12.5% non-denaturing PAGE for 1.5鈥塰. The smallest telomerase product band was 50鈥塨p, and the internal control band was 36鈥塨p. After electrophoresis, the gel was stained with argentation (Tianz, China).37RNA FISHTissue samples harvested from GC patients were immediately frozen in liquid nitrogen. The tissues were cut into 5-渭m-thick sections at 鈭?0鈥壜癈 and adhered to slides. Then, tissue sections were washed with phosphate-buffered saline and fixed in 3.7% formaldehyde for 10鈥塵in. We washed slides with a solution of 10% formamide in 2 脳 sodium citrate buffer, and then added 4鈥壩糽 fluorescent probes in a hybridization solution containing 10% formamide, 2 脳 sodium citrate buffer and 10% dextran sulfate (w/v). Hybridization was performed in a humid chamber at 37鈥壜癈 overnight. Slides were washed twice for 20鈥塵in at 37鈥壜癈 with 10% formamide in 2 脳 sodium citrate buffer. DAPI was added during the second wash. LncRNA and miRNA were labeled with rhodamine fluorophore and FAM fluorophore, respectively. Slides were then rinsed twice with 2 脳 sodium citrate buffer before detecting by confocal microscopy.Dot blot analysislncRNA BC032469, hTERT-3鈥?UTR, hTERT-3鈥?UTR (miR-1207-5p MREs were depleted), hTERT-3鈥?UTR (miR-1207-5p seed region were mutant), hTERT-3鈥?UTR (miR-1266 MREs were depleted) and hTERT-3鈥?UTR (miR-1266 seed region were mutant) were constructed by Sangon (Shanghai, China). These plasmids were identified by enzyme digestion and sequencing. The DNA insert was cloned into the multiple cloning site of the pcDNA3.1 vector containing T7 RNA polymerase (Promega). The DNA was ligated and bacteria were transformed with the construct. The colonies were then screened for the vector. The plasmid was cut with the Eori I restriction enzyme to linearize the DNA. The DNA template was transcribed into RNA in vivo using T7 RNA Production System (Promega). The dot blots were performed by using a threefold dilution, and six concentrations of RNA were used (the highest concentration is 100 ng/渭l). The lncRNA BC032469 (lnc), wild hTERT-3鈥?UTR (tert), hTERT-3鈥?UTR deletion mutation (tert m1) and hTERT-3鈥?UTR seed-size mutation (tert m2) were transcribed into RNA templates. The miR-1207-5p probe, miR-1266 probe and negative probe (Signosis, Santa Clara, CA, USA) were labeled with biotin. The RNAs were loaded directly on a positively charged nylon membrane to avoid variations in dot size when using the same amounts of samples. The membrane was dried and cross-linked by ultraviolet light. The hybridization and detection were performed according to a previously described protocol.38Statistical analysisAll data are presented as the means卤s.d. and were analyzed with Prism 5.0 software (GraphPad, La Jolla, CA, USA). The paired sample t-test was used to assay the differences of the lncRNA expressions in the paired groups. 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Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression. BMC Biotechnol 2004; 4: 12.Article聽Google Scholar聽 Download referencesAcknowledgementsThis study was supported by the National Natural Science Foundation of China (No. 81201950) and Luzhou Medical University Funding (No. 2014ZD-001). We thank Professor Xia Zhang for suggestions on the manuscript.Author informationAuthor notesM-H L眉 and B Tang: These authors contributed equally to this work.AffiliationsDepartment of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, ChinaM-H L眉,聽B Tang,聽S Zeng,聽C-J Hu,聽R Xie,聽Y-Y Wu,聽S-M Wang聽 聽S-M YangDepartment of Gastroenterology, The Affiliated Hospital of Luzhou Medical College, Luzhou, ChinaM-H L眉Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing, ChinaF-T HeAuthorsM-H L眉View author publicationsYou can also search for this author in PubMed聽Google ScholarB TangView author publicationsYou can also search for this author in PubMed聽Google ScholarS ZengView author publicationsYou can also search for this author in PubMed聽Google ScholarC-J HuView author publicationsYou can also search for this author in PubMed聽Google ScholarR XieView author publicationsYou can also search for this author in PubMed聽Google ScholarY-Y WuView author publicationsYou can also search for this author in PubMed聽Google ScholarS-M WangView author publicationsYou can also search for this author in PubMed聽Google ScholarF-T HeView author publicationsYou can also search for this author in PubMed聽Google ScholarS-M YangView author publicationsYou can also search for this author in PubMed聽Google ScholarCorresponding authorCorrespondence to S-M Yang.Ethics declarations Competing interests The authors declare no conflict of interest. Additional informationSupplementary Information accompanies this paper on the Oncogene websiteSupplementary information Supplementary Figure Legends (DOC 39 kb) Supplementary Figure 1 (JPG 1441 kb) Supplementary Figure 2 (JPG 392 kb) Supplementary Figure 3 (JPG 279 kb)Rights and permissionsReprints and PermissionsAbout this articleCite this articleL眉, MH., Tang, B., Zeng, S. et al. Long noncoding RNA BC032469, a novel competing endogenous RNA, upregulates hTERT expression by sponging miR-1207-5p and promotes proliferation in gastric cancer. Oncogene 35, 3524鈥?534 (2016). https://doi.org/10.1038/onc.2015.413Download citationReceived: 25 January 2015Revised: 18 August 2015Accepted: 28 September 2015Published: 09 November 2015Issue Date: 07 July 2016DOI: https://doi.org/10.1038/onc.2015.413 Md. 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