The long non-coding RNA AK001796 contributes to tumor growth via regulating expression of p53 in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the prevalent and deadly cancers worldwide, especially in China. Considering the poor prognosis of ESCC, the aim of this study is to dissect the effects of long non-coding RNA (lncRNA) AK001796 on cell proliferation and cell cycle in vitro and tumorigenicity in vivo, providing therapeutic targets for ESCC. METHODS: We conducted quantitative real time PCR to detect the expression level of lncRNA AK001796 in human ESCC tumor and adjacent non-tumor tissues, and analyzed the correlation between lncRNA AK001796 expression and clinicopathologic feature of ESCC patients. Then we knocked down the expression of lncRNA AK001796 in human ESCC cell lines Eca-109 and TE-1, and next inspected cell cycle and apoptosis condition in these cells using flow cytometry. Subsequently, we used CCK-8 assay to test proliferation ability of the lncRNA AK001796-silenced ESCC cells, and the MDM2/p53 signaling pathway in these cells was analyzed by western blot analysis. At last, the ESCC xenograft models were established to verify the role of lncRNA AK001796 on the occurrence and development of ESCC. RESULTS: In this study, we demonstrated that lncRNA AK001796 was significantly upregulated in ESCC tumor tissues compared to adjacent non-tumor tissues. Knockdown of lncRNA AK001796 inhibited ESCC cell growth, cell cycle, and tumor growth in a xenograft mouse model via regulating MDM2/p53 signal pathway. The expression of lncRNA AK001796 was positively correlated with MDM2 levels in human ESCC samples. CONCLUSIONS: Overall, lncRNA AK001796 regulates cell proliferation and cell cycle via modulating MDM2/p53 signaling in ESCC, which provides a new insight into the treatment targets for ESCC.Trial registration This study was registrated in the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (Trial registration: 2012-SR-127, Registered 20 January 2012).

Genetic variants reducing MTR gene expression increase the risk of congenital heart disease in Han Chinese populations.

AIMS: Elevated homocysteine levels are known to be a risk factor for congenital heart disease (CHD), but the mechanism underlying this effect is unknown. During early embryonic development, homocysteine removal is dictated exclusively by the MTR activity. To examine the role of MTR in CHD risk, we identified genetic variants in MTR and investigated the mechanisms that affect its expression levels and that increase the risk of CHD in Chinese populations. METHODS AND RESULTS: The association between regulatory variants of the MTR gene and CHD was examined in three independent case-control studies in a total of 2340 patients with CHD and 2270 controls. The functional consequences of these variants were demonstrated using dual-luciferase assays, real-time polymerase chain reaction (PCR), electrophoretic mobility shift assays, surface plasma resonance, chromatin immunoprecipitation, and bisulfite sequencing, as well as by a group of predicted microRNAs using a gene reporter system. Two regulatory variants of MTR, -186T>G and +905G>A, were associated with an increased risk of CHD in both the separate and combined case-control studies (-186GG P = 1.32 × 10(-9); +905AA P = 6.35 × 10(-14)). Compared with the major allele, the -186G allele exhibited significantly lower promoter activity, decreased hnRNA and mRNA levels, reduced transcription factor binding affinity, and a more highly methylated promoter. The +905A allele exhibited a statistically stronger binding affinity to functional microRNAs that down regulate MTR expression at the translational level. Both of the minor alleles were correlated with elevated plasma homocysteine concentrations, indicating a genetic component for hyperhomocysteinaemia. CONCLUSIONS: Regulatory variants of the MTR gene increase CHD risk by reducing MTR expression and inducing the homocysteine accumulation and elevation.

miR-550a-5p Functions as a Tumor Promoter by Targeting LIMD1 in Lung Adenocarcinoma.

Lung adenocarcinoma accounts for half of all lung cancer cases in most countries. Mounting evidence has demonstrated that microRNAs play important roles in cancer progression, and some of them can be identified as potential biomarkers. This study aimed to explore the role of miR-550a-5p, a lung adenocarcinoma-associated mature microRNA screened out from the TCGA database via R-studio and Perl, with abundant expression in samples and with 5-year survival prognosis difference, as well as having not been studied in lung cancer yet. Potential target genes were predicted by the online database. Gene ontology enrichment, pathway enrichment, protein-protein interaction network, and hub genes-microRNA network were constructed by FunRich, STRING database, and Cytoscape. Then, LIMD1, a known tumor suppressor gene reported by multiple articles, was found to have a negative correlation with miR-550a-5p. The expression of miR-550a-5p was up-regulated in tumor samples and tumor-associated cell lines. Its high expression was also correlated with tumor size. Cell line A549 treated with miR-550a-5p overexpression promoted tumor proliferation, while H1299 treated with miR-550a-5p knockdown showed the opposite result. Mechanically, miR-550a-5p negatively regulated LIMD1 by directly binding to its 3'-UTR validated by dual luciferase assay. In summary, a new potential prognostic and therapeutic biomarker, miR-550a-5p, has been identified by bioinformatics analysis and experimental validation in vitro and in vivo, which promotes lung adenocarcinoma by silencing a known suppressor oncogene LIMD1.

Long noncoding RNA RP11-766N7.4 functions as a tumor suppressor by regulating epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

BACKGROUND: Numerous studies have proved that long non-coding RNAs participate in the initiation and metastasis of various cancers including esophageal squamous cell carcinoma (ESCC). Recently, a novel long non-coding RNA RP11-766N7.4 was discovered in a variety of human tissues. However, its role in oncogenesis and tumor metastasis remains unknown. METHODS: To investigate the function of long noncoding RNA RP11-766N7.4 in ESCC, RT-qPCR was used to monitor the expression level of long non-coding RNA RP11-766N7.4 in ESCC cell lines and 50 paired ESCC tissues. Moreover, the association between long non-coding RNA RP11-766N7.4 expression level and clinicopathological characteristics as well as 5-year survival rate of ESCC patients was evaluated. Furthermore, function assays containing cell proliferation assay, flow cytometry, Colony Formation, wound healing assay and Transwell assays were conducted to investigate the role of long noncoding RNA RP11-766N7.4 in ESCC. Western blotting assay were used to explore the regulation mechanism. RESULTS: In this study, we found that long noncoding RNA RP11-766N7.4 was downregulated in ESCC tissues and cell lines and correlated with lymph node metastasis, tumor stage and survival rate. Results also revealed that long noncoding RNA RP11-766N7.4 had no significant effect on cell proliferation, cell cycle or cell apoptosis of ESCC cells. In addition, long noncoding RNA RP11-766N7.4 knockdown promoted cellular migration and invasion via inducing EMT process, and overexpression of long noncoding RNA RP11-766N7.4 inhibited cellular migration and invasion by suppressing EMT process. CONCLUSION: Our study suggested that long noncoding RNA RP11-766N7.4 acts as a tumor suppressor in ESCC carcinogenesis and metastasis, and may be a potential prognostic mark and a therapeutic target for ESCC.

Long non­coding RNA AK001796 contributes to cisplatin resistance of non­small cell lung cancer.

Cisplatin (DDP)­based chemotherapy is the most widely used therapy for non­small cell lung cancer (NSCLC). However, the existence of chemoresistance has become a major limitation in its efficacy. Long non­coding RNAs (lncRNAs) have been shown to be involved in chemotherapy drug resistance. The aim of the present study was to investigate the biological role of lncRNA AK001796 in cisplatin­resistant NSCLC A549/DDP cells. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis was performed to monitor the differences in the expression of AK001796 in cisplatin-resistant (A549/DDP) cells and parental A549 cells. Cellular sensitivity to cisplatin and cell viability were examined using an MTT assay. Cell apoptosis and cell cycle distribution were measured using flow cytometry. The expression levels of cell cycle proteins cyclin C (CCNC), baculoviral IAP repeat containing 5 (BIRC5), cyclin­dependent kinase 1 (CDK1) and G2 and S phase­expressed 1 (GTSE1) were assessed using RT­qPCR and western blot analyses. It was found that the expression of AK001796 was increased in A549/DDP cells, compared with that in A549 cells. The knockdown of AK001796 by small interfering RNA reduced cellular cisplatin resistance and cell viability, and resulted in cell­cycle arrest, with a marked increase in the proportion of A549/DDP cells in the G0/G1 phase. By contrast, the knockdown of AK001796 increased the number of apoptotic cancer cells during cisplatin treatment. It was also shown that the knockdown of AK001796 positively induced the expression of cell apoptosis­associated factors, CCNC and BIRC5, and suppressed the expression of cell cycle­associated factors, CDK1 and GTSE5. Taken together, these findings indicated that lncRNA AK001796 increased the resistance of NSCLC cells to cisplatin through regulating cell apoptosis and cell proliferation, and thus provides an attractive therapeutic target for NSCLC.

Long non-coding RNA-Low Expression in Tumor inhibits the invasion and metastasis of esophageal squamous cell carcinoma by regulating p53 expression.

Long non-coding RNAs (lncRNAs) are involved in governing fundamental biological processes, and, in many lncRNAs, the expression level is altered and likely to have a functional role in tumorigenesis, including apoptosis, migration and invasion. The lncRNA­Low Expression in Tumor (LET), a recently identified lncRNA, was demonstrated to be downregulated in hepatocellular and gallbladder cancer. However, its role in esophageal squamous cell carcinoma (ESCC) requires investigation. The expression level of lncRNA­LET mRNA in primary ESCC and matched healthy tissues (48 cases) was determined by reverse transcription­quantitative polymerase chain reaction. In addition, the effects of lncRNA-LET on cell apoptosis were evaluated by flow cytometric analysis, the regulatory effect of lncRNA­LET on migration was detected using a wound healing assay and cellular invasion was analyzed by Matrigel­coated transwell assay. Furthermore, the effect of lncRNA­LET on cell proliferation was investigated by 5­ethynyl­2'-deoxyuridine cell proliferation assay and protein levels of lncRNA-LET targets were analyzed by western blotting. lncRNA-LET expression was decreased in primary ESCC tissues when compared with paired healthy tissues, and was identified to be associated with the clinical features. Overexpression of lncRNA­LET was observed to inhibit the migration and invasion of ESCC cells, and modulate p53 expression levels in human ESCC cell lines in vitro. These results establish that lncRNA-LET is significant in the regulation of tumor progression and metastasis, and serves as a tumor suppressor in, and therefore has therapeutic potential for, the treatment of human ESCC.

Long Noncoding RNA-LET Suppresses Tumor Growth and EMT in Lung Adenocarcinoma.

Recently, many studies showed that long noncoding RNAs (lncRNAs) are involved in tumor progression. It is reported that lncRNA-LET is downregulated and has antitumor effect on several types of cancer. This study focuses on the role of lncRNA-LET on lung adenocarcinoma (LAC) progression. RT-PCR results indicated that frequent downregulation of lncRNA-LET in LAC tissues was related to clinicopathologic factors. lncRNA-LET knockdown significantly promoted LAC cell proliferation, invasion, and migration while lncRNA-LET overexpression obviously inhibited LAC cell proliferation, invasion, and migration, indicating a tumor inhibition of lncRNA-LET in LAC progression. Besides, lncRNA-LET inhibited EMT and negatively regulated Wnt/ß-catenin pathway in part. Our study suggests that lncRNA-LET exhibits an important tumor-suppressive effect on LAC progression by inhibiting EMT and Wnt/ß-catenin pathway, which provides potential therapeutic targets for LAC.

A modified tricuspid valve annuloplasty technique for functional tricuspid regurgitation.

BACKGROUND: Functional tricuspid regurgitation often occurs in patients with concomitant left sided, valve disease. Several types of tricuspid valve annuloplasty have been described, but there is no consensus on the management of functional tricuspid regurgitation. We report a modified annuloplasty technique and compare its efficacy with the conventional Kay technique. METHODS: A retrospective review was made of 60 patients who received tricuspid valve annuloplasty (group A, modified method; group B, Kay technique) and the early and midterm outcomes of modified method and Kay technique were compared. RESULTS: Three patients underwent ring annuloplasty using a semirigid Carpentier-Edwards ring due to failing suture annuloplasty. All patients were completely cured when they left the hospital. The follow-up time was (32 ± 7) months in group A and (30 ± 7) months in group B. After three years, tricuspid regurgitation decreased by more than two grades in 13 patients in group A and 11 in group B. The mean postoperative regurgitation grade in group A was lower than group B at 12, 24 and 36 months but not significantly. Three of 28 patients developed recurrent tricuspid regurgitation in group A and five of 26 patients in group B during the follow-up period (three deaths and three ring annuloplasties excluded). Freedom from recurrent tricuspid regurgitation in group A was higher than that group B at all follow-up points. Postoperative right atrium diameter, right ventricle endodiastolic dimension and tricuspid regurgitation area decreased obviously in both groups. The right ventricle endodiastolic dimension and tricuspid regurgitation area improved more significantly in group A than group B over three years of follow-up, CONCLUSIONS: The modified annuloplasty technique achieved the same outcomes as the conventional Kay annuloplasty over the first three years postoperation. As this modified technique is simple and less expensive, it is another option for correction of functional tricuspid regurgitation.

A functional variant in the cystathionine ß-synthase gene promoter significantly reduces congenital heart disease susceptibility in a Han Chinese population.

Homocysteine is an independent risk factor for various cardiovascular diseases. There are two ways to remove homocysteine from embryonic cardiac cells: remethylation to form methionine or transsulfuration to form cysteine. Cystathionine ß-synthase (CBS) catalyzes the first step of homocysteine transsulfuration as a rate-limiting enzyme. In this study, we identified a functional variant -4673C>G (rs2850144) in the CBS gene promoter region that significantly reduces the susceptibility to congenital heart disease (CHD) in a Han Chinese population consisting of 2 340 CHD patients and 2 270 controls. Individuals carrying the heterozygous CG and homozygous GG genotypes had a 15% (odds ratio (OR) = 0.85, 95% confidence interval (CI) = 0.75-0.96, P = 0.011) and 40% (OR = 0.60, 95% CI = 0.49-0.73, P = 1.78 × 10(-7)) reduced risk to develop CHD than the wild-type CC genotype carriers in the combined samples, respectively. Additional stratified analyses demonstrated that CBS -4673C>G is significantly related to septation defects and conotruncal defects. In vivo detection of CBS mRNA levels in human cardiac tissues and in vitro luciferase assays consistently showed that the minor G allele significantly increased CBS transcription. A functional analysis revealed that both the attenuated transcription suppressor SP1 binding affinity and the CBS promoter hypomethylation specifically linked with the minor G allele contributed to the remarkably upregulated CBS expression. Consequently, the carriers with genetically increased CBS expression would benefit from the protection due to the low homocysteine levels maintained by CBS in certain cells during the critical heart development stages. These results shed light on unexpected role of CBS and highlight the importance of homocysteine removal in cardiac development.Cell Research advance online publication 18 September 2012; doi:10.1038/cr.2012.135.

CD133 is a temporary marker of cancer stem cells in small cell lung cancer, but not in non-small cell lung cancer.

Lung cancer is the most common cause of cancer-related death worldwide. Current investigations in the field of cancer research have intensively focused on the 'cancer stem cell' or 'tumor-initiating cell'. While CD133 was initially considered as a stem cell marker only in the hematopoietic system and the nervous system, the membrane antigen also identifies tumorigenic cells in certain solid tumors. In this study, we investigated the human lung cancer cell lines A549, H157, H226, Calu-1, H292 and H446. The results of real-time PCR analysis after chemotherapy drug selection and the fluorescence-activated cell sorting analysis showed that CD133 only functioned as a marker in the small cell lung cancer line H446. The sorted CD133+ subset presented stem cell-like features, including self-renewal, differentiation, proliferation and tumorigenic capacity in subsequent assays. Furthermore, a proportion of the CD133+ cells had a tendency to remain stable, which may explain the controversies arising from previous studies. Therefore, the CD133+ subset should provide an enriched source of tumor-initiating cells among H446 cells. Moreover, the antigen could be used as an investigative marker of the tumorigenic process and an effective treatment for small cell lung cancer.