Linc-ROR regulates apoptosis in esophageal squamous cell carcinoma via modulation of p53 ubiquitination by targeting miR-204-5p/MDM2.

The long intergenic noncoding RNA, regulator of reprogramming (linc-ROR) has been reported to participate in tumorigenesis, while its functions and fundamental mechanisms in esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, gain-of-function assays showed that linc-ROR upregulation enhanced cell viability, promoted cell proliferation, and inhibited apoptosis. Mechanistically, the regulatory network of linc-ROR/miR-204-5p/MDM2 was established with bioinformatics analysis and online databases, then validated via dual-luciferase reporter assays, RNA immunoprecipitation assays in ESCC cells. Linc-ROR positively regulates the expression of MDM2 as a molecular sponge of miR-204-5p. Moreover, results of western blot and coimmunoprecipitation indicated that linc-ROR overexpression enhanced the ubiquitination level of p53, and its downstream apoptosis-related genes have showed higher bcl-2 expression, lower bax, and cleaved caspase-3 expressions, while miR-204-5p could counteract with this effect. Finally, small interfering RNAs tailored to linc-ROR were established to further evaluate its effects on ESCC comprehensively. In summary, this study revealed that linc-ROR modulated cell apoptosis and regulated p53 ubiquitination via targeting miR-204-5p/MDM2 axis, which provides a novel therapeutic insight into treatments for ESCC.

LincRNA-p21 leads to G1 arrest by p53 pathway in esophageal squamous cell carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC) is the fourth most common cause of cancer death in China. Long noncoding RNAs have emerged as critical regulators in cancer. Long intergenic noncoding RNA-p21, a kind of Long noncoding RNAs, LincRNA-p21 have been discussed dysregulated in several cancers, but its role in ESCC remains unknown. This study investigated the role of LincRNA-p21 in ESCC. Materials and methods: The LincRNA-p21 expression level and its association with esophageal cancer was determined in 64 tumor tissues of esophageal squamous cell carcinoma patients and cells using quantitative real-time reverse transcription PCR. Fluorescence in situ hybridization of single-RNA molecular probes was used to determine subcellular localization of LincRNA-p21. CCK8 and EdU assays were used for proliferation assay, flow cytometry was performed for apoptosis and cell-cycle distribution, and 24-well Mill cell chamber was made for measuring the abilities of migration and invasion after transfected with lentivirus-expressing LincRNA-p21 in EC109 cells. Then, quantitative real-time reverse transcription PCR and Western blot detected the expression of p21. Further, UC2288, an inhibitor of p21, was used to decrease the level of p21, and flow cytometry was used to detect cell cycle. Finally, screening for differential pathways from microarray analysis and expression of p53 and cyclin D were detected by Western blot. Results: LincRNA-p21 expression level was remarkably lower in tumor tissues versus nontumor tissues and lower in EC109 cells versus Het-1A cells. Statistical analysis found that LincRNA-p21 might enhance the risk of ESCC. We observed that LincRNA-p21 was expressed both in the nucleus and cytoplasm, and a larger proportion of LincRNA-p21 was observed in the cytoplasm. The results demonstrated that upregulating the expression of LincRNA-p21 could inhibit cell proliferation, migration, invasion, and the transition of cell cycle from G1 and promoted apoptosis of EC109. Then, we found that LincRNA-p21 promotes the expression of p21. Decreasing the level of p21 revealed that cell-cycle arrest was restored. Pathway analysis found p53 pathway was downregulated, and upregulation of LincRNA-p21 inhibited the expression of cyclin D. Conclusion: Our study suggests that LincRNA-p21 plays as a tumor inhibitor in ESCC development and LincRNA-p21 might induce G1 arrest through p53 signal pathway.

LincRNA-ROR promotes metastasis and invasion of esophageal squamous cell carcinoma by regulating miR-145/FSCN1.

BACKGROUND AND OBJECTIVE: In an attempt to discover a new biomarker for early diagnosis and prognosis of esophageal squamous cell carcinoma (ESCC), the regulation mechanism of large intergenic non-coding RNA-regulator of reprogramming (lincRNA-ROR) as a microRNA (miRNA) sponge was studied. PATIENTS AND METHODS: ROR expression in 91 pairs of ESCC tissue samples and matched adjacent tissues was quantified with real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The ROR-miRNA-mRNA regulatory network was built with 161 esophageal cancer (EC) tissues and 11 adjacent tumor tissues from The Cancer Genome Atlas (TCGA) database. A total of 96 cases of ESCC from TCGA database were collected for analysis on survival rates. The regulatory relationship between ROR, miR-145 and FSCN1 was verified in ESCC cells via qRT-PCR, dual luciferase reporter (DLR) assay, RNA immunoprecipitation (RIP) and Western blotting. The transwell method was used to detect cell migration and invasion. RESULTS: ROR expression in ESCC tumor tissues was significantly higher than in the adjacent tissues, p<0.001. The survival rate of ESCC patients with high ROR expression levels was lower than that of patients with low ROR expression levels (p<0.001). ROR overexpression could downregulate miR-145 by up to 50% was proven by RIP, DLR assay, and qRT-PCR. Two effective binding sites of ROR to miR-145 were verified by DLR assay. One of the sites has never been cited in the literature. The Western blotting results showed that FSCN1 was a downstream target of ROR/miR-145 (p<0.05). Transwell assays were used to show that overexpression of ROR enhanced migration and invasion behavior of ESCC and miR-145 hindered these effects. CONCLUSION: ROR acted as a competitive endogenous RNA (ceRNA) of miR-145 in ESCC. A novel, effective miR-145 binding site of ROR was discovered. The ROR/miR-145/FSCN1 pathway was shown to take part in the metastasis of ESCC. ROR is likely an oncogene biomarker for ESCC early diagnosis and prognosis.

lncRNA UCA1 inhibits esophageal squamous-cell carcinoma growth by regulating the Wnt signaling pathway.

Esophageal squamous-cell carcinoma (ESCC) is one of the most common tumors worldwide. Recent studies suggested that long noncoding RNAs (lncRNAs) might play a key role in regulating cellular processes and cancer progression. One of the lncRNAs, urothelial carcinoma associated 1 (UCA1), is known to be dysregulated in several cancers, including bladder carcinoma, colorectal, melanoma, breast, gastric, and ESCC. However, contributions of UCA1 to ESCC remain largely undiscovered. In order to understand the role and mechanisms underlying UCA1 in ESCC, the association of UCA1 expression with risk of esophageal cancer development was determined in 106 esophageal cancer tissues of ESCC patients and adjacent normal tissues using real-time reverse-transcription polymerase chain reaction (PCR). The relative expression of UCA1 was significantly reduced in cancer versus adjacent normal tissues suggesting an enhanced risk of esophageal cancer. To investigate the biological functions of UCA1 in ESCC, it was of interest to examine whether overexpression of UCA1 might influence cell proliferation, apoptosis, cell cycle distribution, migration, and invasion in vitro using EC109 cells. Our results demonstrated that UCA1 decreased cell proliferation, migration, invasion, and cell cycle progression of EC109 cells. Further, mRNA microarray analysis of overexpressed UCA1 in EC109 cells revealed that abnormal expression of UCA1 also inhibited the Wnt signaling pathway. Gene levels of DKK1 were elevated while C-myc fell significantly in overexpressed UCA1 EC109 cells. Interestingly, Western blot demonstrated no significant differences in relative expression of CTNNB1 (ß-catenin) but marked reduction in ß-catenin (active form) levels in both total and nuclear proteins. These results suggest that UCA1 may inhibit ESCC growth by regulating the Wnt signaling pathway. In conclusion, UCA1 may be a novel biomarker involved in ESCC development that may provide a potential therapeutic target for ESCC.