Colon Cancer-associated Transcript 2 Expression Research Articles

Assessment of long noncoding RNA CCAT1 using real time-polymerase chain reaction in colorectal cancer patients

Colorectal cancer (CRC) is linked to high mortality, mainly when discovered in its advanced stages. Several studies have pointed to the role of epigenetic factors in CRC and other cancers. Long non-coding RNAs (lncRNAs) are involved in the initiation, progression, metastasis, and modulation of the response to chemotherapeutic modalities of CRC as vital contributors to epigenetic mechanisms. Colon cancer-associated transcript-1 (CCAT1) is one of the lncRNAs that have been dysregulated in serum samples, providing a non-invasive route for diagnosing CRC patients. This study aimed to determine the role of CCAT1 expression as diagnostic and prognostic markers. We tested the associations of CCAT1 expression with serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). The study included three groups: 41 patients with colorectal cancer, 39 patients with precancerous benign colorectal diseases, and 20 normal control individuals. CEA and CA 19-9 were measured by an immunoassay automated system. The expression level of CCAT1 was assessed by a real-time polymerase chain reaction. There was a statistically significant elevation of serum CEA levels in patients with CRC compared to patients with precancerous benign colorectal diseases. Furthermore, there was no statistically significant difference in serum CA 19-9 levels between all groups (p = 0.102). Interestingly, CCAT1 expression was significantly upregulated in the blood of CRC patients compared to the precancerous benign colorectal diseases group (p = 0.009) and the control group (p<0.001). Also, expression of CCAT1 was significantly elevated in patients with precancerous benign colorectal diseases compared to the control group (p=0.004). In conclusion, measuring the expression level of CCAT1 is more advised than assessment of CEA and CA 19-9 for the early diagnosis and prognosis of colorectal cancer.

LncRNA CCAT1 participates in pancreatic ductal adenocarcinoma progression by forming a positive feedback loop with c-Myc.

Long noncoding RNAs (lncRNAs) play fundamental roles in cancer development; however, the underlying mechanisms for a large proportion of lncRNAs in pancreatic ductal adenocarcinoma (PDAC) have not been elucidated. The expression of colon cancer-associated transcript-1 (CCAT1) in PDAC specimens and cell lines was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The function of CCAT1 was examined in vitro and in vivo. The interactions among CCAT1, miR-24-3p and c-Myc were determined by bioinformatics analysis, RNA immunoprecipitation (RIP), dual-luciferase reporter assay, and rescue experiments. CCAT1 was significantly increased in PDAC, positively correlated with PDAC progression and predicted a worse prognosis. Furthermore, CCAT1 enhanced Adenosine triphosphate (ATP) production to facilitate PDAC cell proliferation, colony formation and motility in vitro and tumor growth in vivo. CCAT1 may serve as an miR-24-3p sponge, thereby counteracting its repression by c-Myc expression. Reciprocally, c-Myc may act as a transcription factor to alter CCAT1 expression by directly targeting its promoter region, thus forming a positive feedback loop with CCAT1. Collectively, these results demonstrate that a positive feedback loop of CCAT1/miR-24-3p/c-Myc is involved in PDAC development, which may serve as a biomarker and therapeutic target for PDAC.

Knockdown of Long Noncoding RNA CCAT2 Suppresses Malignant Phenotype in Human Laryngeal Squamous Cell Carcinoma.

This study aimed to explore the biological role and mechanism underlying the effects of colon cancer-associated transcript 2 (CCAT2), a long noncoding RNA (lncRNA) in human laryngeal squamous cell carcinoma (LSCC). CCAT2 expression levels in clinical LSCC samples and TU-212 cell line were evaluated by quantitative real-time PCR. The correlation of CCAT2 expression level with clinical-pathological characteristics of patients and their prognosis was analyzed. The functional role of CCAT2 in human LSCC was assessed by Cell Counting Kit-8, Transwell assay, flow cytometric analysis, and LSCC xenograft experiment in vivo. The expression of potential targeted proteins was detected by Western blotting and immunohistochemistry. We found that expression of CCAT2 was significantly elevated in LSCC tissues and TU-212 cells (p<0.05). Survival analysis showed that LSCC patients with high expression of CCAT2 had a shorter 5-year overall survival rate than those with low expression (p<0.05). In addition, CCAT2 silencing with short hairpin RNA significantly decreased the proliferative and invasive potential of TU-212 cells (p<0.05) and promoted their apoptosis. In Nude mice, CCAT2 knockdown suppressed the growth of tumor and decreased its volume and weight in comparison with the controls (p<0.05). In TU-212 cells, CCAT2 silencing with short hairpin RNA significantly down-regulated the expression of β-catenin and CDK8 (p<0.05). Thus, knockdown of CCAT2 suppresses proliferation and invasion of the cells and inhibits Wnt/β-catenin signaling pathway in LSCC, which indicates novel therapeutic targets and prognostic indicators in patients with LSCC.

Abstract 1486: The regulatory mechanism of LncRNA CCAT1 via three-dimensional genome structure in colorectal cancer cells

Abstract Kruppel-like factor 5 (KLF5) belongs to zinc finger transcription factors and its expression is up-regulated in specific cancer types, such as gastrointestinal cancer and squamous carcinoma. It is reported that KLF5 is associated with the maintenance of stemness and essential for the integrity of intestinal epithelium and oncogenesis. Colon Cancer Associated Transcript 1 (CCAT1) is a long noncoding RNA that was initially found to be up-regulated in colorectal cancer (CRC), and studies have reported that high CCAT1 expression is associated with poor prognosis in CRC patients. In this study, we performed in vitro enChIP (engineered DNA-binding molecule-mediated chromatin immunoprecipitation)-sequencing to identify the KLF5 enhancer region which interacts with KLF5 promoter region. We successfully identified the KLF5 enhancer region in colorectal cancer cells and further found that KLF5 promoter region interacted with the transcription start site (TSS) region of CCAT1 gene, which is located at the different chromosome. Our results indicated that KLF5 protein participated in the core regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which contributes to construction of the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. We also found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples and that KLF5 regulated CCAT1 expression. In conclusion, our data propose the mechanistic insight that KLF5 protein constructs the core regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC. Citation Format: Yuhki Yokoyama, Hirofumi Yamamoto. The regulatory mechanism of LncRNA CCAT1 via three-dimensional genome structure in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1486.

Clinicopathological and prognostic value of long non-coding RNA CCAT1 expression in patients with digestive system cancer.

Colon cancer associated transcript-1 (CCAT1) is known to play an important role in numerous types of human cancer, including bladder, prostate and ovarian cancer. However, a consistent perspective has not been established in digestive system cancer (DSC). To explore the prognostic value of CCAT1 in patients with DSC, a meta-analysis was performed. A systematic search of PubMed, Embase, Web of Science, China National Knowledge Infrastructure, Chinese Biological Medical Literature database, Cochrane Library and WanFang database was applied to select eligible articles. Pooled odds ratios (ORs) or hazard ratios (HRs) with corresponding 95% confidence intervals (CIs) were calculated to estimate the effects of CCAT1 on pathological or clinical features. A total of 1,719 patients from 12 eligible articles were enrolled in the meta-analysis. The results revealed that elevated CCAT1 expression was significantly related to larger tumor size (OR, 1.81; 95% CI, 1.31-2.48), poorer differentiation (OR, 0.45; 95% CI, 0.31-0.64), earlier lymph node metastasis (OR, 3.14; 95% CI, 2.34-4.22) and advanced TNM stage (OR, 3.08; 95% CI, 2.07-4.59). In addition, high CCAT1 expression predicted a poorer outcome for overall survival rate (HR, 2.37; 95% CI, 2.11-2.67) and recurrence-free survival rate (HR, 2.16, 95% CI, 1.31-3.57). High expression levels of CCAT1 were therefore related to unfavorable clinical outcomes of patients with DSC. These results demonstrated that CCAT1 could serve as a prognostic predictor in human DSC.

Effect of long noncoding RNA CCAT2 on drug sensitivity to 5-fluorouracil of breast cancer cells through microRNA-145 meditated by p53.

The current study was set out to investigate the mechanism by which silenced long noncoding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) modulates the cell growth, migration, invasion, and drug sensitivity of breast cancer (BC) cells to 5-fluorouracil (5-Fu) with the involvement of miR-145 and p53. First, high CCAT2 expression was presented in BC cells and tissues. Subsequently, the links between CCAT2 expression and BC clinicopathological features were analyzed. Highly-expressed CCAT2 was linked to lymph node metastasis, positive progesterone receptor, estrogen receptor, and Ki-67 of BC cells. Then, the gain- and loss-of-function approaches were performed to measure the regulatory role of CCAT2 in the biological processes of BC cells. Silencing of CCAT2 suppressed in vitro cell growth, proliferation, invasion, migration abilities, and epithelial-mesenchymal transformation, increased cell apoptosis, and enhanced drug sensitivity of BC cells. Silencing of CCAT2 upregulated miR-145, which was poorly expressed in drug-resistant BC cells. p53 can bind to the miR-145 promoter region and increase miR-145 expression. Upregulation of miR-145 induced by silencing of CCAT2 can be invalidated by p53-siRNA. To conclude, p53-induced activation of miR-145 could be inhibited by CCAT2, while overexpression of CCAT2 could improve the drug resistance of BC cells to 5-Fu.

LncRNA-CCAT1/miR-152-3p is involved in CSE-induced inflammation in HBE cells via regulating ERK signaling pathway

Emerging studies have noted that dysregulated long non-coding RNAs (lncRNAs) are implicated in the pathological processes of chronic obstructive pulmonary disease (COPD). LncRNA colon cancer-associated transcript 1 (CCAT1) plays well-defined roles in the inflammatory progression. The study aims to figure out the effect and regulatory mechanism of CCAT1 in the cigarette smoke induced inflammation in COPD. The results showed that CCAT1 was highly expressed in lung tissues of smokers with COPD compared with never-smokers without COPD. In human bronchial epithelial (HBE) cells, cigarette smoke extract (CSE) treatment led to an increase in CCAT1 expression in a dose- and time- dependent manner. Functional experiments showed that knockdown of CCAT1 amelioratedCSE-inducedinflammation. Mechanistically, CCAT1 directly targeted miR-152-3p, and miR-152-3p overexpression reversed the pro-inflammatory effects of CCAT1 on HBE cells. Subsequently, miR-152-3p was found to regulate ERK signaling pathway. PD98059, an ERK specific inhibitor, reversed miR-152-3p knockdown mediated inflammation in HBE cells. In addition, CCAT1 acted as a sponge for miR-152-3p to positively regulate ERK signaling pathway. Overall, current findings suggest that CCAT1 promoted inflammation by activating ERK signal pathway via sponging miR–152–3p in CSE–treated HBE cells. These results may provide a novel therapeutic target for alleviating cigarette smoke mediated airway inflammation.

Dexmedetomidine protects H9C2 rat cardiomyocytes against hypoxia/reoxygenation injury by regulating the long non-coding RNA colon cancer-associated transcript 1/microRNA-8063/Wnt/β-catenin axis

ABSTRACT Dexmedetomidine (Dex) protects the heart from ischemia/reperfusion (I/R) injury. The differential expression of long non-coding RNAs (lncRNAs) is associated with myocardial injury, but whether the lncRNA colon cancer-associated transcript 1 (CCAT1) is associated with Dex-mediated myocardial protection remains unclear. In this study, a hypoxia/reoxygenation (H/R) H9C2 model was established to simulate the in vitro characteristics of I/R. CCAT1 and microRNA (miR)-8063 expression levels in H/R H9C2 cells pretreated with Dex were determined via quantitative reverse transcription-polymerase chain reaction. The survival and apoptotic rates of H9C2 cells were determined via cell counting kit-8 and flow cytometry assays. Wnt3a, Wnt5a, and β-catenin protein levels were measured via western blotting. Luciferase and RNA immunoprecipitation assays were used to explore the binding relationship between miR-8063 and CCAT1. Dex pretreatment increased H/R H9C2 cell viability and CCAT1 expression, while decreasing the cell apoptosis and Wnt3a, Wnt5a, and β-catenin protein levels. Knockdown of CCAT1 abolished the protective effects of Dex on H/R H9C2 cells, and the downregulation of miR-8063 expression eliminated the effect of CCAT1 knockdown. These results revealed that CCAT1, a sponge for miR-8063, is involved in Dex-mediated H9C2 cell H/R injury by negatively targeting miR-8063 and inactivating the Wnt/β-catenin pathway. Dex protects H9C2 cells from H/R impairment by regulating the lncRNA CCAT1/miR-8063/Wnt/β-catenin axis.

LncRNA CCAT2 expression at diagnosis predicts imatinib response in chronic phase chronic myeloid leukemia patients

Long noncoding RNAs (lncRNAs) are identified as key players in the initiation, development, and prognosis of chronic myeloid leukemia (CML). Some lncRNAs are expected to serve as diagnostic biomarkers, predictors of clinical outcomes and therapeutic targets. We aimed to examine the expression of lncRNA colon cancer-associated transcript 2 (CCAT2) in CML patients, as well as to correlate CCAT2 expression with response to imatinib therapy. 43 newly diagnosed patients with chronic phase CML were included, and 30 healthy persons were selected as controls. Real-time reverse transcription PCR was performed to analyze the expression of CCAT2 in peripheral blood mononuclear cells. Our results reported for the first time the upregulated expression of CCAT2 in CML patients as compared with controls (P < 0.001). We demonstrated significant association between CCAT2 expression and therapy response at 3 months, and at 6 months (P = 0.004, and P = 0.005; respectively). Moreover, CCAT2 expression was significantly associated with spleen size (P = 0.006) and EUTOS sore (P = 0.030). LncRNA CCAT2 is highly expressed in the peripheral blood of CML patients, and the enhanced expression at diagnosis is linked to imatinib resistance. CCAT2 is expected to become a reliable molecular marker for predicting imatinib response in chronic phase CML patients.

Long Non-Coding RNA CCAT2 Activates RAB14 and Acts as an Oncogene in Colorectal Cancer.

Here, we investigated the clinicopathological and prognostic potential of the long noncoding RNA Colon Cancer-Associated Transcript 2 (CCAT2) in human colorectal cancer (CRC). We used qPCR to quantify CCAT2 levels in 44 pairs of CRC tissues and adjacent nontumor and healthy colon mucosa tissues, and in several CRC cell lines (SW620, SW480, HT-29, LOVO, HCT116 and DLD-1) and normal human colorectal epithelial cells (HFC). We assessed the effects of CCAT2 overexpression or knockdown on the proliferation, migration and invasion by SW620 and LOVO cells using CCK-8, transwell, and wound−healing assays, respectively. We also investigated the potential interaction between CCAT2 and TAF15 through RNA pull down and rescue experiments. Lastly, we evaluated the expression of the cell cycle progression markers and GSK3β signaling pathway proteins using Western blotting. Our results showed that CCAT2 was upregulated in CRC tissues and cell lines as com-pared to controls. Ectopic expression of CCAT2 promoted CRC cell proliferation, migration and invasion, likely through direct interaction with TAF15, transcriptional activation of RAB14, and activation of the AKT/GSK3β signaling pathway. In vivo, CCAT2 promoted CRC cell growth and metastasis in nude mice. Taken together, these results highlight the actions of CCAT2 as a CRC oncogene.

A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells

BackgroundKLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC).MethodsIn vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter.ResultsWe revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples.ConclusionsOur data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.

LncRNA CCAT1 downregulation increases the radiosensitivity of non-small cell lung cancer cells.

This study aims to investigate if the radiosensitivity of non-small cell lung cancer (NSCLC) cells can be regulated by long noncoding RNA (lncRNA) colon cancer associated transcript1 (CCAT1). CCAT1 was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in NSCLC cells (A549, H1299, SK-MES1, H460, and H647) and human bronchial epithelial cells (16HBE). H460 and A549 cells were then selected for the determination of CCAT1 expression after exposure to radiation (0, 2, 4, 6Gy) at different time points (0, 6, 12, 24 h). Colony forming assay was performed to evaluate the effects of CCAT1 siRNA or pcDNA3.1-CCAT1 vector on the radiosensitivity of H460 and A549 cells. Then, flow cytometry, western blotting and qRT-PCR were also conducted. CCAT1 was increased in NSCLC cells when compared with 16HBE cells, which was declined in a time- and dosage-dependent manner after exposure to radiation. The H460 and A549 cell colonies were decreased and the γ-H2AX expression was elevated with the increase of radiation dosage, which was more obvious in those transfected with CCAT1 siRNA. CCAT1 downregulation arrested NSCLC cells at G2/M phase. Moreover, the enhanced apoptosis of radiotherapy-treated NSCLC cells with reductions of p-p38/p38, p-ERK/ERK, and p-JNK/JNK was promoted by siCCAT1, but it was reversed by pcDNA3.1-CCAT1 vector. Inhibiting CCAT1 regulated cell cycle, DNA damage and apoptosis of NSCLC cells, and affected MAPK pathway, eventually improving the radiosensitivity of NSCLC.

Knockdown of long non-coding RNA CCAT2 suppresses growth and metastasis of esophageal squamous cell carcinoma by inhibiting the β-catenin/WISP1 signaling pathway.

ObjectiveLong non-coding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) plays oncogenic roles in several cancers, including esophageal squamous cell carcinoma (ESCC). However, the specific mechanism of how CCAT2 influences ESCC tumorigenesis is still unknown.MethodsUsing RT-qPCR, the mRNA expression levels of CCAT2 in 33 paired ESCC and adjacent non-cancer tissues and cell lines were measured. Lentiviral vector sh-CCAT2 was designed and transfected into TE10 cells. CCK-8 and transwell assays were employed to detect the effects of CCAT2 knockdown on cell proliferation and invasion, respectively. RT-qPCR and western blots were used to detect the effects of CCAT2 knockdown.ResultsCCAT2 was overexpressed in ESCC tissues compared with corresponding adjacent tissues. CCAT2 knockdown could suppress cell proliferation and invasion in vitro. Furthermore, knockdown of CCAT2 could suppress the mRNA and protein levels of β-catenin and Wnt-induced-secreted-protein-1 (WISP1), as well as the mRNA levels of their downstream targets VEGF-A, MMP2, and ICAM-1. High expression of CCAT2 and WISP1 were associated with poor prognosis of ESCC patients.ConclusionsIn conclusion, a novel CCAT2/β-catenin/WISP1 axis was revealed in ESCC progression and may provide a promising therapeutic target against ESCC. CCAT2 and WISP1 are potential molecular biomarkers for predicting prognosis of ESCC.

RETRACTED: Pancreatic cancer cells-derived exosomal long non-coding RNA CCAT1/ microRNA-138-5p/HMGA1 axis promotes tumor angiogenesis

Pancreatic cancer (PC) cells-derived exosomes could mediate angiogenesis of human microvascular endothelial cells (HUVECs) in PC. Considering that, this research was implemented to figure out the concrete role of PC cells-derived exosomal long non-coding RNA colon cancer-associated transcript-1 (CCAT1) in PC with its regulation on microRNA-138-5p/high mobility group A1 (miR-138-5p/HMGA1) axis. PC tissues and normal tissues were resected. PC cells (PANC-1) were interfered with plasmids to change CCAT1 and/or miR-138-5p expression. Exosomes were isolated from PANC-1 cells and co-cultured with HUVECs. The proliferation and apoptosis of PANC-1 and HUVECs were examined. The angiogenic ability of HUVECs was tested in vivo in xenografted tumors and in vitro. CCAT1, miR-138-5p and HMGA1 expression were determined, as well as their interactions. CCAT1 and HMGA1 expression were raised while miR-138-5p expression was reduced in PC. Silencing CCAT1 disrupted cell proliferation and stimulated apoptosis of PANC-1 cells. Knocked down CCAT1 from PANC-1 cells-derived exosomes promoted apoptosis and repressed proliferation of HUVECs. Down-regulated/up-regulated CCAT1 from PANC-1 cells-derived exosomes destroyed/enhanced the angiogenic ability of HUVECs in vivo and in vitro. CCAT1 mediated HMGA1 through competitively binding to miR-138-5p. Overexpression of miR-138-5p antagonized the effects of up-regulated CCAT1 on angiogenesis of HUVECs in vitro. It is informative that PANC-1 cells-derived exosomal CCAT1 strengthens angiogenesis of HUVECs through binding to miR-138-5p to elevate HMGA1 expression.

Clinical value of lncRNA CCAT1 in serum extracellular vesicles as a potential biomarker for gastric cancer.

Long non-coding RNAs (lncRNAs) in extracellular vesicles (EVs) are considered to be novel non-invasive biomarkers for gastric cancer (GC). lncRNA colon cancer-associated transcript 1 (CCAT1) is aberrantly expressed in certain types of cancer. However, the role of EV lncRNA CCAT1 in patients with GC remains unclear. The current study aimed to assess the expression levels of lncRNA CCAT1 in the serum EVs of patients with GC and evaluate its potential clinical value. EVs were isolated from serum using a commercial kit and ultracentrifugation, and were identified by transmission electron microscopy, nanoparticle tracking analysis and western blotting. Serum EV lncRNA CCAT1 levels in patients with GC, chronic gastritis or atypical hyperplasia and healthy control subjects were detected by reverse transcription-quantitative PCR. Additionally, lncRNA CCAT1 was detected in GC and adjacent non-cancerous tissue samples. Serum EVs were successfully isolated and identified in all patients. The results revealed that serum EV lncRNA CCAT1 levels in patients with GC were significantly higher compared with those in healthy controls, patients with chronic gastritis or atypical hyperplasia (all P<0.05). Additionally, EV lncRNA CCAT1 expression levels were significantly different among various groups based on the depth of invasion, distant metastasis and the Tumor-Node-Metastasis stage. The area under the curve (AUC) value of EV lncRNA CCAT1 was 0.890 [95% confidence interval (CI), 0.826–0.937] with 79.6% sensitivity and 92.6% specificity. The combination of EV lncRNA CCAT1 and carcinoembryonic antibody produced an AUC value of 0.910 (95% CI, 0.849–0.951) with the sensitivity and specificity of 80.5 and 92.6%, respectively. In addition, lncRNA CCAT1 was determined to be stable in serum EVs. The expression levels of lncRNA CCAT1 in GC tissue were positively correlated with those in serum EVs, and high levels of lncRNA CCAT1 were associated with a low disease-free survival rate in patients with GC. The results of the present study demonstrated that serum EV lncRNA CCAT1 levels were upregulated in patients with GC compared with those healthy subjects and patients with other illnesses, and may therefore be used as a novel biomarker for this type of cancer.

Expression of lncRNA CCAT2 in children with neuroblastoma and its effect on cancer cell growth.

The aim of this study was to determine the expression of long-chain non-coding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) in children with neuroblastoma and its effect on cancer cell growth. A polymerase chain reaction assay was carried out to quantify lncRNA CCAT2 miRNA in neuroblastoma cells, corresponding paracancerous cells, SH-SY5Y and SK-N-SH cells, and human umbilical vein endothelial cells (HUVEC), and two groups of children with different lncRNA CCAT2 expression were compared in clinical pathological parameters and prognosis. CCAT2-NC and si-CCAT2 were transfected into SH-SY5Y cells, separately. Then a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was carried out to analyze the cell proliferation, migration, and invasion ability, a flow cytometry to detect cell apoptosis, and a Western blotting (WB) assay to quantify p53 and Bcl-2 proteins. lncRNA CCAT2 expression in cancer tissues of children with neuroblastoma was notably higher than that in corresponding paracancerous tissues (P < 0.05), and children with different tissue differentiation, tumor staging, and lymph node metastasis (LNM) showed notably different lncRNA CCAT2 expression (P < 0.05). In addition, children with neuroblastoma in the high lncRNA CCAT2 expression group showed lower 3-year survival rate than those in the low expression group (P < 0.05). Multivariate analysis revealed that tissue differentiation, tumor-node-metastasis staging, LNM, and lncRNA CCAT2 expression were all independent risk factors affecting the prognosis of children with neuroblastoma (all P < 0.05). Compared with HUVEC cells, SH-SY5Y and SK-N-SH cells showed notably up-regulated lncRNA CCAT2, and the expression of it in SH-SY5Y was higher than that in SK-N-SH cells (P < 0.05). Compared with the CCAT2-NC group, the si-CCAT2 group presented notably down-regulated CCAT2 (P < 0.05). Moreover, according to the MTT assay, the si-CCAT2 group showed notably weakened cell viability and proliferation than the CCAT2-NC group (both P< 0.05), and SH-SY5Y cells in the former group were less active than those in the latter group in terms of migration and invasion. The cell apoptosis rate of SH-SY5Y cells in the si-CCAT2 was higher than that in the CCAT2-NC. The results suggested that knock down of lncRNA CCAT2 could improve the apoptosis activity of neuroblastoma cells in children. According to the WB assay, the si-CCAT2 group showed notably higher p53 expression and notably lower Bcl-2 protein expression than the CCAT2-NC group (both P < 0.05). LncRNA CCAT2 can inhibit the proliferation of neuroblastoma cells and promote their apoptosis, which provides a basis for the treatment of neuroblastoma.

Long coding RNA CCAT2 enhances the proliferation and epithelial-mesenchymal transition of cervical carcinoma cells via the microRNA-493-5p/CREB1 axis

ABSTRACT Cervical cancer (CC) is one of the most common malignancies among women. It has been demonstrated that long coding RNAs (lncRNAs) play a crucial role in CC. The purpose of this study was to investigate the role of the colon cancer associated transcript 2 (CCAT2) lncRNA in CC and elucidate its possible mechanisms of action. The expression of CCAT2, the miR-493-5p microRNA (miRNA), and mRNA was detected using qRT-PCR. Cell viability, proliferation, and migration and invasion were determined using the MTT, colony formation, and transwell assays, respectively. The interactions between miR-493-5p and CCAT2 or cAMP response element-binding protein 1 (CREB1) were verified using the luciferase and RNA pull-down assays. The effects of CCAT2 knockdown on in vivo tumor growth were determined using tumor xenografts and immunohistochemistry assays. The expression of CCAT2 was upregulated in CC cells and tissues. However, the knockdown of CCAT2 inhibited the proliferation and epithelial-mesenchymal transition (EMT) of CC cells in vitro and suppressed tumor growth in vivo. Mechanistically, CCAT2 functions as a competing endogenous RNA (ceRNA) to upregulate the expression of CREB1 by binding to miR-493-5p. The overexpression of CREB1 or downregulation of miR-493-5p antagonized the effect of CCAT2 knockdown on the proliferation and EMT of CC cells. The knockdown of CCAT2 suppressed the aggressiveness of CC via the miR-493-5p/CREB1 axis. Therefore, CCAT2 is likely to be a promising therapeutic target for CC.

Erratum.

Long noncoding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) has been demonstrated to play an important role in diverse tumorigenesis. However, the biological function of lncRNAs in glioma is still unknown. In this study, we found that lncRNA CCAT2 was overexpressed in glioma tissues and cell lines and associated with tumor grade and size. Furthermore, patients with high levels of lncRNA CCAT2 had poorer survival than those with lower levels of lncRNA CCAT2. Knocking down lncRNA CCAT2 expression significantly suppressed the glioma cell growth, migration, and invasion, as well as induced early apoptosis of glioma cells in vitro. Moreover, lncRNA CCAT2 regulated epithelial–mesenchymal transition (EMT)-associated gene expression. In conclusion, lncRNA CCAT2 plays an important role in glioma tumorigenesis and progression and may act as a potential biomarker for therapeutic strategy and prognostic prediction.

CCAT2 contributes to progression and treatment resistance of thyroid carcinoma.

The aim of this study is to uncover the correlations of the expression of colon cancer associated transcript 2 (CCAT2) in the clinical papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC) specimens with the prognosis and chemoresistance of patients. The expression level of CCAT2 in the PTC and ATC specimens was determined using Real-Time quantitative Polymerase Chain Reaction (RT-qPCR), and the correlations of CCAT2 expression with the clinical features of patients were detected via χ2 test. Besides, survival analysis was conducted to verify the relation between CCAT2 expression and patients' survival. After knockdown or overexpression of CCAT2, the changes in the proliferation ability of human thyroid carcinoma cells were examined via Cell Counting kit-8 (CCK-8) assay, and the half maximal inhibitory concentration (IC50) values of doxorubicin and cisplatin were measured by methyl thiazolyl tetrazolium (MTT) assay. According to the χ2-test results, the expression of CCAT2 was notably correlated with the capsular invasion and lymph node metastasis of PTC, and the capsular invasion, tumor size, and lymph node metastasis of ATC. It was discovered through the survival analysis that the expression of CCAT2 was notably associated with the poor prognosis of ATC patients. After knockdown of CCAT2, both the proliferation ability and the IC50 values of doxorubicin and cisplatin substantially declined in human thyroid carcinoma cells. The opposite conditions were found after CCAT2 was overexpressed in human thyroid carcinoma cells. CCAT2 potentiates the proliferation ability and chemoresistance of cells, promotes the progression of thyroid carcinoma, and hinders the prognosis of ATC.

[Long non-coding RNA colon cancer associated transcript-2 from nasopharyngeal carcinoma-derived exosomes promotes angiogenesis].

Objective: To evaluate the role of exosomal long non-coding RNA (lncRNA) colon cancer associated transcript-2(CCAT2) in angiogenesis in nasopharyngeal carcinoma(NPC). Methods: Human umbilical vein endothelial cells (HUVECs) were divided into CNE2 supernatant coculture group and NP69 supernatant coculture group. The proliferation ability of HUVEC in each group was examined by CCK8. The lncRNA CCAT2 level in HUVEC was detected using real-time fluorescent quantitative PCR (qRT-PCR). HUVECs were divided into coculture group of serum in patients with NPC and coculture group of serum in healthy donors. The proliferation ability of HUVEC in each group was examined by CCK8. The lncRNA CCAT2 level in each group was detected using qRT-PCR. Exosomes from CNE2,NP69 supernatant were obtained by ultracentrifugation. HUVECs were divided into coculture group of CNE2's supernatant exosomes and coculture group of NP69's supernatant exosomes. Biological experiments like CCK8 were conducted to compare the angiogenesis ability of HUVEC in each group. shRNA transfection was used in HUVEC to suppress the expression of lncRNA CCAT2. Biological experiments like CCK8were conducted to compare the HUVEC's function with different expression of lncRNA CCAT2. CNE2 was transfected with shRNA, the total supernatant exosomal RNA was extracted, and the difference of exosomal lncRNA CCAT2 in CNE2 supernatant with low expression and normal expression of lncRNA CCAT2 was detected by qRT-PCR. The above mentioned two groups of exosomes were cocultured with HUVEC respectively. The difference of lncRNA CCAT2 expression in each group's HUVEC was detected by qRT-PCR and the function of HUVEC was examined with migration assay. GraphPad Prism 8 software was used for graphing and statistical analysis. Results: Compared with those in the NP69 supernatant coculture group, HUVECs in the CNE2 supernatant coculture group showed a higher proliferation ability and higher expression of lncRNA CCAT2(1 vs. 1.40±0.01, t=42.23, P=0.000). Compared with those in serum of healthy donors, HUVECs in serum of patients with NPC showed a higher proliferation ability and the expression of lncRNA CCAT2 increased (1 vs. 1.25±0.03, t=14.43,P=0.001). Compared with those in the coculture group of NP69 cells' supernatant exosomes, HUVEC in the coculture group of CNE2 cells' supernatant exosomes showed a higher proliferation ability and there were more cells that had passed the well during the migration experiment (53.12±2.13 vs. 154.74±4.17, t=37.73, P=0.000) and more knots formed in the tube-formation experiment(10.72±1.02 vs. 53.65±3.21, t=22.63, P=0.000). Compared with those in the sh-NC group, HUVEC in the sh-CCAT2 group showed a lower proliferation ability, and there were less cells that had passed the cell during the migration experiment(401.34±22.15 vs.138.25±6.85, t=23.19, P=0.000) and less MVC in the experiment of subcutaneous stroma embolization in nude mice(41.00±0.32 vs. 27.15±0.23, t=61.53, P=0.000). Compared with the supernatant exosomes in the sh-NC exosome group, the expression of lncRNA CCAT2 was lower in the supernatant exosomes of sh-CCAT2 group (1 vs. 0.65±0.02, t=30.31, P=0.000). Compared with those of the exosomes of sh-NC exosome group, the expression of lncRNA CCAT2 was lower in the HUVEC cocultured with exosomes of the sh-CCAT2 exosome group (1 vs. 0.73±0.01, t=46.77, P=0.000) and the exosomes of sh-CCAT2 exosome group showed a lower migration ability (389.73±26.34 vs. 190.54±8.36, t=12.47, P=0.001) after cocultured with HUVEC. Conclusion: Nasopharyngeal carcinoma-derived exosomal lncRNA CCAT2 could promote angiogenesis.