Retracted: microRNA-145 regulates tumor suppressor candidate 3 and mitogen-activated protein kinase pathway to inhibit the progression of colorectal cancer.

BACKGROUND: It has been reported that microRNA-145 (miR-145) is downregulated in various cancers, including colorectal cancer (CRC). However, the role of miR-145 in progress of CRC and its mechanism remains unclear. METHODS: The expressions of miR-145 and tumor suppressor candidate 3 (TUSC3) were determined in CRC tissues and cells by real-time quantitative polymerase chain reaction and Western blot analysis. The effects of miR-145 and TUSC3 on cell viability, migration, and invasion of CRC cells were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-trtrazolium bromide assay and trans-well chamber experiment, respectively. The interaction between miR-145 and TUSC3 was explored by bioinformatics analysis, luciferase reporter assay, and Western blot analysis. The abundances of mitogen-activated protein kinase (MAPK) signaling pathway-related proteins were measured by Western blot analysis. RESULTS: miR-145 expression was downregulated in CRC tissues and cell lines, and TUSC3 was upregulated in CRC tissues and correlated inversely with miR-145 abundance. Overexpression of miR-145 and knockdown of TUSC3 suppressed cell viability, migration, and invasion in LS174T and HCT116 cells. Moreover, TUSC3 was indicated as a novel target of miR-145 and its expression was negatively regulated by miR-145. Restoration of TUSC3 can partially reverse the inhibitory effects of miR-145 on phosphorylation of extracellular signal-regulated kinases 1 and 2 in CRC cells. CONCLUSION: miR-145 can inhibit the viability, migration, and invasion through addressing MAPK signaling pathway by targeting TUSC3 in CRC cells, providing a novel biomarker for treatment of CRC.

Effects of RNAi-mediated TUSC3 silencing on radiation-induced autophagy and radiation sensitivity of human lung adenocarcinoma cell line A549 under hypoxic condition.

This study examined the effects of RNAi-mediated TUSC3 silencing on radiation-induced autophagy and radiation sensitivity of human lung adenocarcinoma cell line A549 under hypoxic condition. Different CoCl2 concentrations were used to treat A549 cells and establish a CoCl2-induced hypoxic model of A549 cells. MTT and clone formation assays were used to determine the effects of different concentrations of CoCl2 on the growth and proliferation of A549 cells treated by different doses of X-ray irradiation. The siRNA-expressing vector was transfected by liposomes and for silencing of TUSC3. Flow cytometry was used to measure cell cycle changes and apoptosis rate. Real-time quantitative polymerase chain reaction (qRT-PCR) assay was performed to detect the expression of TUSC3 mRNA. Western blotting was applied to detect the changes of TUSC3, LC3, and p62 proteins under different CoCl2 concentrations and after siRNA silencing of TUSC3. The TUSC3 levels in A549 cells increased under hypoxic conditions in a dose-dependent manner (P < 0.05). Hypoxia inhibited the growth and proliferation of A549 cells and promoted apoptosis (P < 0.05). With an increasing dose of X-ray irradiation, A549 cells showed significantly increased growth and proliferation and decreased apoptosis (P < 0.05). After siRNA-TUSC3 was transfected by liposome, the TUSC3 level was substantially inhibited (P < 0.05). Silencing TUSC3 inhibited A549 cell growth and proliferation after radiotherapy under hypoxic condition, promoted apoptosis, increased G0/G1 phase cells, and reduced S phase cells (all P < 0.05). Hypoxia and radiation along with different CoCl2 concentrations could induce cell autophagy, which increased with concentration and dose, while silencing the TUSC3 gene inhibited autophagy (all P < 0.05). RNAi silencing of TUSC3 inhibited growth and proliferation, while enhanced apoptosis and radiation sensitivity of hypoxic A549 lung adenocarcinoma cells.

Decreased Tumor Suppressor Candidate 3 Predicts Poor Prognosis of Patients with Esophageal Squamous Cell Carcinoma.

TUSC3 was recently identified as a potential tumor suppressor gene in a variety of human malignancies. However, no data are currently available regarding the expressions of TUSC3 in esophageal cancer (ESCC).The purposes of this study was to investigated the expressions of TUSC3 in ESCC tissues and assess the relationship between TUSC3 levels and clinico-pathological characteristics of ESCC patients. TUSC3 protein expressions were evaluated by immunohistochemistry (IHC) on tissue microarray slides in esophageal cancer, which included 95 esophageal squamous carcinoma specimens (ESCC), and 75 normal esophageal mucosa (NEM). We found that TUSC3 in ESCC was significant lower than that in NEM (P=0.000). According to multi-clinical classifications, TUSC3 level varied significantly with TNM stage, T stage, and N stage (p<0.001, p=0.0368, p<0.0001, respectively). Univariate analysis showed that gender, TNM stage, T stage, N stage, TUSC3 expression were prognostic factors for survival. Multivariate analysis showed that in our study, only TUSC3 expression was independent prognostic factors for ESCC. Our results indicated for the first time, a combined analysis of TUSC3 expressions as well as the clinical variables will help predict the prognosis of ESCC patients. Further large-sample validation and functional analysis should be performed to evaluate its potential prognostic and therapeutic values for ESCC patients.

MiR-320d Inhibits Progression of EGFR-Positive Colorectal Cancer by Targeting TUSC3.

Background: The mechanism of miR-320d in EGFR-positive colorectal cancer (CRC) has not been fully elucidated. The aim of the present study was to explore the molecular mechanism of miR-320d in CRC. Methods: The miRNA microarray analysis was conducted to identify differential expressed miRNAs. The expression of miR-320d was validated using quantitative real-time PCR. EGFR-positive CRC cells were transfected with miR-320d mimic and inhibitor, after which cell proliferation, migration, and invasion were assayed. The relationship between miR-320d and TUSC3 was confirmed using bioinformatics and dual-luciferase reporter gene assays. Proteins involved in signaling pathways and the epithelial-mesenchymal transition were detected with Western blot. Results: We found that the miR-320d expression is associated with tumor size and distant metastasis in colorectal cancer. Overexpression of miR-320d in EGFR-positive HCT-116 and SW480 cells decreased not only the proliferation ability but also the invasion and migration ability. In addition, miR-320d had the ability to inhibit epithelial-to-mesenchymal transition. Luciferase assays revealed that miR-320d directly targets the 3'-UTR of TUSC3. TUSC3 was downregulated by miR-320d at both the protein and mRNA levels in EGFR-positive CRC cell lines. Conclusion: Generally, our results demonstrated that miR-320d could inhibit the malignant phenotype of EGFR-positive CRC through targeting TUSC3. The miR-320d might be a potential therapeutic target for EGFR-positive CRC.

Downregulation of microRNA-320a inhibits proliferation and induces apoptosis of retinoblastoma cells via targeting TUSC3.

MicroRNA (miR)-320a is specific to vertebrates and has been indicated to serve a role in a number of cancer types, such as gastric, colorectal, pancreatic and ovarian cancer. miR-320a has been reported to be expressed at high levels in retinoblastoma tissues; however its role and mechanism of function in retinoblastoma remain to be elucidated. The aim of the present study was to investigate the role of miR-320a in retinoblastoma cells and the underlying mechanisms. The expression of miR-320a in retinoblastoma cell lines Y79 and WERI-Rb-1, and normal human retinal pigment epithelial cell line ARPE-19 was examined via reverse transcription-quantitative PCR (RT-qPCR). TargetScan bioinformatics analysis and dual-luciferase reporter assay were used to predict and reveal the target gene of miR-320a. Target gene expression was detected via RT-qPCR in retinoblastoma cell lines and ARPE-19 cells. Subsequently, gain- or loss-of-function experiments for miR-320a and tumor suppressor candidate 3 (TUSC3) were performed to study the role of miR-320a/TUSC3 in retinoblastoma cells. Cell viability and apoptosis were assessed via MTT and flow cytometry analysis, respectively. Compared with ARPE-19 cells, miR-320a was prominently expressed in retinoblastoma cell lines. TUSC3 was predicted to be a target gene of miR-320a. Compared with ARPE-19 cells, the expression of TUSC3 in retinoblastoma cell lines was reduced. The results of MTT and flow cytometry analysis revealed that overexpression of TUSC3 reduced the viability of retinoblastoma cells and induced apoptosis. Additional analysis indicated that miR-320a inhibitor enhanced the expression of the target gene TUSC3, thereby inhibiting retinoblastoma cell viability and inducing apoptosis. The effects of miR-320a inhibitor on retinoblastoma cells were inhibited by TUSC3-short hairpin RNA. miR-320a regulated the viability and apoptosis of retinoblastoma cells via targeting TUSC3. Therefore, the present study provided a reference for investigating a potential target for the clinical treatment of retinoblastoma.

TUSC3 as a potential biomarker for prognosis in clear cell renal cell carcinoma.

The aim of the present study was to explore the expression levels of tumor suppressor candidate 3 (TUSC3) in human clear cell renal cell carcinoma (ccRCC) and its clinical value. Immunohistochemical staining, western blotting and reverse transcription-quantitative polymerase chain reaction were used to detect TUSC3 expression in paracancerous normal tissues and ccRCC tissues. The tissues were derived from the pathological specimens of 54 patients with ccRCC. Additionally, associations among TUSC3 expression and histological grade and clinicopathological staging of ccRCC were investigated. The results of these comparisons revealed that TUSC3 expression in ccRCC tissues was significantly lower than that in paracancerous tissues (P<0.05). TUSC3 expression in the high differentiation group was higher than that in the median and low differentiation groups (P<0.05). Expression levels of TUSC3 in stage I and II tissues were higher than those in stage III and IV tissues (P<0.05). The expression levels of TUSC3 in the lymph node metastasis group were lower than those in the non-lymph node metastasis group (P<0.05). In conclusion, the expression levels of TUSC3 in human ccRCC tissues were downregulated compared with those found in normal human renal tissue, and TUSC3 may inhibit the progression of ccRCC. Furthermore, the TUSC3 gene may be used as a promising tumor marker for the early diagnosis and prognosis of ccRCC.

Tumor suppressor candidate 3: A novel grading tool and predictor of clinical malignancy in human gliomas.

For several years, the cause of autosomal recessive mental retardation has been attributed to the deletion or mutation of a gene named tumor suppressor candidate 3 (TUSC3). Previous research has identified that TUSC3 is a potential tumor suppressor gene in oral epidermoid carcinoma, lung cancer and esophageal cancer. However, to the best of our knowledge, no previously published data has existed on the expression of TUSC3 in gliomas. The present study focused on the expression of TUSC3 in brain gliomas. Additionally, the present study sought to identify he association between TUSC3 expression and the typical clinical and pathological disease manifestations of gliomas. TUSC3 levels were evaluated using a western blot assay and immunohistochemistry on tissue microarray slides. Results indicated a significant decrease in TUSC3 expression in glioma tissues compared with the normal adjacent tissues. Furthermore, TUSC3 expression and World Health Organization grade demonstrated an inverse association in patients with glioma. This revealed that lower levels of TUSC3 in gliomas may be associated with a poorly-differentiated (high grade) tumor and thus a higher malignancy. Through the combination of the results of the present study and future research projects, TUSC3 may be a novel grading tool that assists with evaluating tumor malignancy and consequently a more active therapeutic regimen may be used in patients with glioma.

TUSC3 induces autophagy in human non-small cell lung cancer cells through Wnt/ß-catenin signaling.

We investigated the effects of tumor suppressor candidate 3 (TUSC3) on autophagy in human non-small cell lung cancer (NSCLC) cells. A total of 118 NSCLC patients (88 males and 30 females) who underwent surgery at our institute were enrolled in the study. Immunohistochemical analysis revealed that TUSC3 protein expression was lower in NSCLC specimens than adjacent normal tissue. Correspondingly, there was greater methylation of TUSC3 in NSCLC than adjacent normal tissue. After transient transfection of A549 NSCLC cells with constructs designed to up-regulate or down-regulate TUSC3 expression, we analyzed the effects of inhibiting the Wnt pathway (XAV939) and autophagy (chloroquine, CQ) on the behavior of NSCLC cells. We also performed TOP/FOP-Flash reporter assays, MTT assays, Annexin V-FITC/propidium iodide staining, and acridine orange staining to evaluate Wnt/ß-catenin signaling, cell proliferation, apoptosis, and autophagy, respectively. Expression of Wnt/ß-catenin pathway components and autophagy-related proteins was analyzed using qRT-PCR and Western blotting. We found that TUSC3 inhibited cell proliferation and promoted both apoptosis and autophagy in A549 cells. In addition, TUSC3 increased expression of autophagy-related proteins. It also increased expression of Wnt/ß-catenin signaling pathway components and promoted nuclear transfer of ß-catenin, resulting in activation of Wnt/ß-catenin signaling. TUSC3 thus induces autophagy in human NSCLC cells through activation of the Wnt/ß-catenin signaling pathway.

Quantitative detection of TUSC3 promoter methylation -a potential biomarker for prognosis in lung cancer.

Aberrant promoter methylation of tumor relevant genes frequently occurs in early steps of carcinogenesis and during tumor progression. Epigenetic alterations could be used as potential biomarkers for early detection and for prediction of prognosis and therapy response in lung cancer. The present study quantitatively analyzed the methylation status of known and potential gatekeeper and tumor suppressor genes [O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1A (RASSF1A), Ras protein activator like 1 (RASAL1), programmed cell death 4 (PDCD4), metastasis suppressor 1 (MTSS1) and tumor suppressor candidate 3 (TUSC3)] in 42 lung cancers and in corresponding non-malignant bronchus and lung tissue using bisulfite-conversion independent methylation-quantification of endonuclease-resistant DNA (MethyQESD). Methylation status was associated with clinical and pathological parameters. No methylation was found in the promoter regions of PDCD4 and MTSS1 of either compartment. MGMT, RASSF1A and RASAL1 showed sporadic (up to 26.2%) promoter methylation. The promoter of TUSC3, however, was frequently methylated in the tumor (59.5%), benign bronchus (67.9%) and alveolar lung (31.0%) tissues from each tumor patient. The methylation status of TUSC3 was significantly associated with smaller tumor size (P=0.008) and a longer overall survival (P=0.013). Pooled blood DNA of healthy individuals did not show any methylation of either gene. Therefore, methylation of TUSC3 shows prognostic and pathobiological relevance in lung cancer. Furthermore, quantitative detection of TUSC3 promoter methylation appears to be a promising tool for early detection and prediction of prognosis in lung cancer. However, additional studies are required to confirm this finding.

Tumor suppressor candidate 3 as a novel predictor for lymph node metastasis in lung cancer patients.

Tumor suppressor candidate 3 (TUSC3) was recently identified as a potential tumor suppressor gene in several cancer types. However, no data are currently available regarding the expression of TUSC3 in lung cancer. The present study investigated the expression of TUSC3 in patients with lung cancer and determined its association with the clinicopathological parameters of the disease. Cytoplasmic TUSC3 expression was evaluated by immunohistochemistry on tissue microarray slides, which included 35 small cell lung cancer (SCLC) specimens, 80 squamous cell lung cancer specimens (SCC), 80 adenocarcinoma lung cancer (ADC) specimens and 37 normal lung tissue specimens. Analysis showed significantly reduced TUSC3 expression in the SCLC patients, but not in the ADC and SCC patients, as compared with the normal controls. Additionally, TUSC3 expression in the patients with a degree of differentiation of 1-2 (well-moderately differentiated) was significantly higher than that in patients with a differentiation degree of 3-4 (poorly differentiated-undifferentiated). Further analysis showed that TUSC3 expression levels were negatively correlated with the degree of differentiation in the ADC and SCC patients. Notably, a marked decrease in TUSC3 expression was identified in the patients who were lymph node metastasis-positive (LNM+) compared with patients who were LNM-. Further analysis showed that significant differences in TUSC3 expression were identified among the different N stages (LNM status) in the SCLC, ADC and SCC patients. Correlation analysis also identified a negative correlation between TUSC3 expression and LNM in all three pathological types of lung cancer tested. Overall, these results indicated that a reduction in TUSC3 may be associated with a poorly-differentiated grade of lung cancer. Importantly, TUSC3 expression may be a useful predictor of LNM in lung cancer patients. A combined analysis of TUSC3 expression and the clinical variables will aid in predicting the incidence of LNM.