LncRNA PCAT6 promotes tumor progression in osteosarcoma via activation of TGF-β pathway by sponging miR-185-5p

Long noncoding RNAs (lncRNAs) play crucial roles in tumor development of osteosarcoma (OS). LncRNA PCAT6 was involved in the progression of multiple human cancers. However, the biological function of PCAT6 in OS remains largely unknown. We found that PCAT6 was elevated in OS tissues relative to that in their adjacent normal tissues. The upregulation of PCAT6 was positively associated with metastasis status and advanced stages and predicted poor overall and progression-free survivals in patients with OS. Functionally, silencing PCAT6 inhibited the proliferation, migration and invasion abilities of OS cells. Mechanistically, PCAT6, acting as a competitive endogenous RNA, upregulated expression of TGFBR1 and TGFBR2 to activate TGF-β pathway via sponging miR-185–5p. This study uncovers a novel underlying molecular mechanism of PCAT6-miR-185-5p-TGFBR1/2-TGF-β signaling axis in promoting tumor progression in OS, which indicates that PCAT6 may serve as a promising prognostic factor and therapeutic target again OS.     

lncRNA PCAT19 promotes the proliferation of laryngocarcinoma cells via modulation of the miR-182/PDK4 axis

The mechanism of tumorigenesis has not been fully identified in laryngeal cancer, which accounts for one fourth of patents with head and neck tumors. Long noncoding RNA PCAT19 has been shown to participate in the prostate cancer progression. However, little is known about the role of PCAT19 in the tumorigenesis of laryngeal cancer. In our study, it was shown that the expression levels of PCAT19 was increased in laryngeal tumor tissues and associated with decreased overall survival. Using laryngeal cancer cells lines HEp-2 and AMC-HN-8, it was demonstrated that knockdown of PCAT19 decreased the cell proliferation, increased the mitochondrial respiration, and inhibited the glycolysis. In detail, it showed that the PDK4 expression and PDHE1α phosphorylation levels were decreased upon the PCAT19 knockdown. Further studies indicated that miR-182 functioned as the bridge between PCAT19 and PDK4, which could also regulate the cellular metabolism thus affecting the cell proliferation. Furthermore, it was shown that the PCAT19/miR-182/PDK4 axis existed and regulated cell proliferation by modulating glycolysis and mitochondrial respiration. Finally, we showed that the PCAT19 knockdown decreased the tumor growth in vivo, possibly through regulating the miR-182/PDK4 axis. In conclusion, we demonstrated that lncRNA PCAT19 promoted cell proliferation and tumorigenesis by modulating the miR-182/PDK4 axis and the metabolism balance. PCAT19 might become a promising new target for laryngeal cancer therapeutics.     

Long non‐coding RNA LINC01535 promotes cervical cancer progression via targeting the miR‐214/EZH2 feedback loop

Long non‐coding RNAs (lncRNAs) have shown critical roles in multiple cancers via competitively binding common microRNAs. miR‐214 has been proved to play tumour suppressive roles in various cancers, including cervical cancer. In this study, we identified that lncRNA LINC01535 physically binds miR‐214, relieves the repressive roles of miR‐214 on its target EZH2, and therefore up‐regulates EZH2 protein expression. Intriguingly, we also found that EZH2 directly represses the expression of miR‐214. Thus, miR‐214 and EZH2 form double negative regulatory loop. Through up‐regulating EZH2, LINC01535 further represses miR‐214 expression. Functional experiments showed that enhanced expression of LINC01535 promotes cervical cancer cell growth, migration and invasion in vitro and cervical cancer xenograft growth in vivo. Reciprocally, LINC01535 knockdown suppresses cervical cancer cell growth, migration and invasion. Activation of the miR‐214/EZH2 regulatory loop by overexpression of miR‐214 or silencing of EZH2 reverses the roles of LINC01535 in promoting cervical canc`er cell growth, migration and invasion in vitro and cervical cancer xenograft growth in vivo. Clinically, LINC01535 is significantly up‐regulated in cervical cancer tissues and correlated with advanced clinical stage and poor prognosis. Moreover, the expression of LINC01535 is reversely associated with the expression of miR‐214 and positively associated with the expression of EZH2 in cervical cancer tissues. In conclusion, this study reveals that LINC01535 promotes cervical cancer progression via repressing the miR‐214/EZH2 regulatory loop.     

SMYD2 facilitates cancer cell malignancy and xenograft tumor development through ERBB2-mediated FUT4 expression in colon cancer

The aim of this study is to assess the expression levels of SMYD2 in human tissue samples and cells of colon cancer, and further explore the potential mechanisms of SMYD2 in colon cancer progression. Quantitative PCR and Immunohistochemical (IHC) assays were performed to detect SMYD2 expression in 76 tissue samples of colon cancer tissues and the corresponding normal tissues. The potential correlations between SMYD2 expression levels and clinical pathological features were assessed. We further detected the effects of SMYD2 on the proliferation, invasion and apoptosis of colon cancer cells and on ERBB2/FUT4 signaling pathway through Brdu assay, transwell assay and flow cytometry assay, respectively. The potential effects of SMYD2 on tumor growth were explored using an animal model. We demonstrated the possible involvement of SMYD2 in the progression of colon cancer. We found the high expression of SMYD2 in human colon cancer tissues and cells, and found the correlations between SMYD2 expression and the clinicopathological features including vascular invasion (P?=?0.007*), TNM stage (P?=?0.016*) and lymph node metastasis (P?=?0.011*), of patients with colon cancer. Our data further confirmed that SMYD2 affects cell proliferation, invasion, and apoptosis of colon cancer cells via the regulation of ERBB2/FUT4 signaling pathway. We also demonstrated SMYD2 contributed to tumor growth of colon cancer cells in vivo. We investigated the potential involvement of SMYD2 in the progression of colon, and therefore confirmed SMYD2 as a possible therapeutic target for colon cancer.

     

Bmi-1和EZH2基因在小细胞肺癌中的表达及其意义

目的探讨Bmi-1和EZH2基因在小细胞肺癌中的表达及其意义。方法收集武汉大学人民医院病理科2006-2009年非小细胞肺癌存档蜡块40例,采用免疫组织化学S-P法检测40例非小细胞肺癌及癌旁组织中Bmi-1和EZH2基因的表达水平,并采用HPIAS-1000高清晰度彩色病理图文报告管理系统对Bmi-1和EZH2基因的表达进行定量分析,用SPSS11.5软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK(q)检验。结果 1.Bmi-1在非小细胞肺癌中呈高表达,癌旁组织中呈低表达。非小细胞肺癌组织Bm i-1的表达明显高于癌旁组织,差异有显著性(P<0.05)。2.EZH2基因的表达EZH2基因在非小细胞肺癌中呈高表达,癌旁组织中呈低表达。非小细胞肺癌组织中EZH2基因的表达明显高于癌旁组织(P<0.05)。结论 EZH2和Bmi-1基因对肺癌的生长有着不同的调节作用,在肺癌的发生、发展中发挥重要的作用。     

The enhancer of zeste homolog 2 (EZH2), a potential therapeutic target, is regulated by miR-101 in renal cancer cells

We investigated a prognostic significance and the mechanism of aberrant nuclear expression of EZH2, a histone methyltransferase, in human renal cell carcinoma (RCC). We found nuclear EZH2 in 48 of 100 RCCs and it was significantly correlated with worse survival in RCC patients. We detected a decreased expression of miR-101 in 15 of 54 RCCs. We found that re-expression of miR-101 resulted in EZH2 depletion and decreased renal cancer cell proliferation. Our results show nuclear EZH2 as a prognostic marker of worse survival in human RCC, and identify miR-101 as a negative regulator of EZH2 expression and renal cancer cell proliferation.     

The polycomb group protein EZH2 inhibits lung cancer cell growth by repressing the transcription factor Nrf2

EZH2 is a key component of the polycomb PRC2 complex and functions as a histone H3 Lys27 (H3K27) trimethyltransferase. Here we show that EZH2 is down-regulated in human non-small cell lung cancer and low EZH2 expression predicts poor survival. Further we demonstrate that EZH2 inhibits lung cancer cell proliferation and colony formation in vitro and growth in vivo. We found that EZH2 binds to the promoter of Nrf2, where it increases H3K27me3 and represses Nrf2 expression. Finally, Nrf2 seems to be essential for the hyper proliferation of lung cancer cells in the absence of EZH2.     

LINC01303 functions as a competing endogenous RNA to regulate EZH2 expression by sponging miR‐101‐3p in gastric cancer

Long non‐coding RNA (lncRNA) is one of the important regulators of many malignancies. However, the biological function and clinical significance of a large number of lncRNAs in gastric cancer remain unclear. Therefore, we analysed the TCGA data to find that LINC01303 is significantly up‐regulated in gastric cancer tissues. However, the biological function of LINC01303 in GC remains unknown. In our study, we found that the expression of LINC01303 was significantly higher in GC tissues than in adjacent tissues by real‐time quantitative PCR. We can significantly inhibit the malignant proliferation, migration and invasion of GC cells by silencing LINC01303 expression. In addition, LINC01303 knockdown can also inhibit GC growth in vivo. After the bioinformatics analysis, we found that LINC01303 can be used as a miR‐101‐3p sponge to competitively adsorb miR‐101‐3p with EZH2. Therefore, our results indicate that LINC01303 promotes the expression of EZH2 by inhibiting miR‐101‐3p activity and promotes GC progression. In summary, in this study, we demonstrated for the first time that the LINC01303/miR‐101‐3p/EZH2 axis promotes GC progression.     

Enhancer of zeste homologue 2 (EZH2) down-regulates RUNX3 by increasing histone H3 methylation

Overexpression of enhancer of zeste homologue 2 (EZH2) occurs in various malignancies and is associated with a poor prognosis, especially because of increased cancer cell proliferation. In this study we found an inverse correlation between EZH2 and RUNX3 gene expression in five cancer cell lines, i.e. gastric, breast, prostate, colon, and pancreatic cancer cell lines. Chromatin immunoprecipitation assay showed an association between EZH2 bound to the RUNX3 gene promoter, and trimethylated histone H3 at lysine 27, and HDAC1 (histone deacetylase 1) bound to the RUNX3 gene promoter in cancer cells. RNA interference-mediated knockdown of EZH2 resulted in a decrease in H3K27 trimethylation and unbound HDAC1 and an increase in expression of the RUNX3 gene. Restoration of RUNX3 expression was not associated with any change in DNA methylation status in the RUNX3 promoter region. RUNX3 was repressed by histone deacetylation and hypermethylation of a CpG island in the promoter region and restored by trichostatin A or/and 5-aza-2'-deoxycytidine. Immunofluorescence staining confirmed restoration of expression of the RUNX3 protein after knockdown of EZH2 and its restoration resulted in decreased cell proliferation. In vivo, an inverse relationship between expression of the EZH2 and RUNX3 proteins was observed at the individual cell level in gastric cancer patients in the absence of DNA methylation in the RUNX3 promoter region. The results showed that RUNX3 is a target for repression by EZH2 and indicated an underlying mechanism of the functional role of EZH2 overexpression on cancer cell proliferation.     

Overexpression of polo-like kinase 1 and its clinical significance in human non-small cell lung cancer

Polo-like kinase 1 is a serine/threonine kinase which plays an essential role in mitosis and malignant transformation. The aim of this study was to investigate the prognostic significance of polo-like kinase 1 expression and determine its possibility as a therapeutic target in non-small cell lung cancer. Semi-quantitative RT-PCR assay was performed to detect polo-like kinase 1 mRNA expression in non-small cell lung cancer cells or tissues. Immunohistochemistry was performed to detect polo-like kinase 1 protein expression in 100 non-small cell lung cancer tissue samples, and the associations of polo-like kinase 1 expression with clinicopathological factors or prognosis of non-small cell lung cancer patients were evaluated. RNA interference was employed to inhibit endogenous polo-like kinase 1 expression and analyzed the effects of polo-like kinase 1 inhibition on the malignant phenotypes of non-small cell lung cancer cells including growth, apoptosis, radio- or chemoresistance. Also, the possible molecular mechanisms were also investigated. The levels of polo-like kinase 1 mRNA expression in non-small cell lung cancer cell lines or tissues were significantly higher than those in normal human bronchial epithelial cell line or corresponding non-tumor tissues. High polo-like kinase 1 expression was significantly correlated with advanced clinical stage, higher tumor classification and lymph node metastasis of non-small cell lung cancer patients (P = 0.001, 0.004 and 0.001, respectively). Meanwhile, high polo-like kinase 1 protein expression was also an independent prognostic molecular marker for non-small cell lung cancer patients (hazard ratio: 2.113; 95% confidence interval: 1.326-3.557; P = 0.017). Polo-like kinase 1 inhibition could significantly inhibit in vitro and in vivo proliferation, induce cell arrest of G₂/M phase and apoptosis enhancement in non-small cell lung cancer cells, which might be activation of the p53 pathway and the Cdc25C/cdc2/cyclin B1 feedback loop. Further, inhibition of polo-like kinase 1 could enhance the sensitivity of non-small cell lung cancer cells to taxanes or irradiation. Thus, polo-like kinase 1 might be a prognostic marker and a chemo- or radiotherapeutic target for non-small cell lung cancer.     

EZH2 and ALDH1 expression in ductal carcinoma in situ: Complex association with recurrence and progression to invasive breast cancer

The diagnosis of ductal carcinoma in situ (DCIS) is an increasingly common event due to widespread use of screening mammography. However, appropriate clinical management of DCIS is a major challenge in the absence of prognostic markers. Tumor-initiating cells may be particularly relevant for disease pathogenesis; therefore, two markers associated with such cells, EZH2 and ALDH1, were evaluated. A cohort of 248 DCIS patients was used to determine the association of EZH2 and ALDH1 with ipsilateral breast event, DCIS recurrence and progression to invasive breast cancer (IBC). In this cohort, high EZH2 expression was associated with the risk of an ipsilateral breast event and DCIS recurrence but not invasive progression. ALDH1 expression was observed in both the tumor and stromal compartment; however, in neither compartment were ALDH1 levels independently associated with evaluated study endpoints. Interestingly, the combination of high EZH2 with high epithelial ALDH1 was associated with disease progression. Therefore, ALDH1 within the DCIS lesion can add to the prognostic significance of EZH2, particularly in the context of risk of development of invasive disease.     

MNX1-AS1 is a novel biomarker for predicting clinical progression and poor prognosis in lung adenocarcinoma

Motor neuron and pancreas homeobox 1-antisense RNA1 (MNX1-AS1) is a novel long noncoding RNA and has been suggested to be overexpressed in human ovarian cancer and glioma. The role of MNX1-AS1 in lung cancer was still unknown. In our study, we observed levels of MNX1-AS1 expression through analyzing The Cancer Genome Atlas and found MNX1-AS1 expression was highly expressed in lung adenocarcinoma tissues compared with normal lung tissues, but there was no statistical difference between lung squamous cell carcinoma tissues and normal lung tissues. Furthermore, we conducted quantitative real-time polymerase chain reaction, and confirmed that the expression of MNX1-AS1 was definitely higher in lung adenocarcinoma tissue samples, but not in lung squamous cell carcinoma tissue samples. In addition, high MNX1-AS1 expression was found to be associated with the low differentiated degree, advanced clinical stage, big tumor size, lymph node metastasis, and distant metastasis in lung adenocarcinoma patients. High expression of MNX1-AS1 was negatively correlated with overall survival time and served as an independent unfavorable prognostic factor in patients with lung adenocarcinoma. The in vitro functional studies suggested that suppression of MNX1-AS1 inhibited lung adenocarcinoma cell proliferation and migration, and promoted apoptosis. In conclusion, MNX1-AS1 is overexpressed in lung adenocarcinoma, and associated with clinical progression and poor prognosis.     

Upregulation of Cleavage and Polyadenylation Specific Factor 4 in Lung Adenocarcinoma and Its Critical Role for Cancer Cell Survival and Proliferation

Cleavage and polyadenylation specific factor 4 (CPSF4), a member of CPSF complex, plays a key role in mRNA polyadenylation and mRNA 3′ ends maturation. However, its possible role in lung cancer pathogenesis is unknown. In this study, we investigated the biological role and clinical significance of CPSF4 in lung cancer growth and survival and elucidated its underlying molecular mechanisms. We found that CPSF4 was highly expressed in lung adenocarcinoma cell lines and tumor tissue but was undetectable in 8 normal human tissues. We also found that CPSF4 overexpression was correlated with poor overall survival in patients with lung adenocarcinomas (P<0.001). Multivariate survival analyses revealed that higher CPSF4 expression was an independent prognostic factor for overall survival of the patients with lung adenocarcinomas. Suppression of CPSF4 by siRNA inhibited lung cancer cells proliferation, colony formation, and induced apoptosis. Mechanism studies revealed that these effects were achieved through simultaneous modulation of multiple signaling pathways. Knockdown of CPSF4 expression by siRNA markedly inhibited the phosphorylation of PI3K, AKT and ERK1/2 and JNK proteins. In contrast, the ectopic expression of CPSF4 had the opposite effects. Moreover, CPSF4 knockdown also induced the cleavage of caspase-3 and caspse-9 proteins. Collectively, these results demonstrate that CPSF4 plays a critical role in regulating lung cancer cell proliferation and survival and may be a potential prognostic biomarker and therapeutic target for lung adenocarcinoma.     

LINC01133 inhibits breast cancer invasion and metastasis by negatively regulating SOX4 expression through EZH2

Mounting evidence highlights long non‐coding RNAs (lncRNAs) as crucial regulators in multiple types of biological processes and contributing to tumourigenesis. LINC01133, located in chromosome 1q23.2, was a recently identified novel lncRNA with a length of 1154nt. It was involved in the development of colorectal cancer and non‐small cell lung cancer. However, its clinical relevance, biological functions and potential molecular mechanism in breast cancer are still unclear. In this study, we found that the LINC01133 expression was significantly down‐regulated in breast cancer samples and was associated with progression and poor prognosis of breast cancer. Further experiments demonstrated that overexpression of LINC01133 inhibited invasion and metastasis in breast cancer both in vitro and in vivo. Mechanistic investigations revealed that LINC01133 repressed SOX4 expression by recruiting EZH2 to SOX4 promoter. Moreover, rescue experiments further confirmed that LINC01133 functional acted as an anti‐oncogene, at least partly, via repressing SOX4 in breast cancer. Taken together, these findings imply that LINC01133 could serve as a novel prognostic biomarker and potential therapeutic target for breast cancer.     

Anosmin-1 involved in neuronal cell migration is hypoxia inducible and cancer regulated

Functional expression of KAL1 gene is critical in the migration of GnRH neurons from the olfactory placode to the hypothalamus in embryogenesis. This gene thus far has not been shown to play a functional role in any other physiological or pathological process either in the developed brain or in peripheral tissues. We show here that KAL1 gene expression is decreased in early stage and increased in later stages of cancers. Screening of colon, lung and ovarian cancer cDNA panels indicated significant decrease in KAL1 expression in comparison to corresponding uninvolved tissues. However, KAL1 expression increased with the progression of cancer from early (I and II) stages to later (III and IV) stages of the cancer. There was a direct correlation between the TGF-β and KAL1 expression in colon cancer cDNA. Using colon cancer cell lines, we showed that TGF-β induces KAL1 gene expression and secretion of anosmin-1 protein (KAL1 coded protein). We further report that hypoxia induces anosmin-1 expression; anosmin-1 protects cancer cells from apoptosis activated by hypoxia and increases cancer cell mobility. Using siRNA technique we found that KAL1 expression following hypoxia is hypoxia-inducible factor (HIF-1) α dependent. Our results suggest that KAL1 gene expression plays an important role in cancer metastasis and protection from apoptosis.     

Aberration of FHIT gene is associated with increased tumor proliferation and decreased apoptosis-clinical evidence in lung and head and neck carcinomas.

BACKGROUND: Human FHIT (fragile histidine triad) gene is highly conserved gene homologous to a group of genes identified in prokaryotes and eukaryotes. Loss of FHIT function may be important in the development and/or progression of various types of cancer. MATERIALS AND METHODS: We undertook a clinical study to analyze the relation between aberrant function of FHIT gene, tumor cell proliferation, and intensity of apoptosis as well as prognostic output in lung and squamous cell head and neck carcinoma (HNSCC). Status of FHIT gene, expression of p21waf1, intensity of apoptosis, and cell proliferation were analyzed in HNSCC and lung carcinoma tissues by molecular genetic methods, immunohistochemistry, [3H]-thymidine labeling method, and FACScan analysis in frozen and paraffin-embedded tissue sections. RESULTS: The majority of the malignant lung and HNSCC lesions displayed aberrant expression of FHIT gene, followed by low or negative expression of p21waf1, and increased intensity of cell proliferation. Similar results were obtained on synchronous combinations of normal, precancerous, and cancerous head and neck tissues. The observed changes increased with progression of these lesions. We examined tumor and corresponding normal tissue samples for microsatellite markers D3S1300 and D3S4103 to evaluate the loss of heterozygosity (LOH) at the FHIT gene loci. We found high percentage of LOH in both lung tumors and HNSCC (75% for D3S1300 and 79% for D3S4103 in lung cancer, and 87% for D3S1300 and 78% for D3S4103 in HNSCC). The median survival time of the patients suffering from lung cancer without FHIT protein expression was 22.46 months and that of the patients with FHIT expression 36.04 months. FHIT-negative cases tended to correlate with a worse prognosis, but this was not statistically significant. Median survival time of HNSCC patients without FHIT protein expression was 30.86 months and that of the patients with FHIT expression was 64.04 months (p < 0.05). CONCLUSIONS: Our results show a correlation between aberrant FHIT expression, a low rate of apoptosis, and high tumor cell proliferation. Aberrant FHIT gene could be a prognostic marker in lung cancer.