The LncRNA MIAT is identified as a regulator of stemness-associated transcript in glioma.

BACKGROUND: Myocardial infarction-associated transcript (MIAT) is a long non-coding RNA (lncRNA) with altered expression in different diseases and malignancies. In this study, the potential expression and function of lncRNA MIAT in intuition and progression of brain cancer was investigated. METHODS AND RESULTS: At first, TCGA data analysis demonstrated that lncRNA MIAT is significantly upregulated in various malignancies, especially its expression is dramatically elevated in brain tumors. In line with the data, we further evaluated the expression of MIAT in a series of brain tumor tissue, and our results revealed that the expression of MIAT was noticeably overexpressed in glioblastoma (p = < 0.0001). We further found that the expression of MIAT was markedly upregulated in low-grade brain tumors rather than high-grade ones. To further investigate the biological function of MIAT in brain cancer cells, its expression was suppressed by si-RNA-mediated knocking down. Inhibition of MIAT resulted in reduced proliferation of brain tumor cells followed by cell cycle arrest at the G1 phase, and significant induction of apoptosis, and senescence, but limited the migration ability and epithelial-mesenchymal-transition (EMT). Moreover, knocking-down of MIAT reduced the expression of stemness factors, followed by upregulation of their downstream miRNAs (micro RNAs), let-7a-5p, and miR-29b-3p. CONCLUSIONS: Altogether, our data demonstrated that lncRNA MIAT could control proliferation, migration, and metastasis of brain cancer cells via regulating the Nanog/ Sox2 / let-7a-5p / miR-29b-3p axis. This data could introduce lncRNA MIAT as a novel oncogene in brain cancer pathogenesis.

Small nucleolar RNA host genes promoting epithelial-mesenchymal transition lead cancer progression and metastasis.

The small nucleolar RNA host genes (SNHGs) belong to the long non-coding RNAs and are reported to be able to influence all three levels of cellular information-bearing molecules, that is, DNA, RNA, and proteins, resulting in the generation of complex phenomena. As the host genes of the small nucleolar RNAs (snoRNAs), they are commonly localized in the nucleolus, where they exert multiple regulatory functions orchestrating cellular homeostasis and differentiation as well as metastasis and chemoresistance. Indeed, worldwide literature has reported their involvement in the epithelial-mesenchymal transition (EMT) of different histotypes of cancer, being able to exploit peculiar features, for example, the possibility to act both in the nucleus and the cytoplasm. Moreover, SNHGs regulation is a fundamental topic to better understand their role in tumor progression albeit such mechanism is still debated. Here, we reviewed the biological functions of SNHGs in particular in the EMT process and discussed the perspectives for new cancer therapies.

Upregulation of pluripotent long noncoding RNA ES3 in HER2-positive breast cancer.

Breast cancer is the second most common cancer and estimates to be responsible for 20% of all cancer patients. Breast cancer has several subtypes including luminal A, luminal B, normal breast-like, basal-like, and human epidermal growth factor receptor 2 (HER2)-enriched. HER2-positive breast cancer cells have higher HER2 expression than other breast cancer subtypes. This subtype is the most aggressive breast cancer subtype and has more ability to metastasis than other breast cancer subtypes. HER2 is a growth-promoting protein that is overexpressed in approximately 20 to 30% of breast cancers and its overexpression is strongly related to poor prognosis. New studies suggested that HER2 expression is correlated with cancer stem cell (CSC) markers in breast cancer. ES3 transcript as a pluripotency long noncoding RNA (lncRNA) is linked to pluripotency transcriptional networks in human embryonic stem cells, but its function in breast cancer is not clarified. In the current research, we found ES3 upregulation in breast cancer and its diagnostic value in breast cancer diagnosis. Furthermore, our findings revealed that ES3 transcript has a high expression in high-grade and high-stage breast tumors. In addition, our data demonstrated that ES3 expression downregulated during neural differentiation. Therefore, its expression may be correlated to breast tumor differentiation status. Notably, a high expression level of ES3 in HER2-positive breast tumor tissues motivated us to investigate the effect of HER2 on ES3 expression by blocking HER2 activity with lapatinib. The results showed that ES3 expression suppressed when HER2 activity was blocked. In summary, for the first time, we found that lncRNA ES3 was significantly upregulated in HER2-positive breast tumors and may contribute to breast cancer proliferation as a downstream target of HER2.

OCT4 spliced variants are highly expressed in brain cancer tissues and inhibition of OCT4B1 causes G2/M arrest in brain cancer cells.

The new claim about the origin of cancer known as Cancer Stem Cell theory states that a somatic differentiated cell can dedifferentiated or reprogrammed for regaining the cancer cell features. It has been recently shown that expression of stemness factors such as Oct4, Sox2, Nanog and Klf4, in a variety of somatic cancers can leads to development of tumorogenesis. Here, the expression of Oct4 variants were evaluated in brain tumor tissues by quantitative RT-PCR and immunohistochemical (IHC) analysis. In next phase of our study, the expression of Oct4B1 was knock-down in brain cancer cell lines and its effect on cell cycle was assessed. Finally, in order to get insights into sequence-structure-function relationships of Oct4 isofroms, their sequences were analysed using bioinformatic tools. Our data revealed that all three variants of Oct4 are expressed in different types of brain cancer. The expression level of Oct4B1, in contast to Oct4B, was much higher in high-grade brain tumors compared with low-grade ones. In line with qPCR, the expression of Oct4A and B isofroms was confirmed with IHC in different types of brain tumors. Moreover, as a result of the suppression of Oct4B1 expression, the brain cancer cells were arrested in G2/M phase of cell cycle. Bioinfromatics data indicated that the predicted Oct4B1 protein have DNA binding properties. All together, our findings suggest that Oct4B1 has a potential role in tumorigenesis of brain cancer and can be considered as a new tumor marker with potential value in diagnosis and treatment of brain cancer.

Concomitant upregulation of nucleostemin and downregulation of Sox2 and Klf4 in gastric adenocarcinoma.

Nucleostemin (NS) is a nucleolar protein involved in stem cell (SC) self-renewal by controlling cell cycle progression. In addition to SCs, NS is also expressed in some highly proliferating cells including several adult stem cells and cancer cell lines. NS knock-down in different cell lines demonstrated its cell type-dependent function in arresting cell cycle in either G1 or G2/M phases. Here, we have evaluated the expression of NS and iPS genes in 36 gastric cancer and their matched marginal nontumor tissues by means of real-time polymerase chain reaction (RT-PCR). We have also examined a potential causative role of NS in gastric tumorigenesis by suppressing its expression in a gastric cancer cell line, AGS. Our data revealed that NS expression level is much higher in tumor tissues (p = 0.046), especially in high-grade ones (p < 0.001), whereas the expression of Klf4 and Sox2 is downregulated in tumor tissues compared to marginal nontumor samples (p < 0.001). Furthermore, NS suppression in the AGS cell line caused some morphological alterations, a cell cycle arrest at G1 phase, and an upregulation of iPS genes: Nanog, Sox2, and Klf4. Based on our results, NS overexpression seems to have a causative role in gastric tumorigenesis and/or progression, and it could be considered as a potential tumor marker for diagnosis, molecular classification, and molecular therapy of gastric adenocarcinoma.

LncRNA PNKY Is Upregulated in Breast Cancer and Promotes Cell Proliferation and EMT in Breast Cancer Cells.

Long non-coding RNAs (lncRNAs) are known to be important regulators in different cellular processes and are implicated in various human diseases. Recently, lncRNA PNKY has been found to be involved in pluripotency and differentiation of embryonic and postnatal neural stem cells (NSCs); however, its expression and function in cancer cells is still unclear. In the present study, we observed the expression of PNKY in various cancer tissues, including brain, breast, colorectal, and prostate cancers. In particular, we demonstrated that lncRNA PNKY was significantly upregulated in breast tumors, especially high-grade tumors. Knock down experiments indicated that the suppression of PNKY in breast cancer cells could restrict their proliferation by promoting apoptosis, senescence, and cell cycle disruption. Moreover, the results demonstrated that PNKY may play a crucial role in the cell migration of breast cancer cells. We further found that PNKY may trigger EMT in breast cancer cells by upregulating miR-150 and restricting the expression of Zeb1 and Snail. This study is the first to provide new evidence on the expression and biological function of PNKY in cancer cells and its potential contribution to tumor growth and metastasis.

LncRNA LOC100507144 acts as a novel regulator of CD44/Nanog/Sox2/miR-302/miR-21 axis in colorectal cancer.

Long noncoding RNAs (lncRNAs) appear as vital regulators and biomarkers in many human cancers. LOC100507144 is a validated lncRNA located in the neighborhood of CD44 in a head-to-head configuration, and its expression and function in cancer cells are still unknown. This research aimed to find out more about the expression and function of this lncRNA in colorectal cancer (CRC). Our expression data represented that the expression of LOC100507144 transcript was substantially higher in tumors with advanced stages, lymph node metastasis, and vascular invasion. Loss-of-function examinations demonstrated that LOC100507144 contributed to CRC cell proliferation by restricting apoptosis, cellular senescence, and promoting cell cycle. Gain-of-function experiments also confirmed these results. Our data illustrated that LOC100507144 enhanced the migration and the epithelial to mesenchymal transition (EMT) of CRC cells, accompanied by the generation of cells with stemness characteristics. Our findings revealed that the knocking-down of LOC100507144 inhibited the expression of crucial stemness factors, including CD44, Nanog, and Sox2, and accordingly resulted in suppressing their targets, miR-302 and miR-21. Overall, the current study's findings for the first time reveal that LOC100507144 could enhance CRC progression and metastasis through regulation of the CD44/Nanog/Sox2/miR-302/miR-21 axis.

Construction of modified screen-printed graphite electrode for the application in electrochemical detection of sunset yellow in food samples.

The current work introduced a novel electrochemical sensor (screen-printed graphite electrode (SPGE) modified with MnO2 nanorods anchored graphene oxide nanocomposite (MnO2 NRs/GO) for sensitive determination of sunset yellow. The characterization of MnO2 NRs/GO nanocomposite synthesized through a simple hydrothermal approach was determined employing varied analytical equipment like Field emission-scanning electron microscopy (FE-SEM), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD). Chronoamperometric measurements, differential pulse voltammetry (DPV), cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were recruited to recognize the electrochemical oxidation of sunset yellow on the MnO2 NRs/GO/SPGE. The results of CV proved that the as-synthesized MnO2 NRs/GO nanocomposite has a good electrocatalytic activity toward sunset yellow. The MnO2 NRs/GO/SPGE electrode under optimized conditions using the DPV possessed a linear response for different concentrations of sunset yellow (between 0.01 and 115.0 µM) with a low limit of detection (LOD) (0.008 µM). Finally, the impressive applicability of this sensor was confirmed via real sample analysis with excellent recoveries (between 97.3 and 104.6%).

Differential expression of nucleostemin, a stem cell marker, and its variants in different types of brain tumors.

Nucleostemin (NS) is implicated in the control of stem and cancer cell proliferation. In the present study, we have examined the expression of NS and its spliced variants in various brain tumors. Total RNA was extracted from 59 brain tumor samples, and the expression of different NS spliced variants was measured by semi-quantitative RT-PCR. The subcellular distribution of NS protein in brain tumors was further examined by immunohistochemistry. Furthermore, to decipher the potential involvement of NS in brain tumorogenesis, its expression was knocked-down by means of RNA interference (RNAi) in two malignant glioma (U-87MG and A172), one astrocytoma (1321N1) and one medulloblastoma (DAOY) cell lines. The alterations in cell-cycle progression of the treated cells were then analyzed by flow cytometry. Our data revealed that NS and its variants are widely expressed in different types of brain tumors. Among the NS spliced variants, variant "1" and variant "3" were detected in the majority of tumor samples, whereas variant "2" was only detectable in few samples. Moreover, the intensity of the expression was correlated with the grade of the tumors (P < 0.05). Accordingly, the expression was much higher in glial tumors compared to that of meningiomas. As expected, a nucleolar/nucleoplasmic localization of NS protein was observed in the examined tumor samples. RNAi results revealed a significant reduction of NS expression along with a moderate blockade of the cell cycle in G(2)/M and S phases of NS-siRNA treated cells. All in all, our data suggest a potential role for NS in tumorogenesis of brain cancers.

Long non-coding RNA ES1 controls the proliferation of breast cancer cells by regulating the Oct4/Sox2/miR-302 axis.

ES1 is a long non-coding RNA (lncRNA) that regulates pluripotency of human embryonic stem cells, which is known to be a downstream target of stemness factors Oct4 and Nanog, and serves as a modular scaffold for Sox2. However, the role of ES1 in cancer biology is not fully characterized. The results of our study show that ES1 transcript is upregulated in both high-grade and P53-mutated breast tumor tissues. Knockdown experiments show that ES1 suppression in breast cancer cells restricts cancer cell proliferation and cell cycle progression. Moreover, ES1 inhibition can also induce apoptosis and cellular senescence. Additionally, our data reveal that ES1 transcript promotes cell migration as well as the epithelial to mesenchymal transition of breast cancer cells. Furthermore, loss of ES1 expression downregulates the expression of Oct4/Sox2 and consequently leads to downregulation of their targets, miR-302 and miR-106b. Altogether, for the first time, our findings reveal that ES1 controls the proliferation and death of breast cancer cells by regulating the Oct4/Sox2/miR-302/miR-106b axis.

SNHG6 is upregulated in primary breast cancers and promotes cell cycle progression in breast cancer-derived cell lines.

BACKGROUND: Long non-coding RNAs (lncRNAs) are known as RNAs that do not encode proteins and that are more than 200 nucleotides in size. Previously, it has been found that LncRNAs play crucial roles in normal cellular processes, including proliferation and apoptosis. A growing body of evidence suggests that lncRNAs may also play regulatory roles in the initiation, progression and metastasis of various malignancies, including breast cancer. SNHG6 is a lncRNA that has previously been found to contribute to the initiation and progression of hepatocellular and gastric carcinomas. In this study, the clinical significance of SNHG6 expression in breast cancer was investigated. METHODS: SNHG6 expression in primary breast cancer tissues was assessed using RT-qPCR. The functional role of SNHG6 was investigated using RNAi-mediated silencing and exogenous overexpression in breast cancer-derived cells. MTT, colony formation, cell cycle, apoptosis and senescence assays were used to determine the impact of SNHG6 expression on breast cancer-derived cells. The effect of SNHG6 on the migration and epithelial-to-mesenchymal transition (EMT) of breast cancer-derived cells was determined using scratch wound healing and immunofluorescence assays, respectively. RESULTS: We found that the expression of SNHG6 was significantly upregulated in primary high-grade and progesterone receptor (PR)-positive breast tumours. Additional siRNA-based experiments revealed that SNHG6 silencing led to G1 cell cycle arrest in SK-BR-3 and MDA-MB-231 breast cancer-derived cells. Moreover, we found that SNHG6 silencing led to suppressed breast cancer cell proliferation by inducing apoptosis and senescence. Our data also indicate that SNHG6 may contribute to the migration and EMT of breast cancer cells. CONCLUSIONS: Our results indicate that lncRNA SNHG6 is involved in breast cancer development and may be considered as a potential biomarker for the diagnosis, prognosis and treatment of breast cancer.

Differential Expression Profile of ZFX Variants Discriminates Breast Cancer Subtypes

Background: ZFX is a transcriptional regulator in embryonic stem cells and plays an important role in pluripotency and self-renewal. ZFX is widely expressed in pluripotent stem cells and is down-regulated during differentiation of embryonic stem cells. ZFX has five different variants that encode three different protein isoforms. While several reports have determined the overexpression of ZFX in a variety of somatic cancers, the expression of ZFX-spliced variants in cancer cells is not well-understood. Methods: We investigated the expression of ZFX variants in a series of breast cancer tissues and cell lines using quantitative PCR. Results: The expression of ZFX variant 1/3 was higher in tumor tissue compared to marginal tissue. In contrast, the ZFX variant 5 was down-regulated in tumor tissues. While the ZFX variant 1/3 and ZFX variant 5 expression significantly increased in low-grade tumors, ZFX variant 4 was strongly expressed in high-grade tumors, demonstrating lymphatic invasion. In addition, our result revealed a significant association between the HER2 status and the expression of ZFX-spliced variants. Conclusion: Our data suggest that the expression of ZFX-spliced transcripts varies between different types of breast cancer and may contribute to their tumorigenesis process. Hence, ZFX-spliced transcripts could be considered as novel tumor markers with a probable value in diagnosis, prognosis, and therapy of breast cancer.

Long noncoding RNA VIM-AS1 promotes colorectal cancer progression and metastasis by inducing EMT.

Emerging evidence indicates that lncRNAs play crucial roles in the initiation and progression of various malignant tumors. VIM-AS1 RNA is an lncRNA that transcribes from a shared bidirectional promoter with vimentin mRNA and its function in cancer cells is largely unknown. This study assessed the clinical significance of VIM-AS1 expression in colorectal cancer (CRC). We found that the VIM-AS1 transcript was significantly upregulated in high-grade, lymph node metastasis and vascular invasion tumors. Loss-of-function experiments revealed that the downregulation of VIM-AS1 could inhibit tumor cell proliferation by inducing apoptosis, cellular senescence and arresting the cell cycle. Moreover, the obtained data demonstrated that VIM-AS1 might play a crucial role in cell migration as well as the epithelial to mesenchymal transition (EMT) of CRC cells. Collectively, for the first time, our data provide novel evidence for the biological and clinical significance of VIM-AS1 expression in CRC. Further, the findings of this study suggest that VIM-AS1 promotes tumor growth and metastasis by inducing EMT in CRC cells and could be considered as a novel tumor marker with probable value in diagnosis and CRC treatment.

Distinctive expression pattern of OCT4 variants in different types of breast cancer.

BACKGROUND: OCT4 is a key regulator of self-renewal and pluripotency in embryonic stem cells which can potentially encode three spliced variants designated OCT4A, OCT4B and OCT4B1. Based on cancer stem cell concept, it is suggested that the stemness factors misexpressed in cancer cells and potentially is involved in tumorigenesis. OBJECTIVE: Accordingly, in this study, we investigated the potential expression of OCT4 variants in breast cancer tissues. METHODS: A total of 94 tumoral and peritumoral breast specimens were evaluated with respect to the expression of OCT4 variants using quantitative RT-PCR and immunohistochemical (IHC) analysis. RESULTS: We detected the expression of OCT4 variants in breast tumor tissues with no or very low levels of expression in peritumoral samples of the same patients. While OCT4B was highly expressed in lobular type of breast cancer, OCT4A and OCTB1 variants are highly expressed in low grade (I and II) ductal tumors. Furthermore, the results of this study revealed a considerable association between the expression level of OCT4 variants and the expression of ER, PR, Her2 and P53 factors. CONCLUSIONS: All data demonstrated a distinctive expression pattern of OCT4 spliced variants in different types of breast cancer and provide further evidence for the involvement of embryonic genes in carcinogenesis.

Anti-carcinogenic and anti-angiogenic properties of the extracts of Acorus calamus on gastric cancer cells.

OBJECTIVE: Acorus calamus (A. calamus) has been used as a medicinal plant in Asia for its effects on digestive system for the last 2000 years. To investigate the anti-cancer activity of rhizome of A. calamus, the ethanolic and methanolic extracts and essential oil of the rhizome were prepared and their effects were assessed on human gastric cancer cell line (AGS). MATERIALS AND METHODS: The viability of cells which were treated with the extracts and the essential oil was assessed by MTT assay. To evaluate the anti-angiogenic property of the extracts, in vitro tube formation assay was done. Cell cycle distribution and the expression of Oct4 and Nucleostemin, after treatments, were checked by flowcytometry and quantitative RT-PCR, respectively. Furthermore, analysis of essential oil from A.calamus was done by GC-MS. RESULTS: Our results showed that the growth of AGS cells was inhibited by the extracts and essential oil and the extracts inhibited the angiogenesis in HUVEC cells. Our data revealed that the extracts and essential oil of A. calamus caused G1 arrest in AGS cells and downregulation of Oct4 and NS after treatment. By GC-MS analysis, we found new compounds such as epiprezizaene, valencene and isocyclocitral in essential oil of A. CONCLUSION: All together, our results showed that the extracts of A. calamus have anti-proliferative and anti-angiogenic effects on cancer cells.

Altered Expression of High Molecular Weight Heat Shock Proteins after OCT4B1 Suppression in Human Tumor Cell Lines.

OBJECTIVE: OCT4B1, a novel variant of OCT4, is expressed in cancer cell lines and tis- sues. Based on our previous reports, OCT4B1 appears to have a crucial role in regulating apoptosis as well as stress response [heat shock proteins (HSPs)] pathways. The aim of the present study was to determine the effects of OCT4B1 silencing on the expression of high molecular weight HSPs in three different human tumor cell lines. MATERIALS AND METHODS: In this experimental study, OCT4B1 expression was suppressed in AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cell lines using RNAi strategy. Real-time polymerase chain reaction (PCR) array was em- ployed for expression level analysis and the fold changes were calculated using RT2 Pro- filer PCR array data analysis software version 3.5. RESULTS: Our data revealed up-regulation of HSPD1 (from HSP60 family) as well as HSPA14, HSPA1L, HSPA4, HSPA5 and HSPA8 (from HSP70 family) following OCT4B1 knock-down in all three cell lines. In contrast, the expression of HSP90AA1 and HSP90AB1 (from HSP90 family) as well as HSPA1B and HSPA6 (from HSP70 family) was down-regulated under similar conditions. Other stress-related genes showed varying ex- pression pattern in the examined tumor cell lines. CONCLUSION: Our data suggest a direct or indirect correlation between the expression of OCT4B1 and HSP90 gene family. However, OCT4B1 expression was not strongly corre- lated with the expression of HSP70 and HSP60 gene families.

A plausible anti-apoptotic role of up-regulated OCT4B1 in bladder tumors.

PURPOSE: To investigate and compare the expression of OCT4B1 between tumor and non-tumor bladder tissues. MATERIALS AND METHODS: We investigated the expression of OCT4B1 in 30 tumor and non-tumor surgical specimens of the bladder, using the TaqMan real-time polymerase chain reaction approach and by carefully designing primers and probes specific for the amplification of the variant. RESULTS: Most tumor and non-tumor samples of the bladder showed OCT4B1 expression, but its expression level was significantly higher in the tumors (P < .002). Moreover, the up-regulation of OCT4B1 was more significant in high-grade tumors compared to the low-grade ones (P < .05). We have also employed the RNA interference strategy to evaluate the functional role of OCT4B1 in a bladder cancer cell line, 5637. Suppression of OCT4B1 caused some changes in cell cycle distribution, and significantly elevated the rate of apoptosis in the cells. CONCLUSION: Our findings suggest that OCT4B1 plays a potential role in tumor initiation and/or progression of the bladder cancer. Additionally, OCT4B1 can be regarded as a new tumor marker for detection, classification, and treatment of the bladder cancer. However, more experimental studies are needed to replicate our findings.