LINC02381 suppresses cell proliferation and promotes apoptosis via attenuating IGF1R/PI3K/AKT signaling pathway in breast cancer.

Identification of the genes and genetic networks involved in breast cancer development is a major need for prevention and therapy. LINC02381 (lncRNA) has already been introduced as a tumor suppressor in colorectal and gastric cancers. Here, we intended to investigate its potential functional effects on breast cancer. In the analysis performed on RNA-Seq and microarray data, the LINC02381 lncRNA was found to be significantly downregulated in the breast tumors and associated with poor survival of the patients. Then, the differential expression of LINC02381 was confirmed in breast tumor tissues and cancer cell lines using RT-qPCR. Overexpression of LINC02381 resulted in reduced IGF1R and p-AKT expression levels which indicates decreased PI3K pathway activity, detected by RT-qPCR and western blotting. At the cellular level, LINC02381 overexpression was followed by a decreased proliferation rate of transfected breast cell lines, detected by PI flow cytometry, RT-qPCR, colony formation, and MTT assays. Consistently, the results of Annexin-V/PI flow cytometry, RT-qPCR, caspase3/7 activity, and AO/EB-H33342/PI dual staining revealed that LINC02381 overexpression induced apoptosis and cell death. The reduced migration rate of these cells was also verified through wound healing assay and RT-qPCR against the EMT-involved genes. Our data show that LINC02381 exerts its tumor suppressor effect at least partly through attenuation of the IGF1R/PI3K/AKT signaling pathway, which originated from IGF1R downregulation.

Can circulating miR-7-1-5p, and miR-33a-5p be used as markers of T2D patients?

PURPOSE: Recent evidence has indicated that miRNAs play an important role in both initiation and progression of many pathologic processes such as diabetes and can be used as an important and more sensitive tool to predict the development of the disease than the currently used biomarkers. This research aimed at comparing miR-7-5p and miR-33a-5p expression levels in the diabetics and pre-diabetics with the control group. METHODS: In this study, we compared expression of miR-7-5p and miR-33a-5p in plasma of three groups including pre-diabetic patients (n = 20), T2D patients (n = 20) and control group (n = 20), using RT-qPCR. Biochemical parameters were measured by auto-analyser. In silico analysis was performed to identify potential target genes of these miRNAs. RESULTS: Compared to the controls, miR-7-1-5p expression was down regulated in the pre-diabetics and the T2D patients; whereas, miR-33a-5p was expressed at higher levels in the T2D patients compared to the control group. Both miRs were correlated with glycaemic status such as FBS and HbA1c levels. The ROC analysis indicated a significant ability for miR-33a-5p in discriminating between the diabetics and the healthy individuals. In silico analysis suggests that both miRs affect biological pathways related to T2DM pathogenesis, such as MAPK, and insulin signalling pathway. CONCLUSION: Our results demonstrated that the miR-7-1-5p and miR-33a-5p expression levels are deregulated in the diabetics and pre-diabetics. Furthermore, miR-33a-5p showed significant ability in discriminating between diabetics and healthy individuals, suggesting a potential diagnostic use of miRNAs in type-2 diabetes detection.

Radiosensitizing effect of dendrosomal nanoformulation of curcumin on cancer cells.

BACKGROUND: Curcumin was found to possess numerous pharmacological activities in clinical research, however, its biological effects together with radiation are yet to be addressed. The present study investigated whether the combined treatment of dendrosomal nanoformulation of curcumin (DNC) and gamma radiation can enhance the radiosensitivity of U87MG and MDA-MB-231 cell lines. METHODS: U87MG and MDA-MB-231 cell lines were exposed to 2 Gray (Gy) and 10 µM DNC determined by MTT assay, then subjected to clonogenic assay, cell cycle assay, and flow cytometric apoptosis analysis. Acridine Orange/Ethidium Bromide (AO/EB) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) stained cells were used to study morphologic changes. The expression evaluation of putative cell cycle genes, i.e., P53, P21, CCND1, and CCNB1 was carried out by RT-qPCR. RESULTS: Our findings indicated that the combined treatment with DNC and radiation might cooperatively augment the efficacy of ionizing radiation in the cancer cells and notably decrease the survival and viability of the cells in a time- and concentration-dependent manner. In addition to a synergistic effect deducted by sensitizer enhancement ratio (SER) assessment, co-treatment resulted in greater apoptotic cells than the individual treatments. Further experiments then indicated that DNC could effectively induce G2/M phase cell cycle arrest and apoptosis following irradiation. Conformably, there was a decrement of CCND1 and CCNB1 expression, and an increment of P53, P21 expression. CONCLUSIONS: The data implied that DNC as a radiosensitizer can enhance the lethal effect of ionizing radiation on cancer cells which could be a promising adjuvant therapy in clinical treatments.

Can miR-145-5p be used as a marker in diabetic patients?

In the light of emerging global epidemics of type 2 diabetes mellitus significant efforts are continuing to discover novel biomarkers for early detection of the disease. Since miRNAs play an important role in both the initiation and progress of many pathologic processes such as diabetes, in this study we aimed to evaluate expression level of plasma miR-145-5p in diabetics and pre-diabetics in comparison to the control group and assess its use as a biomarker in diagnosis of T2D. The plasma level of miR-145-5p was assessed in three groups including 20 prediabetic patients, 20 T2D patients and 20 healthy controls using RT-qPCR. Biochemical parameters were also measured by the auto-analyzer. Expression level of miR-145-5p was down-regulated in the prediabetics and the T2D patients compared to the controls. In the control group miR-145-5p showed a borderline correlation with FBS (p = .06), while in the prediabetic group miR-145 showed a significant negative correlation with FBS and finally in the T2D patients miR-145 was negatively correlated with HbA1c and TC and showed a negative borderline correlation with FBS. The ROC analysis indicated a significant ability for miR-145-5p in discriminating between the diabetics and pre-diabetics from the healthy subjects. Our results demonstrated that the miR-145-5p expression level is deregulated in the diabetics and the prediabetics. Furthermore miR-145-5p displayed a significant ability to discriminate the diabetics from the healthy subjects. These results suggest that miR-145-5p may be a useful biomarker for the diagnosis of T2DM.

MiRNA-Wnt signaling regulatory network in colorectal cancer.

Colorectal cancer (CRC) is one of the common malignancies worldwide and the Wnt signaling pathway is recognized as the main disrupted pathway in this malignancy. MicroRNAs (miRNAs) are recognized to contribute to the pathogenesis of CRC by triggering or impeding the Wnt signaling pathway. In addition, transcriptional regulation of miRNAs by canonical Wnt signaling also participates in CRC cell progression. In this review, we present comprehensive literature of the existing data on the interaction of miRNAs and Wnt signaling that could be useful in future studies in the field of CRC management.

Long Noncoding RNA LOC400043 (LINC02381) Inhibits Gastric Cancer Progression Through Regulating Wnt Signaling Pathway.

Gastric cancer is one of the common causes of cancer mortality worldwide, with a low survival rate for the affected people. Recent studies have revealed the key role of long non-coding RNAs (lncRNAs) in the development and progression of many cancers, including gastric cancer. Looking for the potential molecular regulators of gastric cancer incidence and progression, LINC02381 was identified as a downregulated lncRNA in gastric cancer tissues by analysis of available microarray and RNA-seq data and RT-qPCR confirmed this differential expression. MiR-21, miR-590, and miR-27a miRNAs were predicted to be sponged by LINC02381, and dual luciferase assay verified LINC02381 as a competitive endogenous RNA (CeRNA), which binds to them. Furthermore, we found that increased expression of LINC02381 attenuates Wnt pathway activity. Also, functional analysis indicates that LINC02381 arrests cell cycle, increases apoptosis and caspase activity, and reduces cell survival and proliferation rate of the human gastric cancer cell lines AGS and MKN45. Moreover, EMT analysis showed that LINC02381 is involved in gastric cancer progression and inhibits metastasis. Overall, this work for the first time introduces LINC02381 as a CeRNA involved in gastric cancer and provides novel insight into the molecular pathogenesis of gastric cancer.

Epigenetically silenced LINC02381 functions as a tumor suppressor by regulating PI3K-Akt signaling pathway.

Recent advances have revealed that lncRNAs play important roles in tumorigenesis. However, only a small number of functional lncRNAs have been well characterized, particularly in colorectal cancer. Therefore, more extensive studies are needed to identify and characterize these lncRNAs to better understand cancer progression. In the present study, using available RNA-seq data, we found that LINC02381 (NR_026656.1) differentially expresses in CRC tissues compared to normal pairs. Consistently, RT-qPCR results showed that LINC02381 was down regulated in CRC tissues and also in different cancerous cell lines. CRC cells treatment with de-methylating and chemotherapy agents indicated that DNA methylation of LINC02381 may be responsible for the transcriptional silencing of LINC02381 in colorectal cancer cells. Then, the functional changes of the cells in response to LINC02381 alteration were assessed and the data indicated that LINC02381 up-regulation suppressed cell viability and proliferation while increasing the apoptosis in CRC-originated cell lines. Mechanistically, LINC02381 overexpression was increased PTEN protein levels but decreased phospho-Akt levels. Finally, we proposed a hypothesized model for PI3K signaling regulation by LINC02381. Altogether, the result of this study suggests that LINC02381 might have suppressive effects on human colorectal cancer tumorigenesis partly by regulating PI3K signaling pathway.

LncRNA HSPC324 plays role in lung development and tumorigenesis.

Lung cancer is a leading cause of cancer-related death in the world. Therefore, identifying the genes and molecular pathways involved in lung development and tumorigenesis can help us improve the therapeutic strategies of lung cancer. Accumulating evidence confirms that long noncoding RNAs, as a novel layer of regulatory RNA molecules, play an important role in various aspects of the cells. Here, using available high throughput gene expression data, we identified an lncRNA (HSPC324) with high expression level in lung tissue that is distinctly expressed in lung tumor tissues relative to normal. Using GO enrichment and KEGG pathway analyses, we further analyzed the functions and pathways involving the HSPC324-correlated genes. Ectopic expression of lncRNA HSPC324 significantly inhibited proliferation, cell cycle and migration; on the other hand, increased apoptosis and ROS production in lung adenocarcinoma cells. Overall, this study introduces HSPC324 as a new player in the development of lung cancer.

MicroRNA-590-3P suppresses cell survival and triggers breast cancer cell apoptosis via targeting sirtuin-1 and deacetylation of p53.

Downregulation of microRNA-590-3p (miR-590-3p) is a frequently occurring, nonphysiological event which is observed in several human cancers, especially breast cancer. However, the significance of miR-590-3p still remain unclear in the progression of this disease. This study explored the role of miR-590-3p in apoptosis of breast cancer cells. Gene expression of miR-590-3p, Sirtuin-1 (SIRT1), Bcl-2 associated X protein (BAX), and p21 was evaluated with real-time polymerase chain reaction (PCR) and SIRT1 protein expression was assessed by Western blot analysis in breast cancer cell lines. Bioinformatics analysis and luciferase reporter assay were used to evaluate targeting of SIRT1 messenger RNA (mRNA) by miR-590-3p. Cells were transfected with miR-590-3p mimic and inhibitor and their effects on the expression and activity of SIRT1 were evaluated. The effects of miR-590-3p upregulation on the acetylation of p53 as well as cell viability and apoptosis were assessed by Western blot analysis, WST-1 assay, and flow cytometry, respectively. miR-590-3p expression was considerably downregulated in breast cancer cells which was accompanied by upregulation of SIRT1 expression. SIRT1 was recognized as a direct target for miR-590-3p in breast cancer cells and its protein expression and activity was dramatically inhibited by the miR-590-3p. In addition, there was an increase in p53 and its acetylated form that ultimately led to upregulation of BAX and p21 expression, suppression of cell survival, and considerable induction of apoptosis in breast cancer cells. These findings suggest that miR-590-3p exerts tumor-suppressing effects through targeting SIRT1 in breast cancer cells, which makes it a potential therapeutic target for developing more efficient treatments for breast cancer.

Hsa-miR-497 as a new regulator in TGFß signaling pathway and cardiac differentiation process.

Cardiosphere-derived cells (CDCs) contain cardiac stem cell subpopulations and are introduced as useful source for cardiac differentiation and therapy. Furthermore, research has highlighted miRNAs important role in various biological processes and cardiogenesis. Here, we intended to investigate the effect of hsa-miR-497 (miR-497) on TGFß signaling pathway genes expression during the process of CDCs differentiation to cardiomyocytes. CDCs were successfully differentiated to the cardiac-like cells. In this study, we found that after cardiac differentiation induction, miR-497 expression was significantly decreased. Computational miRNA target prediction analyses revealed that TGFß signaling pathway is a possible target of miR-497. Therefore, miR-497 was overexpressed in CDCs before the induction of differentiation. TGFß1, TGFßR1, TGFßR2, and SMAD3 genes expression level was decreased after miR-497 overexpression. Also, immunocytochemistry and cell morphology analysis indicated that miR-497 overexpression affecting cardiac differentiation process. Finally, direct interaction of miR-497 with 3'-UTR sequence of TGFßR1 was supported through dual luciferase assay, consistent with miR-497 reported negative effect on SMAD3 expression. Accordingly, here a model of miR-497 involvement in regulation of TGFß signaling pathway is introduced in which, side branches of TGFß signaling pathway downregulate miR-497 to ensure upregulation of TGFßR1 and TGFßR2 and finally stronger TGFß signaling.

Down-regulation of NAMPT expression by mir-206 reduces cell survival of breast cancer cells.

Nicotinamide adenine dinucleotide (NAD) is a critical coenzyme for all living cells. Nicotinamide phosphoribosyltransferase (NAMPT) functions as a key enzyme in the salvage pathway of NAD biosynthesis. Cancer cells have higher rate of NAD consumption and therefore NAMPT is essential for their survival. Thus, we investigated the effect of NAMPT inhibition by miR-206 on breast cancer cell survival. Breast cancer cells were transfected with miR-206 mimic, inhibitor and their negative controls. NAMPT levels were assessed by real-time PCR as well as western blotting. Cell survival assay and quantification of NAD level were performed by using colorimetric methods. Apoptosis assay was performed by labeling cells with Annexin V-FITC and propidium iodide followed by the flow cytometric analysis. Bioinformatics analysis was done to assess whether NAMPT 3'-UTR is a direct target of miR-206 and the results were confirmed by the luciferase reporter assay. NAMPT 3'-UTR was shown to be a direct target of miR-206. miR-206 reduced NAMPT expression at the protein level, leading to a significant decrease in the intracellular NAD level and subsequent decline in cell survival and induction of apoptosis. Targeting of NAMPT-mediated NAD salvage pathway by miR-206 might provide a new insight in the possible molecular mechanism of breast cancer cell growth regulation. This pathway might provide a new approach for breast cancer therapy.

A novel microRNA located in the TrkC gene regulates the Wnt signaling pathway and is differentially expressed in colorectal cancer specimens.

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis, differentiation, and tumorigenesis. TrkC diverse functions might be attributed to the hypothetical non-coding RNAs embedded within the gene. Using bioinformatics approaches, a novel microRNA named TrkC-miR2 was predicted within the TrkC gene capable of regulating the Wnt pathway. For experimental verification of this microRNA, the predicted TrkC-premir2 sequence was overexpressed in SW480 cells, which led to the detection of two mature TrkC-miR2 isomiRs, and their endogenous forms were detected in human cell lines as well. Later, an independent promoter was deduced for TrkC-miR2 after the treatment of HCT116 cells with 5-azacytidine, which resulted in differential expression of TrkC-miR2 and TrkC host gene. RT-quantitative PCR and luciferase assays indicated that the APC2 gene is targeted by TrkC-miR2, and Wnt signaling is up-regulated. Also, Wnt inhibition by using small molecules along with TrkC-miR2 overexpression and TOP/FOP flash assays confirmed the positive effect of TrkC-miR2 on the Wnt pathway. Consistently, TrkC-miR2 overexpression promoted SW480 cell survival, which was detected by flow cytometry, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, and crystal violate analysis. RT-qPCR analysis revealed that TrkC-miR2 is significantly up-regulated (∼70 times) in colorectal tumor tissues compared with their normal pairs. Moreover, the TrkC-miR2 expression level discriminated grades of tumor malignancies, which was consistent with its endogenous levels in HCT116, HT29, and SW480 colorectal cancer cell lines. Finally, an opposite expression pattern was observed for TrkC-miR2 and the APC2 gene in colorectal cancer specimens. In conclusion, here we introduce TrkC-miR2 as a novel regulator of Wnt signaling, which might be a candidate oncogenic colorectal cancer biomarker.

Introduction of novel splice variants for CASC18 gene and its relation to the neural differentiation.

BACKGROUND: CASC18 along with APPL2, OCC-1 and NUAK1 flanking genes are located in 12q23.3 locus which is known as a potential cancer predisposition locus. Only an uncharacterized EST was initially reported for CASC18 and it was crucial to find its full length sequence and function. METHODS AND RESULTS: In an attempt to search for the CASC18's full-length gene sequence, other related ESTs were bioinformatically collected and four novel splice variants (designated as; CASC18-A, -B, -C and -D) were deduced and some were experimentally validated. Two transcription start sites and two alternative polyadenylation sites were deduced for CASC18 gene, using EST data mining and RACE method. CASC18-A and CASC18-D were exclusively expressed in neural cell lines and CASC18-D expression level was gradually increased during the NT2 differentiation to the neuron-like cells. Consistently, overexpression of CASC18-D variant in NT2 cells resulted in remarkable up-regulation of PAX6 neural differentiation marker, suggesting a crucial role of this variant in neural differentiation. CONCLUSION: Here, we introduced seven novel transcription variants for human CASC18 gene in which CASC18-D has the potential of being used as a neural cell differentiation marker.

Hsa-miR-590-5p Interaction with SMAD3 Transcript Supports Its Regulatory Effect on The TGFß Signaling Pathway.

OBJECTIVE: SMAD proteins are the core players of the transforming growth factor-beta (TGFß) signaling pathway, a pathway which is involved in cell proliferation, differentiation and migration. On the other hand, hsa-miRNA-590-5p (miR-590-5p) is known to have a negative regulatory effect on TGFß signaling pathway receptors. Since, RNAhybrid analy- sis suggested SMAD3 as a bona fide target gene for miR-590, we intended to investigate the effect of miR-590-5p on SMAD3 transcription. MATERIALS AND METHODS: In this experimental study, miR-590-5p was overexpressed in different cell lines and its increased expression was detected through quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Western blot analysis was then used to investigate the effect of miR-590-5p overexpression on SMAD3 protein level. Next, the direct interaction of miR-590-5p with the 3´-UTR sequence of SMAD3 transcript was investigated using the dual luciferase assay. Finally, flow cytometery was used to inves- tigate the effect of miR-590-5p overexpression on cell cycle progression in HeLa and SW480 cell lines. RESULTS: miR-590-5p was overexpressed in the SW480 cell line and its overexpression resulted in significant reduction of the SMAD3 protein level. Consistently, direct interaction of miR-590-5p with 3´-UTR sequence of SMAD3 was detected. Finally, miR-590-5p over- expression did not show a significant effect on cell cycle progression of Hela and SW480 cell lines. CONCLUSION: Consistent with previous reports about the negative regulatory effect of miR-590 on TGFß receptors, our data suggest that miR-590-5p also attenuates the TGFß signaling pathway through down-regulation of SMAD3.

Experimental evidences for hsa-miR-497-5p as a negative regulator of SMAD3 gene expression.

The SMAD family comprises of transcription factors that function as signal transducers of transforming growth factor (TGFß) superfamily members. MiRNAs are a class of small noncoding RNAs that may play a major role in post transcriptional regulation of SMAD genes. Here, we intended to investigate if hsa-miR-497-5p is capable of regulating SMAD3 gene expression. Hsa-miR-497-5p was bioinformatically predicted as a candidate regulator of SMAD3 gene expression and then, hsa-miR-497-5p expression status was analyzed in different cell lines using RT-qPCR. Overexpression of hsa-miR-497-5p in HEK293t cells resulted in downregulation of SMAD3 which was detected by RT-qPCR and western analysis. Further, dual luciferase assay results supported direct interaction of hsa-miR-497-5p with 3'-UTR sequences of SMAD3 transcript. Overexpression of hsa-miR-497-5p in HEK293t cells resulted in cell cycle arrest in G0/G1 phase, detected by flow cytometry. Overall, accumulative results indicated that hsa-miR-497-5p by targeting SMAD3 is potentially one of the regulators of the TGFß signaling pathway.