Novel Approach to Overcome Defects of Cell-SELEX in Developing Aptamers against Aspartate ß-Hydroxylase.

Cell-based aptamer selection (Cell-SELEX) against predefined protein targets that benefits using the native form of the targets is the most promising approach to achieve aptamer probes capable of recognizing targets under both in vitro and in vivo conditions. The major disadvantages in Cell-SELEX are the imperfectness of the negative selection step and the lengthy procedure of selection. Here, we introduced the Counter-SELEX as part of our modified Cell-SELEX and implemented deep sequencing to overcome these shortcomings in developing aptamers against aspartate ß-hydroxylase (ASPH) as a known tumor marker. In parallel with the conventional Cell-SELEX, five consecutive cycles of counter selection were accomplished using sequences bound to negative cells (the Counter-SELEX) to detect oligos that are not specific for ASPH. After high-throughput sequencing, the representative of each promising achieved family was subjected to further confirmatory analysis via flow cytometry, followed by the fluorescence immunostaining of histopathological sections. Implementing our innovative complementary method, annoying mis-selected sequences in Cell-SELEX enriched pools were effectively identified and removed. According to the affinity assay on the cells displaying ASPH, three aptamers, AP-Cell 1, AP-Cell 2, and AP-Cell 3, with K d values of 47.51, 39.38, and 65.23 nM, respectively, were obtained, while AP-Cell 1 and 3 could then successfully spot ASPH displayed on the tissues. Our study showed that the Counter-SELEX could be considered as a complementary method for Cell-SELEX to overcome the imperfectness of the negative selection step. Moreover, high-throughput nucleotide sequencing could help to shorten the overall process.

15-Hydroxy-8(17),13(E)-labdadiene-19-carboxylic acid (HLCA) inhibits proliferation and induces cell cycle arrest and apoptosis in ovarian cancer cells.

AIM: 15-Hydroxy-8(17),13(E)-labdadiene-19-carboxylic acid (HLCA) isolated from Juniperus foetidissima, has been recently identified as an antiproliferative agent; however, the molecular basis of antiproliferative effects of HLCA remains unknown. To investigate it, the current study has emphasized the hypothesis that HLCA induced cell death is a consequence of intracellular reactive oxygen species (ROS) production followed by cell cycle arrest and apoptosis. MAIN METHODS: Human ovarian OVCAR-3 and Caov-4 cells were treated with various concentrations of HLCA (48 h) and the measurement of intracellular ROS was considered. Then, the potential of HLCA in promoting apoptosis was investigated via flow cytometry, western blot, and caspase activity assay. Also, the inhibitory effect of HLCA on the cell cycle was evaluated using flow cytometry and western blot analysis. KEY FINDINGS: We found intracellular (ROS) accumulation in HLCA-treated cells. Subsequent observation of the increment in pro-apoptotic Bax as well as the decrement in antiapoptotic Bcl2 revealed that the HLCA-induced cytotoxicity may be triggered by the intrinsic pathway of apoptosis. Our subsequent experiments suggested that caspase-9 and -3 were activated and led the cells to apoptosis during the process. Cell cycle disruption at the G1 phase via down-regulation of cyclin D1 and Cyclin-dependent kinase 4 (CDK4) was another proved mechanism by which HLCA exerts its antiproliferative effects on the ovarian cell lines, OVCAR-3 and Caov-4, especially at relatively lower concentrations. SIGNIFICANCE: This is the first study that reveals the apoptotic effects of HLCA, suggesting its therapeutic potential as an effective anti-tumor agent. However, further in vivo studies are required to confirm these effects.

An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl ß-hydroxylase.

BACKGROUND AND PURPOSE: Aspartyl/asparaginyl ß-hydroxylase (ASPH) is abundantly expressed in malignant neoplastic cells. The establishment of a human cell line overexpressing ASPH could provide the native-like recombinant protein needed for developing theranostic probes. In the process of transfection, the obtained cells normally contain a range of cells expressing the different levels of the target of interest. In this paper, we report on our simple innovative approach in the selection of best-transfected cells with the highest expression of ASPH using subclone selection, quantitative real-time polymerase chain reaction, and gradual increment of hygromycin concentration. EXPERIMENTAL APPROACH: To achieve this goal, human embryonic kidney (HEK 293T) cells were transfected with an ASPH-bearing pcDNA3.1/Hygro(+) vector. During antibiotic selection, single accumulations of the resistant cells were separately cultured and the ASPH mRNA levels of each flask were evaluated. The best subclones were treated with a gradually increasing amount of hygromycin. The ASPH protein expression of the obtained cells was finally evaluated using flow cytometry and immunocytochemistry. FINDINGS / RESULTS: The results showed that different selected subclones expressed different levels of ASPH. Furthermore, the gradual increment of hygromycin (up to 400mg/mL) improved the expression of ASPH. The best relative fold change in mRNA levels was 57.59 ± 4.11. Approximately 90.2% of HEKASPH cells overexpressed ASPH on their surface. CONCLUSION AND IMPLICATIONS: The experiments indicated that we have successfully constructed and evaluated a recombinant human cell line overexpressing ASPH on the surface. Moreover, our innovative selection approach provided an effective procedure for enriching highly expressing recombinant cells.

Aptamer-based approaches for in vitro molecular detection of cancer.

Cancer is typically associated with abnormal production of various tumor-specific molecules known as tumor markers. Probing these markers by utilizing efficient approaches could be beneficial for cancer diagnosis. The current widely-used biorecognition probes, antibodies, suffer from some undeniable shortcomings. Fortunately, novel oligonucleotide-based molecular probes named aptamers are being emerged as alternative detection tools with distinctive advantages compared to antibodies. All of the existing strategies in cancer diagnostics, including those of in vitro detection, can potentially implement aptamers as the detecting moiety. Several studies have been performed in the field of in vitro cancer detection over the last decade. In order to direct future studies, it is necessary to comprehensively summarize and review the current status of the field. Most previous studies involve only a few cancer diagnostic strategies. Here, we thoroughly review recent significant advances on the applications of aptamer in various in vitro detection strategies. Furthermore, we will discuss the status of diagnostic aptamers in clinical trials.

Expression and clinicopathological significance of lipin-1 in human breast cancer and its association with p53 tumor suppressor gene.

Breast cancer (BC) is an important cause of female cancer-related death. It has recently been demonstrated that metabolic disorders including lipid metabolism are a hallmark of cancer cells. Lipin-1 is an enzyme that displays phosphatidate phosphatase activity and regulates the rate-limiting step in the pathway of triglycerides and phospholipids synthesis. The objective of this study was to evaluate lipin-1 expression, its prognostic significance, and its correlation with p53 tumor suppressor in patients with BC. In this study, 55 pairs of fresh samples of BC and adjacent noncancerous tissue were used to analyze lipin-1, using quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) staining. The expression of other clinicopathological variables and p53 was also examined using IHC technique. The cell migration was studied in MCF-7 and MDA-MB231 cells following the inhibition of lipin-1 by propranolol. Our results show that the relative expression of lipin-1 messenger RNA was significantly higher in BC tissues compared with the adjacent normal tissue and its inhibition reduced cell migration in cancer cells. This upregulation was negatively correlated with histological grade of tumor and p53 status (p = .001 and p = .034) respectively and positively correlated with the tumor size (p = .006). Our results also seem to indicate that the high lipin-1 expression is related to a good prognosis in patients with BC. The expression of lipin-1 may be considered as a novel independent prognostic factor. The inhibition of lipin-1 may also have therapeutic significance for patients with BC. The correlation between lipin-1 and p53 confirms the role of p53 in the regulation of lipid metabolism in cancer cells.

The insulin-like growth factor binding protein-3 and its death receptor in pancreatic ductal adenocarcinoma poor prognosis.

Insulin-like growth factor binding protein-3 (IGFBP-3) and its newly discovered death receptor (IGFBP-3R) have been reported to involve in a wide variety of cancers. However, their role in pancreatic ductal adenocarcinoma (PDAC) has not been elucidated yet. Here, 478 pancreatic cancers were screened for primary PDAC tumors. The samples were evaluated using quantitative reverse-transcriptase polymerase chain reaction, western blotting, and immunohistochemistry staining. The results indicated that relative IGFBP-3 mRNA expression and its protein level were reduced stage dependently in the PDAC tumors (p < .001 and p < .05, respectively). The subcellular distribution of IGFBP-3 was mainly nuclear only in Stage 0 + 1 (about 150% compared to adjacent normal tissues [p < .05]). The value for IGFBP-3R messenger RNA (mRNA) and protein were also reduced in tumors in compared to adjacent normal pancreatic tissues (p < .05). The Kaplan-Meier analysis also showed that mRNA expression of IGFBP-3 and IGFBP-3R was positively associated with survival, (p = .001). In addition, there is a strong association between low expression of IGFBP-3 and tumor size (p = .032), the lymphatic invasion (p = .001), the TNM (tumor, node, metastasis) staging (p = .001), tumor differentiation (p = .001), and PNI status (p = .021). Down-regulation of IGFBP-3R was also correlated with the tumor size (p = .01), the lymphatic invasion (p = .012) TNM staging (p = .001), tumor differentiation (p = .021) and PNI status (p = .038). In conclusion, IGFBP-3 and its receptor were down-regulated and their expression was associated with poor prognosis of PDAC.

Serum-based microRNA biomarkers for major depression: MiR-16, miR-135a, and miR-1202.

BACKGROUND: Depression is a common medical condition with a high prevalence leading to emotional abnormality. Despite some drawbacks, depression currently diagnosed using a combination of patient interviews and self-report questionnaires. Recently, there is emerging emphasis to establish biomarkers to diagnosis and clinical management of depression. This case-control study was designed to develop microRNA (miRNA)-based serum biomarker for depression. MATERIALS AND METHODS: In this study, 39 patients with depression and 36 healthy controls were enrolled. Serum miRNAs gene expression was measured using real-time polymerase chain reaction (PCR) analysis; finally, the data represent as the 2-ΔCt followed by further statistical analysis. RESULTS: The serum level of miR-16 was significantly (P < 0.001) down-regulated (mean: 0.9123 and standard deviation [SD]: 0.06) in compared to normal individuals (mean: 1.6848 and SD: 0.09). The concentration of miR-135a was also catastrophically decreased (P < 0.001) in the patients (mean: 1.160 and SD: 0.07) in compared to control (mean: 1.819 and SD: 0.09). The relative miR-1202 expression levels were significantly lower (P < 0.001) in the patients (mean: 0.1755 and SD: 0.01) than in the healthy individuals (mean: 0.2939 and SD: 0.01). The receiver operating characteristic curve analysis indicated the obvious separation between patient and healthy control, with an AUC of 0.75 (95% confidence interval [CI] = 0.642-0.858, P < 0.001), 0.72 (95% CI = 0.607-0.834, P < 0.001), and 0.74 (95% CI = 0.630-0.861, P < 0.001) for miR-16, miR-135a, and miR-1202, respectively. The data suggest that these miRNAs have a potential to be used as a biomarker of depression with sensitivity 77.8% and specificity of 61.5% for miR-16, 94.4% and 41.0% for miR-135a as well as 86.1% and 61.5% for miR-1202, respectively (P < 0.001). CONCLUSION: Our findings showed that these miRNA can be used as a biomarker of depression diagnosis. MiR-135a and miR-1202 exhibited better sensitivity and specificity, respectively.