Expression, Purification and Functional Assessment of Smallest Isoform of Human Interleukin-24 in Escherichia coli

ABSTRACT Interleukin-24 (IL-24) is a novel tumor-suppressor gene that has different alternative splice isoforms. It has been shown that new smallest isoform of human IL-24 gene, lacking three exons, induces higher levels of cytotoxicity than all the isoforms, indicating shortest isoform of IL-24 may be a new promising anti-cancer agent. In this study, we aimed to provide a reproducible method for recombinant production of the smallest isoform of IL-24 (sIL-24). The Structure of sIL-24 was analyzed using bioinformatics tools (I-TASSER, Prosa, RAMPAGE and SPDBV version 4.1). The DNA sequence encoding sIL-24 was chemically synthesized and sub-cloned into the pET-32a (+) vector for further protein expression in Escherichia coli BL21 (DE3) strain. Upon IPTG induction, sIL-24 peptide was expressed as a thioredoxin fusion protein. The recombinant sIL-24 was released from the fusion by TEV protease cleavage followed by nickel affinity chromatography. The yield of the purified sIL-24 was estimated about 380 μg/ml. MTT assay showed that sIL-24 peptide inhibited the proliferation of PC-3 cancer cells more effectively than full length IL-24 protein, while none affect the survival of MRC-5 normal cells. These results indicate that the presented expression system is an efficient system for the production of small functional recombinant sIL-24 peptide.This functional peptide may have cancer therapeutic application.

Is There a Role for Genetic Information in Risk Assessment and Decision Making in Prostate Cancer?

OBJECTIVES: Prostate cancer is a neoplasm with a variable natural history and clinical behavior. There is much debate on the use of inherited genetic information in clinical application including risk assessment and treatment decisions. This study was performed to evaluate the relationship between clinical parameters of prostate cancer (PSA, Gleason score, and metastasis) and expression of NKX3.1, AMACR, TMPRSS2-ERG, ERG, and SPINK1 genes. METHODS: Newly diagnosed cases of prostate cancer were selected for this study. Thirty four tissue samples were obtained via open radical prostatectomy and 9 samples were obtained via needle biopsy. Each tissue sample was sectioned into two parts, one used for detection of malignant changes and Gleason score determination, and the other immersed in RNA later solution (Qiagen). The expression of NKX3.1, AMACR, TMPRSS2-ERG, ERG, and SPINK1 genes were assessed by real-time PCR assay. Correlation between expression of each gene and PSA level, Gleason score, and presence of metastasis were examined. RESULTS: A total number of 43 specimens were studied, from which 9 were obtained from patients with metastatic prostate cancer. The expression of five examined genes had no correlation with PSA level and Gleason score. The expression of AMACR decreased in metastatic prostate cancer (P = 0.02). The expression of other genes showed no difference between metastatic and non-metastatic tumors (P > 0.1). CONCLUSIONS: Genetic information combined with clinical data can be useful in risk assessment and treatment planning. Based on the results of the current study, the decreased expression of AMACR was a sign of poor prognosis.

Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis.

Development of a rapid and accurate quantification method for the detection of microRNAs (miRNAs) has been desired, in particular, when they are differently expressed in normal and pathological conditions. However, various methods for the quantification of small non-coding RNAs as well as miRNAs have been described. These methods mainly include hybridization-based approaches such as primer extension, northern blotting, microarray profiling, and reverse transcription (RT) PCR. Here, we developed a simple and rapid method based on stem-loop primer-based real-time PCR assay for sensitive and accurate detection of mature miRNAs. Initially, a miRNA-specific stem-loop RT primer is used for RT, which is followed by TaqMan real-time PCR assay using specific forward primer in combination with universal reverse primer and TaqMan probe. The assay has shown high sensitivity (≤50 copies/reaction) for miRNA detection in two breast cancer cell lines, MCF-7 and MDA-MB-231. This assay might be implicated as a rapid and cost effective method for the detection of small non-coding RNAs.

Development of a quantitative real-time PCR assay for detection of unknown alpha-globin gene deletions.

BACKGROUND: Alpha-Thalassemia is the most common inherited disorder of hemoglobin (Hb) synthesis in the world. Unlike beta-thalassemia, in which non-deletional mutations predominate, most of recognized alpha-thalassemia mutations include deletion of one or both alpha-globin genes. The importance of alpha-thalassemia detection is mainly due to its shared blood parameters with beta-thalassemia and its impact on discrimination between unknown alpha-thalassemia and normal HbA2 beta-thalassemia during thalassemia prevention program. MATERIALS AND METHODS: Cases with hematologic profile of low MCV, MCH, and normal HbA2 were enrolled in this study. Common alpha-globin deletional mutations including alpha(3.7)kb, alpha(4.2)kb, alpha(20.5)kb, and alpha(MED) and point mutation including 5 nt, Constant Spring (CS), and C19 were checked using either GAP-PCR or ARMS-PCR. Cases with unknown molecular defects were investigated further by direct gene sequencing. Finally, further study was done for probable unknown deletions by gene dosage analysis using real-time PCR. For this, five pairs of primers were used spanning from theta-globin gene up to the 3' upstream of alpha(2) gene. RESULTS: After validation of primers specificity and performing serial dilution analysis in order to calculate PCR efficiency, the assay was performed on normal samples and cases with known alpha-globin gene deletions as positive and negative controls, respectively. The assay was able to diagnose the control groups successfully. In 21 out of 29 unknown cases (72.4%), the assay showed various patterns of deletions in at 2 to 5 screened regions (theta gene up to the upstream of alpha2 gene). In 8 (27.6%) cases, deletions were seen in all regions. CONCLUSION: Gene dosage study by quantitative real-time PCR can be suggested as a rapid and reliable assay to screen probable carrier of alpha-thalassemia for unknown alpha-globin gene deletions.