Molecular cloning and characterization of heat-responsive LcOPR1, a gene encoding oxophytodienoic acid reductase in lentil.

Improving crop plants using biotechnological implications is a promising and modern approach compared to traditional methods. High-temperature exposure to the reproductive stage induces flower abortion and declines grain filling performance, leading to smaller grain production and low yield in lentil and other legumes. Thus, cloning effective candidate genes and their implication in temperature stress tolerance in lentil (Lens culinaris Medik.) using biotechnological tools is highly demandable. The 12-oxophytodienoic acid reductases (OPRs) are flavin mononucleotide-dependent oxidoreductases with vital roles in plants. They are members of the old yellow enzyme (OYE) family. These enzymes are involved in the octadecanoid pathway, which contributes to jasmonic acid biosynthesis and is essential in plant stress responses. Lentil is one of the vital legume crops affected by the temperature fluctuations caused by global warming. Therefore, in this study, the LcOPR1 gene was successfully cloned and isolated from lentils using RT-PCR to evaluate its functional responses in lentil under heat stress. The bioinformatics analysis revealed that the full-length cDNA of LcOPR1 was 1303 bp, containing an 1134 bp open reading frames (ORFs), encoding 377 amino acids with a predicted molecular weight of 41.63 and a theoretical isoelectric point of 5.61. Bioinformatics analyses revealed that the deduced LcOPR1 possesses considerable homology with other plant 12-oxophytodienoic acid reductases (OPRs). Phylogenetic tree analysis showed that LcOPR1 has an evolutionary relationship with other OPRs in different plant species of subgroup I, containing enzymes that are not required for jasmonic acid biosynthesis. The expression analysis of LcOPR1 indicated that this gene is upregulated in response to the heat-stress condition and during recovery in lentil. This study finding might be helpful to plant breeders and biotechnologists in LcOPR1 engineering and/or plant breeding programs in revealing the biological functions of LcOPR1 in lentils and the possibility of enhancing heat stress tolerance by overexpressing LcOPR1 in lentil and other legume plants under high temperature.

Dihydrotestosterone Induces Chemo-Resistance of Triple-Negative Breast MDA-MB-231 Cancer Cells Towards Doxorubicin Independent of ABCG2 and miR-328-3p.

BACKGROUND: Androgens potentially have an important role in the biology of breast cancer, particularly triple-negative breast cancer (TNBC). Androgen receptor (AR) may offer a novel therapeutic strategy, including the use of microRNA (miRNA) molecules. We have previously shown that AR agonist, dihydrotestosterone (DHT), increases the expression of miR-328-3p in the TNBC MDA-MB-231 cells. One target of the latter miRNA is ATP-binding cassette subfamily G member 2 (ABCG2), which modulates the chemo-response of cancer cells by pumping out xenobiotics. OBJECTIVE: Using MDA-MB-231 cells as a model system for TNBC, we hypothesized that DHT would induce cell sensitivity towards doxorubicin via increasing levels of miR-328-3p and, consequently, reducing ABCG2 levels. METHODS: Chemo-response of cells towards doxorubicin, tamoxifen, and mitoxantrone was evaluated using cell viability MTT assay. Cells were transfected with both miR-328-3p mimic or antisense molecules. Real-time PCR was utilized to assess RNA levels and immunoblotting was performed to investigate levels of ABCG2 protein. PCR arrays were used to assess changes in the expression of drug response regulatory genes. RESULTS: Contrary to our hypothesis, treating MDA-MB-231 cells with DHT no effect towards tamoxifen or mitoxantrone, increased cell resistance towards doxorubicin was noted, concomitant with decreased expression of ABCG2. This under-expression of ABCG2 was also found in MCF-7 and MDA-MB-453 cells treated with DHT. Although miR-328-3p decreased ABCG2 mRNA and protein levels, the miRNA did not alter the chemo-response of cells towards doxorubicin and did not affect DHT-induced chemo-resistance. AR activation slightly decreased the expression of 5 genes, including insulin-like growth factor 1 receptor that may explain the mechanism of DHT-induced chemo-resistance of cells. CONCLUSION: DHT regulates chemo-response via a mechanism independent of ABCG2 and miR-328-3p.

Role of CD44 in breast cancer.

Breast cancer (BC) is among the most prevalent type of malignancy affecting females worldwide. BC is classified into different types according to the status of the expression of receptors such as estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR). Androgen receptor (AR) appears to be a promising therapeutic target of BC. Binding of 5α-dihydrotestosterone (DHT) to AR controls the expression of microRNA (miRNA) molecules in BC, consequently, affecting protein expression. One of these proteins is the transmembrane glycoprotein cluster of differentiation 44 (CD44). Remarkably, CD44 is a common marker of cancer stem cells in BC. It functions as a co-receptor for a broad diversity of extracellular matrix ligands. Several ligands, primarily hyaluronic acid (HA), can interact with CD44 and mediate its functions. CD44 promotes a variety of functions independently or in cooperation with other cell-surface receptors through activation of varied signaling pathways like Rho GTPases, Ras-MAPK, and PI3K/AKT pathways to regulate cell adhesion, migration, survival, invasion, and epithelial-mesenchymal transition. In this review, we present the relations between AR, miRNA, and CD44 and their roles in BC.

Cloning and molecular characterization of a flavin-dependent oxidoreductase gene from barley.

Oxophytodienoate reductases (OPRs) are a small group of flavin-dependent oxidoreductases in plants. In this study, a new member of the OPR gene family (HvOPR2) was cloned from barley (Hordeum vulgare L.) using reverse transcription polymerase chain reaction (RT-PCR). The full-length cDNA of HvOPR2 was 1,206 bp with an open reading frame of 1,101 bp, encoding a 366 amino acids long polypeptide with a predicted molecular weight of 40.52 and a theoretical isoelectric point of 6.21. The corresponding genomic clone of HvOPR2 was isolated using the PCR amplification technique and was found to consist of five exons and four introns. Bioinformatic analysis revealed that the deduced HvOPR2 has a considerable homology with other plant OPRs and possessed the flavin oxidoreductase/NADH oxidase substrate-binding domain. Phylogenetic analysis showed that HvOPR2 codes for the OPR of subgroup I, which contains enzymes that are not required for jasmonic acid biosynthesis. Time-course transcriptional profiling of HvOPR2 was analyzed in response to a variety of abiotic stresses and hormonal treatments by semi-quantitative RT-PCR. The HvOPR2 gene was induced in response to drought, hydrogen peroxide, and wounding. Moreover, the corresponding mRNA transcripts were increased in response to jasmonic acid and salicylic acid, but not in response to abscisic acid. These results strongly suggested a role for HvOPR2 in barley defense/response to abiotic stresses and signaling molecules.