Iranian Journal of Biotechnology
Volume:21 Issue: 1, Winter 2023

Laryngeal cancer (LC) remains one of the most common tumors of the respiratory tract, the exact pathogenesis remains unclear. MiRNA-106a-5p is aberrantly expressed in a variety of cancers and plays a pro- or anti-cancer role, but is indistinct in LC. Showing the role of miRNA-106a-5p in the development of LC. Quantitative reverse transcription-polymerase chain reaction was used for miR-106a-5p measurement in clinical samples and LC cell lines (AMC-HN8 and TU212), first. The expression of miR-106a-5p was inhibited by inhibitor, then followed clonogenic and flow cytometric assays for cell proliferation; wood healing, and Transwell assays for cell migration. Dual luciferase reporter assay was performed for interaction verification, and the activation of the signal pathway was detected by western blots. MiR-106a-5p was significantly over-expressed in LC tissues and cell lines. The proliferation ability of the LC cells was significantly reduced after miR-106a-5p inhibition, and most LC cells were stagnated in the G1 phase. The migration and invasion ability of the LC cells was decreased after the miR-106a-5p knockdown. Further, we found that miR-106-5a is bound with 3’-UTR of AKT interacting protein (AKTIP) mRNA specifically, and then activate PI3K/AKT/m-TOR pathway in LC cells. A new mechanism was uncovered that miR-106a-5p promotes LC development via AKTIP/PI3K/AKT/m-TOR axis, which guides clinical management and drug discovery.

Overexpression of miR-141 and miR-200a is known to be associated with the differentiation of T helper 17 (Th17) cells, which are key players in the pathophysiology of autoimmune disorders. However, the function and governing mechanism of these two microRNAs (miRNAs) in Th17 cell skewing are poorly defined. The aim of the present study was to identify the common upstream transcription factors and downstream target genes of miR-141 and miR-200a to obtain a better insight into the possible dysregulated molecular regulatory networks driving miR-141/miR-200a-mediated Th17 cell development. A consensus-based prediction strategy was applied for in-silico identification of potential transcription factors and putative gene targets of miR-141 and miR-200a. Thereafter, we analyzed the expression patterns of candidate transcription factors and target genes during human Th17 cell differentiation by quantitative real-time PCR and examined the direct interaction between both miRNAs and their potential target sequences using dual-luciferase reporter assays. According to our miRNA-based and gene-based interaction network analyses, pre-B cell leukemia homeobox 1 (PBX1) and early growth response 2 (EGR2) were respectively taken into account as the potential upstream transcription factor and downstream target gene of miR-141 and miR-200a. There was a significant overexpression of the PBX1 gene during the Th17 cell induction period. Furthermore, both miRNAs could directly target EGR2 and inhibit its expression. As a downstream gene of EGR2, the suppressor of cytokine signaling 3 (SOCS3) was also downregulated during the differentiation process. These results indicate that activation of the PBX1/miR-141-miR-200a/EGR2/SOCS3 axis may promote Th17 cell development and, therefore, trigger or exacerbate Th17-mediated autoimmunity.

Tumor necrosis factor (TNF)-α induces changes in the glucocorticoid receptor (GR) isoforms’ expression in human nasal epithelial cells (HNECs) in chronic rhinosinusitis (CRS). However, the underlying mechanism of TNF-α induced GR isoforms’ expression in HNECs remains unclear. Here, we explored changes in inflammatory cytokines and glucocorticoid receptor alpha isoform (GRα) expression in HNECs. To explore the expression of TNF-α in nasal polyps and nasal mucosa of CRS, fluorescence immunohistochemical analysis was employed. To investigate changes in inflammatory cytokines and GRα expression in HNECs, RT-PCR and western blotting were performed following the cells’ incubation with TNF-α. Cells were pretreated with the nuclear factor-κB gene binding (NF-κB) inhibitor QNZ, the p38 inhibitor SB203580, and dexamethasone for one hour, then a TNF-α. Western blotting, RT-PCR, and immunofluorescence had been utilized for the cells’ analysis and the ANOVA for the data analysis. The TNF-α fluorescence intensity was mainly distributed in nasal epithelial cells of nasal tissues. TNF-α prominently inhibited the expression of GRα mRNA from 6 to 24 h in HNECs. GRα protein was decreased from 12 to 24 h. Treatment with QNZ, SB203580, or dexamethasone inhibited the TNF-α and interleukin (IL)-6 mRNA expression and increased the GRα levels. TNF-α induced changes in the GR isoforms’ expression in HNECs, and it was mediated through p65-NF-κB and p38-MAPK signal transduction pathways, which could be considered a promising neutrophilic CRS treatment.

Tumor cells proliferation and apoptosis inhibition are the mechanisms through which the Colorectal Cancer (CRC) progression, metastasis and chemoresistance are promoted pathologically, offering clinical advantages for characterizing their molecular regulators.

In this study, to unravel the role of PIWIL2 as a potential CRC oncogenic regulator, we examined the effect of its overexpression on proliferation, apoptosis and colony formation of SW480 colon cancer cell line.

Established SW480-P (overexpression of PIWIL2) and SW480-control (SW480-empty vector) cell lines were cultured in DMEM containing 10% FBS with 1% penicillin-streptomycin. The total DNA and RNA was extracted for further experiments. Real-Time PCR and western blotting assay were performed to measure the differential expression of proliferation associated genes including the expression of cell cycle and anti-apoptotic genes as well as Ki-67 and PIWIL2 in both cell lines. Cell proliferation was determined using MTT assay, doubling time assay and the colony formation rate of transfected cells was measured with the 2D colony formation assay.

At the molecular level, PIWIL2 overexpression was associated with significant up-regulation of cyclin D1, STAT3, BCL2-L1, BCL2-L2 and Ki-67 genes. MTT and doubling time assay showed that PIWIL2 expression induced time-related effects on proliferation rate of SW480 cells. Moreover, SW480-P cells had markedly greater capacity to form colonies.

PIWIL2 plays important roles to promote cancer cell proliferation and colonization via the cell cycle acceleration and inhibition of apoptosis, the mechanisms through which this gene seems to contribute to CRC development, metastasis and chemoresistance, hence potentially highlighting PIWIL2 targeted therapy as a valuable tool for CRC treatment.

Troponin T1 (TNNT1) is implicated in human carcinogenesis. However, the role of TNNT1 in ovarian cancer (OC) remains unclear. To investigate the effect of TNNT1 on the progression of ovarian cancer. The level of TNNT1 was evaluated in OC patients based on The Cancer Genome Atlas (TCGA). Knockdown or overexpression of TNNT1 using siRNA targeting TNNT1 or plasmid carrying TNNT1 was performed in the ovarian cancer SKOV3 cell, respectively. RT-qPCR was performed to detect mRNA expression. Western blotting was used to examine protein expression. Cell Counting Kit-8, colony formation, cell cycle, and transwell assays were performed to analyze the role of TNNT1 on the proliferation and migration of ovarian cancer. Besides, xenograft model was carried out to evaluate the in vivo effect of TNNT1 on OC progression. Based on available bioinformatics data in TCGA, we found that TNNT1 was overexpressed in ovarian cancer samples comparing to normal samples. Knocking down TNNT1 repressed the migration as well as the proliferation of SKOV3 cells, while overexpression of TNNT1 exhibited opposite effect. In addition, down-regulation of TNNT1 hampered the xenografted tumor growth of SKOV3 cells. Up-regulation of TNNT1 in SKOV3 cells induced the expression of Cyclin E1 and Cyclin D1, promoted cell cycle progression, and also suppressed the activity of Cas-3/Cas-7. In conclusion, TNNT1 overexpression promotes SKOV3 cell growth and tumorigenesis by inhibiting cell apoptosis and accelerating cell-cycle progression. TNNT1 might be a potent biomarker for the treatment of ovarian cancer.

Dopamine (DA) is one of the most important catecholamine neurotransmitters in the central nervous system. The degeneration and deletion of dopaminergic neurons are closely linked to Parkinson's disease (PD) and other psychiatric or neurological diseases. Several studies have been suggesting that intestinal microorganisms are associated with the occurrence of central nervous diseases, including diseases that are closely related to dopaminergic neurons. However, the intestinal microorganisms regulation of dopaminergic neurons in the brain is largely unknown. This study aimed to investigate the hypothetical differences of DA and its synthase tyrosine hydroxylase (TH) expression in different parts of the brain of germ free (GF) mice. Several studies in recent years have shown that commensal intestinal microbiota promotes changes in DA receptor expression, DA levels, and affects this monoamine turnover. Germ free (GF) and specific pathogen free (SPF) C57b/L male mice were used to analyze TH mRNA and expression levels, and DA levels in the frontal cortex, hippocampus, striatum, and cerebellum, using real time PCR, western blotting, and ELISA tools. Compared with SPF mice, the TH mRNA levels were decreased in the cerebellum of GF mice, while the TH protein expression was tended to increase in the hippocampus, and conversely showed significant decrease in the striatum. The average optical density (AOD) of TH immunoreactive nerve fibers and the number of axons in striatum of mice in GF group were significantly lower than that in SPF group. Compared with SPF mice, the DA concentration in the hippocampus, striatum and frontal cortex of GF mice was decreased in GF mice. The changes of DA and its synthase TH in the brain of GF mice showed that the absence of conventional intestinal microbiota had certain regulatory effects on central dopaminergic nervous system, which is considered helpful for studying the effect of commensal intestinal flora on diseases related to impaired dopaminergic nerve system.

Long noncoding RNAs (lncRNAs) play an important role in cellular mechanisms including transcription, translation, and apoptosis. NEAT1 is one of the essential types of lncRNAs in humans that can bind to active genes and modify their transcription. NEAT1 upregulation in various forms of cancer such as kidney cancer has been reported. Kidney cancer accounts for approximately 3% of all cancers worldwide and occurs almost twice as often in men as in women. This study has been performed to knockout the NEAT1 gene using the CRISPR/Cas9 technique in the Renal Cell Carcinoma ACHN cell line and to evaluate its effects on cancer progression and apoptosis. Two specific (single guide RNA (sgRNA) sequences for the NEAT1 gene were designed by CHOPCHOP software. These sequences were then cloned into plasmid pSpcas9, and recombinant vectors PX459-sgRNA1 and PX459-sgRNA2 were generated. ACHN cells were transfected using recombinant vectors carrying sgRNA1 and sgRNA2. The expression level of apoptosis-related genes was assessed by real-time PCR. Annexin, MTT and cell scratch tests were performed to evaluate the survival, proliferation, and migration of the knocked out cells, respectively. The results have shown successful knockout of the NEAT1 gene in the cells of the treatment group. Expressions of P53, BAK, BAX and FAS genes in the cells of the treatment group (NEAT1 knockout) showed significant increases in expression compared to the cells of the control group (P <0.01). Additionally, decreased expression of BCL2 and survivin genes was observed in knockout cells compared to the control group (p <0.05). In addition, in the cells of the treatment group compared to control cells, a significant decrease in cell viability, ability to migrate and cell growth and proliferation was observed. Inactivation of the NEAT1 gene in ACHN cell line using CRISPR/Cas9 technology elevated apoptosis and reduced cell survival and proliferation which makes it a novel target for kidney cancer therapeutics.

Reteplase (recombinant plasminogen activator, r-PA) is a recombinant protein designed to imitate the endogenous tissue plasminogen activator and catalyze the plasmin production. It is known that the application of reteplase is limited by the complex production processes and protein’s stability challenges. Computational redesign of proteins has gained momentum in recent years, particularly as a powerful tool for improving protein stability and consequently its production efficiency. Hence, in the current study, we implemented computational approaches to improve r-PA conformational stability, which fairly correlates with protein’s resistance to proteolysis.

The current study was developed in order to evaluate the effect of amino acid substitutions on the stability of reteplase structure using molecular dynamic simulations and computational predictions.

Several web servers designed for mutation analysis were utilized to select appropriate mutations. Additionally, the experimentally reported mutation, R103S, converting wild type r-PA into non-cleavable form, was also employed. Firstly, mutant collection, consisting of 15 structures, was constructed based on the combinations of four designated mutations. Then, 3D structures were generated using MODELLER. Finally, 17 independent 20-ns molecular dynamics (MD) simulations were conducted and different analysis were performed like root-mean-square deviation (RMSD), root-mean-square fluctuations (RMSF), secondary structure analysis, number of hydrogen bonds, principal components analysis (PCA), eigenvector projection, and density analysis.

Predicted mutations successfully compensated the more flexible conformation caused by R103S substitution, so, improved conformational stability was analyzed from MD simulations. In particular, R103S/A286I/G322I indicated the best results and remarkably enhanced the protein stability.

The conformational stability conferred by these mutations will probably lead to more protection of r-PA in protease-rich environments in various recombinant systems and potentially enhance its production and expression level.

Microbial phytase is one of the most widely used enzymes in food industries like cattle, poultry, and aquaculture food. Therefore, understanding the kinetic properties of the enzyme is very important to evaluate and predict its behavior in the digestive system of livestock. Working on phytase is one of the most challenging experiments because of some problems, including free inorganic phosphate (FIP) impurity in phytate (substrate) and interference reaction of the reagent with both phosphates (product and phytate impurity).

In the present study, FIP impurity of phytate was removed, and then it was shown that the substrate (phytate) has a dual role in enzyme kinetics: substrate and activator.

phytate impurity was decreased by two-step recrystallization prior to the enzyme assay. The impurity removal was estimated by the ISO30024:2009 method and confirmed by Fourier-transform infrared (FTIR) spectroscopy. The kinetic behavior of phytase activity was evaluated using the purified phytate as substrate by non-Michaelis-Menten analysis, including Eadie-Hofstee, Clearance, and Hill plots. The possibility of an allosteric site on phytase was assessed by molecular docking.

The results showed a 97.2% decrease in FIP due to recrystallization. The phytase saturation curve had a sigmoidal appearance, and Lineweaver-Burk plot with a negative y-intercept indicated the positive homotropic effect of the substrate on the enzyme activity. A right-side concavity of Eadie-Hofstee plot confirmed it. Hill coefficient was calculated to be 2.26. Molecular docking also showed that Escherichia coli phytase molecule has another binding site for phytate very close to the active site, called “allosteric site”.

The observations strongly propose the existence of an intrinsic molecular mechanism in Escherichia coli phytase molecules to be promoted for more activity by its substrate, phytate (positive homotropic allosteric effect). In silico analysis showed that phytate binding to the allosteric site caused new substrate-mediated inter-domain interactions, which seems to lead to a more active conformation of phytase. Our results provide a strong basis for animal feed development strategies, especially in the case of poultry food and supplements, regarding a short food passage time in their gastrointestinal tract and variable concentration of phytate along with it. Additionally, the results strengthen our understanding of phytase auto-activation as well as allosteric regulation of monomeric proteins in general.

Tomato mosaic disease, mainly caused by Tomato mosaic virus (ToMV), is one of the devastating viral diseases which adversely affects tomato yield, globally. Plant growth-promoting rhizobacteria (PGPR) have been recently used as bio-elicitors to induce resistance against plant viruses.

The goal of this research was to apply PGPR in the tomato rhizosphere and to determine the response of plants challenged with ToMV infection, under greenhouse conditions.

Two different strains of PGPR, Pseudomonas fluorescens SM90 and Bacillus subtilis DR06, in single- and double-application methods applied to evaluate their effectiveness in inducing defense-related genes, viz., NPR1, COI1, and PR1-a before (induced systemic resistance [ISR]-prime) and after (ISR-boost) ToMV challenge. Additionally, to investigate the biocontrol potential of PGPR-treated plants against viral infection, plant growth indices, ToMV accumulation, and disease severity were compared in primed and non-primed plants.

Analysis of expression patterns of putative defense-related genes before and after ToMV infection indicated that studied PGPR trigger defense priming through different signaling pathways acting at the transcriptional level and in a species-dependent manner. Moreover, the biocontrol efficacy of consortium treatment did not differ significantly from the single bacteria treatments, even though their mode of action differed in transcriptional changes of ISR-induced genes. Instead, simultaneous application of Pseudomonas fluorescens SM90 and Bacillus subtilis DR06 led to more significant growth indices than the single treatments suggesting that integrated application of the PGPR could additively reduce the disease severity and virus titer and promote the growth of the tomato plant.

These results suggested that enhanced defense priming via activation of the expression pattern of defense-related genes is responsible for biocontrol activity and growth promotion in PGPR-treated tomato plants challenged with ToMV compared to non-primed plants, under greenhouse conditions.