Mina Lashkarboloki; Amin Jahanbakhshi; Seyed Javad Mowla; Bahram Mohammad Soltani
Abstract
Cancer is one of the most challenging diseases in the world. It is widely accepted that knowing the molecular aspects of diseases, including cancers, helps to develop methods for their therapy and diagnosis. Long non-coding RNAs (lncRANs) are a novel category of regulatory genes known to be involved ...
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Cancer is one of the most challenging diseases in the world. It is widely accepted that knowing the molecular aspects of diseases, including cancers, helps to develop methods for their therapy and diagnosis. Long non-coding RNAs (lncRANs) are a novel category of regulatory genes known to be involved in cancer incidence. The expression of these genes is said to be suitable of using in prognosis, diagnosis, targeted therapy, etc. The RT-qPCR method that is widely used for analyzing the gene expression requires the application of appropriate reference genes as the internal control. The expression status of a proper housekeeping reference gene is not supposed to change under experimental circumstances. This study aimed to find a suitable reference gene in the U87 cells after overexpression of a gene of interest. To this aim, the expression status of four common reference genes (ACTB, β2M, GAPDH, and HPRT1) was examined in the transfected U87 cells. The U87 cells were transfected with a vector overexpressing YWHAE-lncRNA and an empty vector (mock). After total RNA extraction and cDNA synthesis, RT-qPCR was applied using the aforementioned internal control genes. Data were analyzed, and their graphs were plotted in GraphPad Prism 8.2 software. Β2M showed the most change; accordingly, GAPDH and HPRT1 expression levels were changed about 5 and 4 times, respectively. Of the candidate genes, only the ACTB gene had a consistent expression level in two different modes of transfection, and therefore, it is suggested as an appropriate reference gene for the study of gene expression in the transfected U87 cell line. It is remained to be tested if β2M, GAPDH, and HPRT1 common internal controls are specifically affected by YWHAE-lncRNA overexpression or other lncRNAs may affect their expression as well.
Hamideh Monfared; Yavar Jahangard; Maryam Nikkhah; Seyed Javad Mirnajafi-Zade; Seyed Javad Mowla
Abstract
There are different subtypes of brain tumors, classified according to the origin of the abnormally proliferated glial cells. Glioblastoma multiforma (GBM) is the grade 4 of brain tumors, gliomas, with the least life expectancy. microRNAs (miRNAs) are small, single stranded, non-coding ...
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There are different subtypes of brain tumors, classified according to the origin of the abnormally proliferated glial cells. Glioblastoma multiforma (GBM) is the grade 4 of brain tumors, gliomas, with the least life expectancy. microRNAs (miRNAs) are small, single stranded, non-coding RNAs with 20-25 nt length with post-transcriptional gene regulatory activities. An altered expression of miRNAs is linked to developmental disorders and some diseases, most importantly cancers. miR-21 is a well-known microRNA, overexpressed in almost all cancer types, including brain tumors. It targets several genes with vital roles in cellular pathways involve in proliferation, invasion and metastatic behaviors. Exosomes are 30-100 nm extracellular vesicles which are packed with various molecules, including miRNAs. Here, we suppressed miR-21 expression level in HEK-293T cells by transfecting them with the miRZip-21 vector. However, when U87-MG cells were cultured in the presence of exosomes isolated from conditioned medium of engineered HEK-293T cells derived exosomes, we did not observe any suppressing effect on host cells’ miR-21 expression level. Moreover, by analyzing the effects of miRZip-21-enriched cell’s conditioned media on three other brain cell lines including 1321N1, A-172 and DAOY, cell type-specific effects of exocrine miRZip-21 were revealed. These data suggested that cell lines from different brain tumor subtypes could exert different responses to microRNA-based therapies, based on their cellular origin and clinical behaviors.
Samaneh Khazaei; Sedigheh Gharbi; Seyed Javad Mowla
Abstract
Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with poor prognosis. In this regard, early diagnosis is of vital importance to cure the tumor in its early stages. Novel cancer diagnostic and therapeutic approaches have been recently introduced based on microRNAs (miRNAs). Also, accurate ...
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Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with poor prognosis. In this regard, early diagnosis is of vital importance to cure the tumor in its early stages. Novel cancer diagnostic and therapeutic approaches have been recently introduced based on microRNAs (miRNAs). Also, accurate normalization using appropriate reference genes is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference genes for miRNA quantification in serum samples of ESCC. In this case and control experimental study, two statistical algorithms including GeNorm and NormFinder were used to evaluate the suitability of miR-16 and 5S rRNA and their geometric mean as reference genes. Then, relative expression of miR-451 and miR-24 were evaluated while different normalizer including miR-16, 5S rRNA and their geometric mean were applied. Both GeNorm and NormFinder analyses showed that geometric mean of miR-16 and 5S rRNA is the most stable reference gene in these samples. Also, our data showed that choosing an inappropriate normalizer could change the relative expression of target genes of miR-451 and miR-24 in ESCC samples which emphasize on the importance of selecting a reliable internal control in expression analyses. We demonstrated that geometric mean of two reference genes could increase the reliability of normalizers and also by using geometric mean as reference gene, relative expression of different target is closer to reality.
zahra bahadori; seyed javad Mowla; Hamid Reza Kalhor
Abstract
Keratinocyte Growth Factor (KGF) is a paracrine-acting and epithelium-specific growth factor produced by cells of
mesenchymal origin. Based on preclinical data, recombinant KGF plays a critical role in protecting and repairing of
damaged epithelial tissues. Despite great efforts to express recombinant ...
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Keratinocyte Growth Factor (KGF) is a paracrine-acting and epithelium-specific growth factor produced by cells of
mesenchymal origin. Based on preclinical data, recombinant KGF plays a critical role in protecting and repairing of
damaged epithelial tissues. Despite great efforts to express recombinant human KGF(rhKGF) in different organisms,
attempts for finding appropriate protein expression system with the ability of producing a properly folded and
processed KGF needs further investigation. Pichia pastoris has been used successfully and extensively for production
of industrial enzymes and pharmaceutical proteins. Herein, we investigated the affect of pro-region-α-factor early
deletion on production and secretion of rhKGF in Pichia pastoris. Initially, expression of human KGF induced in
MCF-7 cell line treated with 1, 25-Dihydroxy vitamin D3. The coding sequence of full-length rhKGF194 was then
cloned into the yeast integrative expression vector, downstream of α-factor and was integrated into P. pastoris
genome. KGF protein was expressed in P. pastoris x33 cells, usingα-factor signal peptide for translocation of KGF
to ER. An internal human signal peptide was also arranged after α-factorfor early removal of the pro-region in ER.
RT-PCR results demonstrated that KGF mRNA was expressed successfully after induction by methanol.
Recombinant KGF protein expression was detected by Western blotting in cell lysats, but not in conditioned media.
A molecular weight of 17 kD for rhKGF194 indicates that the α-factor and internal human signal peptideshad been
removed in x33 cells. The results indicate that in the absence of pro-region-α-factor, the recombinant KGF protein
was not efficiently processed and transported within the biosynthesis-secretory pathway. As KGF protein is an
unstable growth factor and tend to aggregate because of some native properties, It seems that presence of a chaperon
molecule fusion with KGF is necessary for efficient secretion of the recombinant protein.
Mahshid Malakootian; Youssef Fouani; Parisa Naeli; Fatemeh Mirzadeh Azad; Seyed Amir Mohsen Ziaee; Seyed Javad Mowla
Abstract
Long non-coding RNAs (lncRNAs) have recently found to have important regulatory roles, and their aberrant expressions and functions are directly linked to carcinogenesis. Both urinary bladder and breast tumors are prevalent neoplasms, with high rates of incidence. To identify a potential expression alteration ...
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Long non-coding RNAs (lncRNAs) have recently found to have important regulatory roles, and their aberrant expressions and functions are directly linked to carcinogenesis. Both urinary bladder and breast tumors are prevalent neoplasms, with high rates of incidence. To identify a potential expression alteration of the recently discovered "anti-differentiation non-coding RNA, (ANCR), during tumorigenesis, we initially assessed its expression in several cancer cell lines (LNCAP, MCF-7, Ht-29, 5637, A549, HepG2, and PC3) and then compared its expression variability in tumor vs. non-tumor samples of bladder and breast. Here, ANCR expression profile was studied by qRT-PCR in paired tumor and marginal non-tumor samples obtained from patients that had been referred to the Labbafi-Nejad and Imam Khomeini Hospitals, respectively. Our data revealed a significant upregulation (p = 0.003) of ANCR in breast tumor tissues, in comparison to non-tumor marginal specimens from same patients. Similar upregulation was also detected in bladder tumor samples, however, this alteration was not statistically significant (p ≥ 0.05), probably due to small number of samples (n = 10). In conclusion, our results suggest a possible role of ANCR in tumorigenesis of bladder and breast tissues, as well as its potential usefulness as a novel diagnostic biomarker for bladder and breast tumors.
Nazila Nouraee; Mohammad Vasei; Shahriar Semnani; Seyed Javad Mowla
Abstract
MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamental cellular
processes, and their disregulations have been linked to several pathologic conditions, mainly cancers.
Determining tissue distribution of miRNAs is a prerequisite for understanding their exact functions ...
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MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamental cellular
processes, and their disregulations have been linked to several pathologic conditions, mainly cancers.
Determining tissue distribution of miRNAs is a prerequisite for understanding their exact functions during
development, tissue homeostasis and abnormality. In situ hybridization is a powerful technique to delineate the
sub-cellular localization and tissue distribution patterns of mRNAs as well as miRNAs. Due to the important
role of miRNAs in tumorigenesis, we optimized an ISH technique for detection of two well-known miRNAs
(miR-302 and miR-21) in formalin-fixed paraffin-embedded (FFPE) tumor samples along with a pluripotent
embryonal carcinoma cell line, NTERA-2 (NT2). After fixation of cells on slides/sectioning of FFPE blocks,
proteinase K digestion, probe concentration, antibody development and light sensitive color reaction were
optimized for both the FFPE samples and cell line. Signals for U6 snRNA, as an internal control, were detected
in the nuclei of the cells. MiR-21 and miR-302 expression was detected in the cytoplasm of FFPE samples of
seminoma carcinoma and in NT2 cell line, respectively. In this study, we optimized ISH for miRNA detection
in FFPE samples and NT2 cell line.
