Fatemeh Mirzadeh azad; Mahshid Malakootian; Seyed Javad Mowla
Abstract
OCT4 is the major regulator of pluripotency in embryonic stem cells and its association with tumorigenesis, cellular stress response, and homeostatic multifactorial diseases have been recently reported. To serve the versatility in its function, OCT4 generates several transcript variants which their expression ...
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OCT4 is the major regulator of pluripotency in embryonic stem cells and its association with tumorigenesis, cellular stress response, and homeostatic multifactorial diseases have been recently reported. To serve the versatility in its function, OCT4 generates several transcript variants which their expression levels are tightly regulated through different mechanisms. PSORS1C3 is a long non-coding RNA with overlapping genomic location with OCT4 gene. Here, we investigated the effect of PSORS1C3 overexpression on OCT4 expression in different cell lines. Our data revealed that ectopic expression of PSORS1C3 did not affect OCT4 transcripts abundance in NT2 cells, as a model of pluripotent cells. However, in HEK293T cells, PSORS1C3 overexpression led to an increase in OCT4B as a homeostatic isoform and a decrease in OCT4A transcript level. We also observed that manipulating PSORS1C3 in HeLa cells, as a model of epithelial carcinoma line, caused an upregulation in OCT4A, OCT4C which could regulate stemness and proliferation and OCT4B transcripts at different time points. Our findings indicated that PSORS1C3 could affect the expression level of OCT4 spliced variants, according to their functions and the cells molecular context as well as genetic background. Considering these diverse regulatory effects and co-expression of OCT4 and PSORS1C3 in some cell lines, it is safe to consider PSORS1C3 as a modulator of OCT4 expression in non-pluripotent cells and in association with homeostatic pathways.
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.
Sedigheh Gharbi; Fatemh Mirzadeh; Shahriar Khatrei; Mohammad Reza Soroush; Mahmood Tavallaie; Mohammad Reza Nourani; mehdi Sahmsara; Seyyed Javad Mowla
Abstract
MicroRNAs constitute a group of small non-coding RNAs that negatively regulate gene expression. Aside from their contribution to biological and pathological pathways, altered expression of microRNAs is reported in bio-fluid samples, such as serum. To employ serum's microRNAs as potential biomarkers, ...
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MicroRNAs constitute a group of small non-coding RNAs that negatively regulate gene expression. Aside from their contribution to biological and pathological pathways, altered expression of microRNAs is reported in bio-fluid samples, such as serum. To employ serum's microRNAs as potential biomarkers, it is crucial to develop an efficient method for microRNA quantification, avoiding pre-analytical and analytical variations which could affect the accuracy of data analysis. Here, we optimized a real-time PCR quantification procedure for microRNA detection in serum samples. Serum's total RNA was extracted using two different RNA isolation methods, one based on phenol-chloroform and the other based on silica column. To investigate a potential PCR inhibitory effect, different RNA amounts were subjected to reverse transcription. Moreover to assess the enzymatic efficiency, synthetic exogenous microRNAs was spiked into the mixture. Moreover, to find a reliable internal control gene for normalizing the microRNA quantification, the amounts of 8 candidate non-coding RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-5p, miR-191, miR-16 and miR-103 were assessed on serum samples. Altogether, our data demonstrated that the silica-based method was more efficient for microRNA recovery. Furthermore, increasing the input volume of the extracted RNA would dramatically increase inhibitors' amounts which could end up in a larger Cq values. Therefore, the best input volume of RNA turned out to be 1.5 microliter/reaction. Among the 8 aforementioned internal controls, U6, SNORD38B and SNORD49A showed low levels of expression, and were undetectable in some samples. Amongst the others, 5s rRNA, had the biggest standard deviation which could significantly affect data analysis. MiR-103 with the least variation appeared to be the best normalizer gene.
