Mitochondrial copy number and d-loop variants in Pompe patients

Objective: Pompe disease is a rare neuromuscular genetic disorder and is classified into two forms of early and late-onset. Over the past two decades, mitochondrial abnormalities have been recognized as an important contributor to an array of neuromuscular diseases. We therefore aimed to compare mitochondrial copy number and mitochondrial displacement-loop sequence variation in infantile and adult Pompe patients

Materials and Methods: in this retrospective study, the mitochondrial D-loop sequence was analyzed by polymerase chain reaction [PCR] and direct sequencing to detect possible variation in 28 Pompe patients [17 infants and 11 adults]. Results were compared with 100 healthy controls and sequences of all individuals were compared with the Cambridge reference sequence. Real-time PCR was used to quantify mitochondrial DNA copy number

Results: among 59 variants identified, 37[62.71%] were present in the infant group, 14[23.333%] in the adult group and 8[13.333%] in both groups. Mitochondrial copy number in infant patients was lower than adults [P<0.05]. A significant frequency difference was seen between the two groups for 12 single nucleotide polymorphism [SNP]. A novel insertion [317-318 ins CCC] was observed in patients and six SNPs were identified as neutral variants in controls. There was an inverse association between mitochondrial copy number and D-loop variant number [r=0.54]

Conclusion: the 317-318 ins CCC was detected as a new mitochondrial variant in Pompe patients

miRNA-21 expression analysis in 35 colorectal cancer

Colorectal cancer is the third most common cancer in the world. Non-coding RNA especially miRNAs have important regulatory roles in cancer. miRNAs are small non coding RNA 21-23 nucleotides long which have different levels of expression between tumors and normal tissues. This study was designed to compare expression level of miRNA-21 between Iranian population colorectal cancer tissues and normal tissue. This case-control study has performed in medical genetics department of Tehran University of Medical Sciences from January to November 2013. We used 35 samples. The samples were isolated from tumor and adjacent normal tissues of colon. Thirty-five samples were divided into different groups according to cliniopathologic features including tumor size [>4 and <4 cm], metastasis [+ and -] and stage. After small RNA extraction from tissues by small RNA purification kit the quality and quantity of extracted RNA was determined using spectrophotometry. cDNAs were synthesized and real-time polymerase chain reaction carried out. Finally expression levels were statistically analyzed by LinRegPCR and REST software. miRNA-21 expression ratio in stages I, II and III were 1/804 and 4/574, respectively, the increase from stage III was statistically significant [P= 0.037]. The expression were also studied according to different clinicopathologic status of colon cancer, tumor size [>4 and <4 cm] and metastatic [+ and -], miRNA-21 over expressed in both groups, however the increase was not statistically significant. In this study, we found miR-21 over-expression in advanced stage in tumoral tissue comparing with normal adjacent tissue. This means perhaps in the future it would be possible to use miRNA-21 as an informative prognostic biomarker to guide for better treatment strategies for colorectal cancer patients. Our findings also indicate that miRNA-21 is a promising new molecular target for designing novel therapeutic strategies to control colorectal cancer

[MIR-520d expression analysis in breast cancer]

Breast cancer is the most common cancer in women. Non-coding RNAs especially miRNAs have important regulatory roles in cancer. MiRNAs are 21-24 nucleotides which have different levels of expression between tumors and normal tissues. In this study, we have analyzed expression level of miR-520d in three different groups of breast cancer. Fifty nine samples were divided into different groups according to their immunohistochemistry [IHC] classification: estrogen receptor [ER] positive and/or progesterone receptor [PR] positive group [as group I]; human epidermal growth factor receptor 2 [HER2] positive group [as group II]; and Triple negative group [as group III]. After small RNA extraction from tissues, cDNAs were synthesized and Real time RTPCR carried out using DNA binding dye. Expression levels were analyzed by LinRegPCR and REST software. MiR-520d under- expressed in all of three different groups. The expression ratio in groups I, II, and III were 0.193, 0.167, 0.21, respectively, but only the result from group II was significant [P=0.017]. According to the different clinicopathological status of breast cancer, miR-520d underexpressed significantly not only in patients with metastatic lymph node [P=0.019] but also in patients which have cancer at stage three [P=0.036]. In this study, we found that miR-520d possibly acts as a tumor suppressor. It may be useful for diagnosis of tumor from normal tissue. In addition, miR-520d significantly underexpressed in HER-2 positive group of breast cancers. Therefore, it may be useful as an additional diagnostic test in this group of breast tumors along with other biomarkers.

Differential expression of human homeodomain TGIFLX in brain tumor cell lines

Glioblastoma is the most common and the most lethal primary brain cancer. This malignancy is highly locally invasive, rarely metastatic and resistant to current therapies. Little is known about the distinct molecular biology of glioblastoma multiforme [GBM] in terms of initiation and progression. So far, several molecular mechanisms have been suggested to implicate in GBM development. Homeodomain [HD] transcription factors play central roles in the expression of genomic information in all known eukaryotes. The TGIFX homeobox gene was originally discovered in human adult testes. Our previous study showed implications of TGIFLX in prostate cancer and azoospermia, although the molecular mechanism by which TGIFLX acts is unknown. Moreover, studies reported that HD proteins are involved in normal and abnormal brain developments. We examined the expression pattern of TGIFLX in different human brain tumor cell lines including U87MG, A172, Daoy and 1321N1. Interestingly, real time RT-PCR and western blot analysis revealed a high level of TGIFLX expression in A172 cells but not in the other cell lines. We subsequently cloned the entire coding sequence of TGIFLX gene into the pEGFP-N1 vector, eukaryotic expressionvector encoding eGFP, and transfected into the U-87 MG cell line. The TGIFLX-GFP expression was confirmed by real time RT-PCR and UV-microscopic analysis. Upon transfection into U87 cells, fusion protein TGIFLX-GFP was found to locate mainly in the nucleus. This is the first report to determine the nuclear localization of TGIFLX and evaluation of its expression level between different brain tumor cell lines. Our data also suggest that TGIFLX gene dysregulation could be involved in the pathogenesis of some human brain tumors

novel in vitro model for cancer stem cell culture using ectopically expressed Piwil2 stable cell line

Piwil2, a member of Ago/Piwi gene family containing Piwi and PAZ domains, has been shown to be ectopically expressed in different cancer cells, especially its remarkable expression in cancer stem cells [CSCs], and is also known to be essential for germ line stem cell self-renewal in various organisms. The hypothesis that CSC may hold the key to the central problem of clinical oncology and tumor relapse leads to more anticancer treatment studies. Due to emerging controversies and extreme difficulties in studying of CSC, like the cells using in vivo models, more attempts have expended to establish different in vitro models. However, the progress was slow owing to the problems associated with establishing proper CSC cultures in vitro. To overcome these difficulties, we prompted to establish a novel stable cell line over-expressing Piwil2 to develop a potential proper in vitro CSC model. In this experimental study, mouse embryonic fibroblasts [MEFs] were isolated and electroporated with a construct containing Piwil2 cDNA under the control of the cytomegalovirus promoter [CMV]. Stable transfectants were selected, and the established MEF-Piwil2 cell line was characterized and designated as CSC-like cells using molecular markers. Functional assays, including proliferation, migration, and invasion assays were performed using characterized CSC like cells in serum-free medium. Additionally, MEF-Piwil2 cell density and viability were measured by direct and indirect methods in normoxic and hypoxic conditions. The results of reverse transcriptase-polymerase chain reaction [RT-PCR], western blot, and immunocytochemistry revealed an overexpression for Piwil2 in the transfected Piwil2 cells both in the RNA and protein levels. Furthermore, analysis of the kinetic and stoichiometric parameters demonstrated that the specific growth rate and the yield of lactate per glucose were significantly higher in the MEF-Piwil2 group compared to the MEF cells [ANOVA, p<0.05]. Also, analysis of functional assays including migration and invasion assays demonstrated a significantly higher number of migrated and invaded cells in the MEF-Piwil2 compared to that of the MEF cells [ANOVA, p<0.05]. The MEF-Piwil2 cells tolerated hypoxia mimetic conditions [CoCl[2]] with more than 95% viability. According to the molecular and functional studies, it has been realized that Piwil2 plays a key role[s] in tumor initiation, progression and metastasis. Therefore, Piwil2 can be used not only as a common biomarker for tumor, but also as a target for the development of new anticancer drug. Finally, the main outcome of our study was the establishment of a novel CSC-like in vitro model which is expected to be utilized in understanding the complex roles played by CSC in tumor maintenance, metastasis, therapy resistance or cancer relapse

Repair of spinal cord injury by co-transplantation of embryonic stem cell-derived motor neuron and olfactory ensheathing cell

The failure of regeneration after spinal cord injury [SCI] has been attributed to axonal demyelination and neuronal death. Cellular replacement and white matter regeneration are both necessary for SCI repair. In this study, we evaluated the co-transplantation of olfactory ensheathing cells [OEC] and embryonic stem [ES] cell-derived motor neurons [ESMN] on contused SCI. OEC cultured from olfactory nerve rootlets and olfactory bulbs. ESMN was generated by exposing mouse ES cells to retinoic acid and sonic hedgehog. Thirty female rats were used to prepare SCI models in five groups. Control and medium-injected groups was subjected to induce lesion without cell transplantation. OEC or ESMN or both were transplanted into the site of the lesion in other groups. The purity of OEC culture was 95%. Motor neuron progenitor markers [Olig2, Nkx6.1 and Pax6] and motor neuron markers [Is11, Is12 and Hb9] were expressed. Histological analysis showed that significantly more [P<0.001] spinal tissue was spared in OEC, ESMN and OEC+ ESMN groups but the OEC+ ESMN group had a significantly greater percentage of spared tissue and myelination than other groups [P< 0.05]. The numbers of ESMN in co-transplanted group were significantly higher than ESMN group [P<0.05]. A significant [P<0.05] recovery of hindlimb function was observed in rats in the transplanted groups. We found that the co-transplantation of ESMN and OEC into an injured spinal cord has a synergistic effect, promoting neural regeneration, ESMN survival and partial functional recovery