Down-regulatory effects of mir-211 on long non-coding rna sox2ot and sox2 genes in esophageal squamous cell carcinoma

Objective: MicroRNAs [miRNAs] are a class of non-coding RNAs [ncRNAs] that tran-scriptionally or post-transcriptionally regulate gene expression through degradation of their mRNA targets and/or translational suppression. However, there are a few reports on miRNA-mediated expression regulation of long ncRNAs [lncRNAs]. We have previ-ously reported a significant upregulation of the lncRNA SOX2OT and its intronic coding gene, SOX2, in esophageal squamous cell carcinoma [ESCC] tissue samples. In this study, we aimed to evaluate the effect of induced overexpression of miR-211 on SOX2OT and SOX2 expression in vitro

Materials and Methods: In this experimental study, we performed both bioinformatic and experimental analyses to examine whether these transcripts are regulated by miRNAs. From the list of potential candidate miRNAs, miR-211 was found to have complementary sequences to SOX2OT and SOX2 transcripts. To validate our finding experimentally, we transfected the NT-2 pluripotent cell line [an embryonal carcinoma stem cell] with an expression vector overexpressing miR-211. The expression changes of miR-211, SOX2OT, and SOX2 were then quantified by a real-time polymerase chain reaction [RT-PCR] approach

Results: Compared with mock-transfected cells, overexpression of miR-211 caused a significant down-regulation of both genes [P<0.05]. Furthermore, flow-cytometry analysis revealed a significant elevation in sub-G1 cell population following ectopic expression of miR-211 in NT-2 cells

Conclusion: We report here, for the first time, the down-regulation of SOX2OT and SOX2 genes by an miRNA. Considering the vital role of SOX2OT and SOX2 genes in pluripotency and tumorigenesis, our data suggest an important and inhibitory role for miR-211 in the aforementioned processes

CD133 is not suitable marker for isolating melanoma stem cells from D10 cell line

Objective: Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells [CSCs] are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture

Materials and Methods: In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting [FACS] based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133- and spheroid cells. Significant differences of the two experimental groups were compared using student's t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold

Results: Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A compared to other groups [P<0.05]

Conclusion: Although CD133+ derived melanoma cells represented stemness features, our findings demonstrated that spheroid culture could be more effective method to enrich melanoma stem cells

Differential expression of OCT4 pseudogenes in pluripotent and tumor cell lines

Objective: The human OCT4 gene, the most important pluripotency marker, can generate at least three different transcripts [OCT4A, OCT4B, and OCT4B1] by alternative splicing. OCT4A is the main isoform responsible for the stemness property of embryonic stem [ES] cells. There also exist eight processed OCT4 pseudogenes in the human genome with high homology to the OCT4A, some of which are transcribed in various cancers. Recent conflicting reports on OCT4 expression in tumor cells and tissues emphasize the need to discriminate the expression of OCT4A from other variants as well as OCT4 pseudogenes

Materials and Methods: In this experimental study, DNA sequencing confirmed the authenticity of transcripts of OCT4 pseudogenes and their expression patterns were investigated in a panel of different human cell lines by reverse transcription-polymerase chain reaction [RT-PCR]

Results: Differential expression of OCT4 pseudogenes in various human cancer and pluripotent cell lines was observed. Moreover, the expression pattern of OCT4-pseudogene 3 [OCT4-pg3] followed that of OCT4A during neural differentiation of the pluripotent cell line of NTERA-2 [NT2]. Although OCT4-pg3 was highly expressed in undifferentiated NT2 cells, its expression was rapidly down-regulated upon induction of neural differentiation

Analysis of protein expression of OCT4A, OCT4-pg1, OCT4-pg3, and OCT4-pg4 by Western blotting indicated that OCT4 pseudogenes cannot produce stable proteins. Consistent with a newly proposed competitive role of pseudogene microRNA docking sites, we detected miR-145 binding sites on all transcripts of OCT4 and OCT4 pseudogenes

Conclusion: Our study suggests a potential coding-independent function for OCT4 pseudogenes during differentiation or tumorigenesis

Aberrant Expression of Breast Development-Related MicroRNAs, miR-22, miR-132, and miR-212, in Breast Tumor Tissues / 한국유방암학회지

PURPOSE: MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that posttranscriptionally regulate the expression of most genes in the human genome. miRNAs are often located in chromosomal fragile sites, which are suscept-ible to amplification or deletion. Chromosomal deletions are frequent events in breast cancer cells. Deletion and loss of heterozygosity at 17p13.3 have been reported in 49% of breast cancers. The aim of the current study was to evaluate potential expression alterations of miR-22, miR-132, and miR-212, which are located on the 17p13.3 locus and are required for mammary gland development. METHODS: A matched case-control study was conducted, which included 36 pairs of tumor and matched nontumor surgical specimens from patients diagnosed with breast invasive ductal carcinoma. Formalin-fixed paraffin-embedded samples from archival collections at the pathology department of Shariati Hospital were prepared for RNA extraction using the xylene-ethanol method before total RNA was isolated with TRIzol Reagent. Specific primers were designed for cDNA synthesis and miRNA amplification. The expression of miRNAs was then evaluated by real-time polymerase chain reaction (RT-PCR). RESULTS: According to our RT-PCR data, the miR-212/miR-132 family was downregulated in breast cancer (0.328-fold, p<0.001), and this reduced expression was the most prominent in high-grade tumors. In contrast, miR-22 exhibited a significant upregulation in breast tumor samples (2.183-fold, p=0.040). CONCLUSION: Consistent with the frequent deletion of the 17p13.3 locus in breast tumor cells, our gene expression data demonstrated a significant downregulation of miR-212 and miR-132 in breast cancer tissues. In contrast, we observed a significant upregulation of miR-22 in breast tumor samples. The latter conflicting result may have been due to the upregulation of miR-22 in stromal/cancer-associated fibroblasts, rather than in the tumor cells.

Verification of ALDH activity as a biomarker in colon cancer stem cells-derived HT-29 cell line

Background: Recent evidence has suggested that epithelial cancers including colorectal cancer [CRC] have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells [CSCs] which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 [ALDH1] activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer

Objectives: The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line

Materials and Methods: In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice

Results: According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers [P < 0.001]. Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells [P < 0.05]. In comparison with the parental cells, expression levels of ''stemness'' genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells [P < 0.05]. Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor

Conclusions: For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn't seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line

Downregulation of plasma MiR-142-3p and MiR-26a-5p in patients with colorectal carcinoma

Colorectal cancer is one of the most commonly diagnosed cancers and cancer- related death worldwide. Identification of new specific biomarkers could be helpful to detection of this malignancy. Altered plasma microRNA expression has been identified in many cancers, including colorectal cancer. The main objective of this study was to identify the circulating microRNAs with the most expression changes in colorectal cancer patients compared with neoplasm free healthy individuals. MicroRNA expression profiling was performed on plasma samples of 37 colorectal cancer patients and 8 normal subjects using microRNA microarray. Quantitative real-time reverse transcription polymerase chain reaction was used to validate the two selected altered microR NAs. Plasma samples from 61 colorectal cancer patients and 24 normal subjects were used in our validation study. In profiling study we found a panel of six plasma microRNAs with significant downregulation. MicroRNA-142-3p and microRNA-26a-5p were selected and validated by polymerase chain reaction. Our results demonstrated that expression levels of plasma microRNA-142-3p and microRNA-26a-5p were significantly downregulated in patients with colorectal cancer when compared to control group. Our findings suggest that downregulation of plasma microRNA-142-3p and microRNA-26a-5p might serve as novel noninvasive biomarkers in the diagnosis of colorectal cancer, although more studies are needed to highlight the theoretical strengths

Identification of Reliable reference genes for quantification of MicroRNAs in serum samples of sulfur mustard-exposed veterans

Objective: In spite of accumulating information about pathological aspects of sulfur mustard [SM], the precise mechanism responsible for its effects is not well understood. Circulating microRNAs [miRNAs] are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims

Materials and Methods: In this case and control experimental study, using quantitative real-time polymerase chain reaction [qRT-PCR], we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war [1980-1988] and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle [Cq] method were employed to find the least variable reference gene

Results: miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that

Conclusion: We demonstrate that non-miRNA reference genes have the least stability in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers

Comparing the effects of small molecules BIX-01294, Bay K8644, RG-108 and valproic acid, and their different combinations on induction of pluripotency marker-genes by Oct4 in the mouse brain

Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid [VPA] are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction [PCR] was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell [NSC] markers. Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog [p<0.05]. Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc [p<0.001]. VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc [p<0.01], Pax6 and Sox1 [p<0.001]. These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4

Enrichment of a rare subpopulation of miR-302-expressing glioma cells by serum deprivation

MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem [ES] cells. The miR-302-367 promoter is functional during embryonic development but is turned off in later stages. Motivated by the cancer stem cell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. In the present experimental study, we have tried to expand our knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma [A-172, 1321N1, U87MG] and medulloblastoma [DAOY] cell lines. To further assess the functionality of miR-302 in these cell lines, we cloned its promoter core region upstream of the enhanced green fluorescent protein [EGFP] or luciferase encoding genes. Our data demonstrated a very low expression of miR-302 in glioma cell lines, compared with that of embryonal carcinoma cell line NT2 being used as a positive control. The expression of miR-302 promoter-EGFP construct in the aforementioned cell lines demonstrated GFP expression in a rare subpopulation of the cells. Serum deprivation led to the generation of tumorospheres, enrichment of miR-302 positive cells and upregulation of a number of pluripotency genes. Taken together, our data suggest that miR-302 could potentially be used as a novel putative cancer stem cell marker to identify and target cancer stem cells within tumor tissues

[Characterization of stemness properties of colonospheres in colon adenocarcinoma patients]

Proliferation and expansion of cancer stem cells as spheroids were proved in previous studies. But, capability of primary tumor-derived stem cells to keep their unique properties in vitro is still disputed. So, the goal of this study was to isolate, expand and characterize of colon cancer-derived stem cells. In the present work, colon cancer stem cells markers including CD44 and EPCAM in spheroid and parental cells were analyzed by flow cytometry. The expression levels of stemness genes in both spheroid and parental cells were investigated using real-time PCR. Tumorigenic potential of spheroid cells was evaluated and used implantation of tumor xenografts into nude mice. Our data shows 79% of spheroids were CD44+/EpCAM+, while parental cells only expressed 20% of CD44/EpCAM markers [p< 0.01]. In compared with the parental cells, the expression levels of "stemness" genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 were significantly increased in spheroid cells [p< 0.05]. Furthermore, as little as 1000 spheroid cells were sufficient to obtain tumor growth in nude mice, while 1x10[6] of parental cells was needed to generate tumor. Sphere formation assay is a useful method to enrich cancer stem cells. Spheroid cells showed increasing expression of stemness genes and tumorigenic activity in nude mice

Evaluating the effects of Escanbil [Calligonum] extract on the expression level of Catsper gene variants and sperm motility in aging male mice

Catsper proteins are responsible for entering Ca[2+] to the cell and play an important role in sperm motility and male fertility. Antioxidants are vital for sperm motility too. Escanbil [Calligonum] extract possess some of the important antioxidant like Catechin and Quercetin. Here we investigated the effects of Escanbil [Calligonum] extract on the sperm parameters and the expressing of Catsper gene in aging male mice. In this animal study, firstly, dose response was performed by using these three doses of Escanbil [Calligonum] [10, 30 and 50 mg/kg]. 5 mice in each group were considered and Intra Peritoneal injection was done for 5 weeks. the sperm parameters analyzed and Terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]staining was done. 30 mg/kg dose was considered as optimum dose. Secondly: fifteen aging male mice [11-13 months] were divided into three groups: control, sham and experiment. The experiments were injected Intra peritonealy with Escanbil [Calligonum] extract [30mg/kg] weekly for up to 5 weeks. The sham group was injected Intra Peritoneal [DMSO]. Sperm parameters were analyzed. Expression of Catsper genes was analyzed by Real time PCR. Our results showed that after Escanbil [Calligonum] treatment [30 mg/kg], the sperm parameters were improved in experimental group [p<0.05]. Our data showed that there was a statistical significance difference between the expressions of Catsper 2, 4 in aging experiment group comparison with aging control group [p<0.05]. We investigated that the Escanbil [Calligonum] extract [30 mg/kg] can improve sperm parameters and change the expression of Catsper genes in aging male mice. This herbal extract can be used as an antioxidant component for clinical usages

[ long non-coding RNA, PSORS1C3, located upstream of the human Oct4 gene is expressed in pluripotent and tumor cell lines]

Long non-coding RNAs [lncRNAs], a vast class of recently discovered noncoding genes in the human genome, have been implicated in the regulation of several biological processes, including the maintenance of stem cell pluripotency and neurogenesis. New evidences have emerged that some long IncRNAs act as enhancers for their neighboring genes. Oct4, also known as POU5F1 and Oct3/4, functions as a master regulator in maintaining the properties of pluripotency and self-renewal of embryonic stem [ES] cells and embryonal carcinoma [EC] cells. Oct-4 expression must be tightly regulated; too much or too little expression can lead to cell differentiation. PSORS1C3, an IncRNA, is located upstream of the Oct4 gene. This IncRNA could potentially impact the level of Oct4 expression. Here, we have investigated potential expression of PSORS1C3 on 23 different human pluripotent and cancer cell lines by means of RT-PCR. Our results revealed a noticeable expression of PSORS1C3 both in a well-known pluripotent cell line [NTera2/NT2] and five different cancer cell lines [AGS, 5637, Ht-29, HepG2 and PC3]. We detected the expression of PSORS1C3 for the first time in both cancer cell lines and stem cells

Evaluating the expression of self-renewal genes in human endothelial progenitor cells

Endothelial progenitor cells [EPCs] have a potential application for cell therapy, however, their biological nature is not well-understood. EPCs also possess some stemness features, such as their clonogenicity and differentiation capacity. The main aim of this study was to evaluate the expression of certain transcription factors regulating self-renewal property of stem cells. In this experimental study, peripheral blood mononuclear cells were isolated from fresh human blood of several volunteers and were cultured in fibronectin- coated plates. EPCs were identified based on their morphology and growth characteristic. Then, the expression of some markers implicated in self-renewal capacity was assessed in the isolated cells using reverse transcription-polymerase chain reaction [RT-PCR] and immunocytochemistry. Expression of the cell surface markers, CD31 and CD34, was determined by RT-PCR and immunocytochemistry. Furthermore, these cells had the ability for Di-AC-LDL incorporation as well as attachment to lectin I. EPCs did not express the main stem cell markers, like OCT4-A, Nanog, and Sox2; nevertheless, they expressed the weaker pluripotent markers, including OCT4B and OCT4-B1 spliced variants, such as Nucleostemin and ZFX. Furthermore, the expression of Nucleostemin and ZFX genes revealed a decreasing pattern from days 4[th] to 11[th]. The main regulators of stem cell self-renewal genes, including OCT4-A, Nanog, and Sox2 are not expressed in EPCs. Forced expression of these genes can elevate the stemness property and clinical application of EPCs

[Dendrosomal curcumin induced apoptosis by suppression of pluripotency genes in 5637 bladder cancer cells]

The anti-cancer properties of curcumin, a poliphenol extract from the rhizome of curry, has been confirmed by many investigators. However, low levels of uptake, tissue distribution and rapid metabolism has limited its application as an anti-cancer drug. This study is aimed at increasing curcumin's water solubility due to a biodegradable, neutral and non-toxic micellar nano-carrier called dendrosome. This study intends to evaluate the role of dendrosomal-curcumin [DNC] in bladder cancer cell growth. We performed the MTT assay, flow cytometry and Annexin V-FLUOS [as an apoptosis detection kit] to evaluate cell death. The genetic mechanism of DNC-induced apoptosis was accomplished by a study of the relative expressions of OCT4A, OCT4B1, SOX-2 and Nanog using real-time PCR. DNC-induced cell death complied with a time and dose-dependent paradigm in the 5637 cell line. Cell cycle analysis revealed that the number of cells increased in pre-G1 and gradually decreased in G1 and S phases. This showed the inhibitory property of dendrosomal-curcumin on DNA synthesis. Data from real-time PCR determined that expressions of OCT4A, OCT4B1, SOX-2 and Nanog could be related to 5637 cancer cell growth. Dendrosomal-curcumin significantly suppressed mRNA expression of the above mentioned genes [p<0.01]. The data showed that DNC induced apoptosis by suppression of pluripotency genes in 5637 bladder cancer cells, which confirmed the useful characteristic of nano-drug in bladder cancer therapy.

[ effects of calligonum extract on sperm parameters and the rate of apoptosis in aged male mice testis issue]

Antioxidants are essential for sperm motility. Calligonum extract possesses the important antioxidants catechin and quercetin. This study investigates the effects of calligonum extract on sperm parameters and the rate of apoptosis in testes of aging male mice. We initially performed a dose response test with using three doses of calligonum [10 mg/kg, 30 mg/kg and 50 mg/kg]. A total of 25 aging male mice [11-13 months] were divided into the following groups of five mice each: control, sham and three experimental groups. The experimental groups received IP injections of calligonum extract [10 mg/kg, 30 mg/kg, 50 mg/kg] weekly for up to five weeks. The sham group received IP injections of DMSO. At the end of the injection period, mice were sacrificed and sperm parameters analyzed. To determine apoptosis in testes, we performed TUNEL staining. Our results showed that after calligonum treatment, there were improved sperm parameters in the 30 mg/kg-treated group compared to the other groups [P

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

Transplanting p75-suppressed bone marrow stromal cells promotes functional behavior in a rat model of spinal cord injury

Bone marrow stromal cells [BMSC] have been successfully employed for movement deficit recovery in spinal cord injury [SCI] rat models. One of the unsettled problems in cell transplantation is the relative high proportion of cell death, specifically after neural differentiation. According to our previous studies, p75 receptor, known as the death receptor, is only expressed in BMSC in a time window of 6-12 hours following neural induction. Moreover, we have recently reported a decreased level of apoptosis in p75-suppressed BMSC in vitro. Therefore, our objective in this research was to explore the functional effects of transplanting p75:siRNA expressing BMSC in SCI rats. Laminectomy was performed at L1 vertebra level to expose spinal cord for contusion using weight-drop method. PBS-treated SCI rats [group one] were used as negative controls, in which cavitations were observed 10 weeks after SCI. pRNA-U6.1/Hygro- [group two, as a mock] and pRNA-U6.1/Hygro-p75 shRNA- [group three] transfected BMSC were labeled with a fluorescent dye, CM-DiI, and grafted into the lesion site 7 days after surgery. The Basso-Beattie-Bresnehan locomotor rating scale was performed weekly for 10 weeks. There was a significant difference [P

Down-regulation of miR-302b, an ESC-specific microRNA, in gastric adenocarcinoma

microRNAs [miRNAs] are a new class of non-coding RNAs involved in regulating various biological processes including proliferation, differentiation, and apoptosis, among others. Alterations in miRNA expression are reported in several human cancers, which suggests their potential roles in tumor initiation and progression. Members of the miR-302 cluster are highly expressed in embryonic stem cells [ESC], where they regulate cell self-renewal and pluripotency. Based on the cancer stem cell [CSC] hypothesis, mis-expression of such genes might contribute to tumorigenicity. This study aims to find a potential link between the expression level of human/homo sapiens miR-302b [has-miR- 302b] and tumor/grade state of gastric tissues. A matched based case-control study was conducted that included tumor and matched marginal non-tumor surgical specimens from 34 patients diagnosed with gastric adenocarcinoma. Randomly selected samples were obtained from the Iran National Tumor Bank. cDNA synthesis was carried out on total RNA, by using the miRCURY LNA[Trade Mark sign]Universal RT microRNA PCR Kit. Real-time reverse transcriptionpolymerase chain reaction [RT-PCR] assays were performed with specific LNA[Trade Mark sign] primers and SYBR Green master mix. The human embryonic carcinoma cell line, NTERA2 [NT2] and a human gastric adenocarcinoma cell line, AGS, were used to optimize the PCR reactions. A comparative evaluation of miR-302b expression in tumor and non-tumor gastric samples was performed by either paired t test or Wilcoxon non-parametric test. The ability of miR-302b to discriminate tumor from non-tumor gastric samples was evaluated using the area under the receiver operating characteristic [ROC] curve. According to our data, miR-302b expression [normalized to that of the U6 snRNA housekeeping gene] in the pluripotent cell line NT2 was more than 500 times greater than that of the AGS cell line. The level of expression was even lower in tumor and non-tumor gastric tissue samples. The data further revealed a down-regulation of miR-302b in gastric tumor samples [p=0.001], particularly in high-grade adenocarcinoma [p=0.009]. However, ROC analysis data demonstrated a low sensitivity and specificity of miR-302b expression to discriminate between the tumor and non-tumor state of the samples [AUC=0.63]. Despite the upregulation of some hESC-specific genes in tumors, our data revealed a down-regulation of miR-302b in high-grade tumors. This data suggested a potential tumor-suppressor role for miR-302b in tumorigenesis of gastric tissue

Investigation in vitro expression of catSper sub fragment followed by production of polyclonal antibody: potential candidate for the next generation of non hormonal contraceptive

CatSper is a voltage-sensitive calcium channel that is specifically expressed in the testis and it has a significant role in sperm performance. CatSper [1-4] ion channel subunit genes, causes sperm cell hyperactivation and male fertility. In this study, we have explored targeting of the extracellular loop as an approach for the generation of antibodies with the potential ability to block the ion channel and applicable method to the next generation of non-hormonal contraceptive. In this experimental study, a small extracellular fragment of Cat-Sper1 channel was cloned in pET-32a and pEGFP-N1 plasmids. Then, subsequent methods were performed to evaluate production of antibody: 1] pEGFP-N1/CatSper was used as a DNA vaccine to immunize Balb/c mice, 2] The purified protein of pET-32a/CatSper was used as an antigen in an enzyme-linked immunosorbent assay [ELISA] and western-blot, and 3] The serum of Balb/-c mice was used as an antibody in ELISA and western-blot. The statistical analysis was performed using the Mann Whitney test. The results showed that vaccination of the experimental group with DNA vaccine caused to produce antibody with [p< 0.05] unlike the control group. This antibody extracted from Balb/c serum could recognize the antigen, and it may be used potentially as a male contraception to prevent sperm motility. CatSpers are the promising targets to develop male contraceptive because they are designed highly specific for sperm; although, no antagonists of these channels have been reported in the literature to date. As results showed, this antibody can be used in male for blocking CatSper channel and it has the potential ability to use as a contraceptive

Autologous transplantation of adult mice spermatogonial stem cells into gamma irradiated testes

We evaluated structural and functional changes of fresh and frozen-thawed adult mouse spermatogonial stem cells following auto-transplantation into gamma-irra-diated testes. In this experimental research, the right testes from adult mice [n=25] were collected, then Sertoli and spermatogonial cells were isolated using two-step enzymatic digestion, lectin immobilization and differential plating. Three weeks after cultivation, the Bromodeoxyuridine [BrdU]-labeled spermatogonial cells were transplanted, via rete testis, into the other testis of the same mouse, which had been irradiated with 14Gy. The mice were transplanted with: fresh cells [control 1], fresh cells co-cultured with Sertoli cells [control 2], the frozen-thawed cells [experimental 1] and frozen-thawed cells co-cultured with Sertoli cells [experimental 2]. The morphological changes between different transplanted testes groups were compared in 8 weeks after transplantation. The statistical significance between mean values was determined by Kruskal Wallis and one-way analysis of variance in efficiency of transplantation. The statistical analysis revealed significant increases in the mean percentage of testis weight and normal seminiferous tubules following spermatogonial stem cells transplantation in the recipient's testes. The normal seminiferous tubules percentage in the co-culture system with fresh cells and frozen-thawed groups were more than those in non-transplanted and fresh cell transplanted groups [p