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.

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

Outcome of subsequent pregnancies in familial molar pregnancy

Familial recurrent molar pregnancy is an exceedingly rare condition, in which complete hydatidiform moles are mostly diploid but biparental in origin and the outcome of subsequent pregnancies is likely to be a hydatidiform mole or other type of reproductive loss. We previously reported a case of familial molar pregnancy [family K] comprising five affected members [four sisters and one of their cousins] each with at least one hydatidiform mole [HM]. In addition to the molar pregnancies, these patients have a total of three miscarriages and 8 normal pregnancies leading to healthy children; but the youngest member of this family has given birth to a boy with Down syndrome. Our second family [case S] includes two sisters with diploid biparental complete moles. They have a total of six molar pregnancies with no living child. Recently the younger sister had a partial molar pregnancy with apparently normal XX fetus accompanying diffuse molar changes of the placenta that led to preeclampsia and preterm delivery. Overall, these families have had 26 pregnancies including 12 molar pregnancies [complete or partial] and three abortions. We concluded that these families are predisposed to various genetic mutations, chromosomal abnormalities and clinical manifestations, which affect their offspring. Further studies of patients are needed to determine any relationship between a history of familial molar pregnancy and trisomy or other chromosomal abnormalities in offspring and genetic mutations in the products of conception to complete the puzzle and manage familial molar pregnancy

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

Alpha 1 antitrypsin deficiency in infants with neonatal cholestasis

Alpha 1-antitrypsin deficiency [A1ATD] is the most important indication for liver transplantation in children. The gene frequencies vary in different ethnic groups. In the present study, we attempt to determine the frequencies of the most common defective alleles, Z and S, in Iranian children suffering from idiopathic neonatal cholestasis. Eighty-seven infants were typed for Z and S alleles. In a single center study, 87 consecutive liver biopsies from infants with cholestasis were reviewed and patients with neonatal cholestasis enrolled in the study and cases with confirmed biliary tract atresia excluded. Formalin fixed paraffin embedded blocks were used for DNA extraction. AAT genotype was determined by polymerase chain reaction [PCR] assay and amplification of the two most common deficiency variants, S and Z alleles, and then sequencing of PCR products. There were 48 [55.2%] males and 39 [44.8%] females, with a median age of 60 days. Out of 87 of the study subject, 2 [2.2%] were heterozygous for the S allele, and no ZZ, SS or MZ individual was found in the patients. No other polymorphism was found in the sequencing results. In comparison to other populations, AAT deficiency seems not to be an important etiologic factor for neonatal cholestatic liver disease in Iran; however, further studies are recommended to estimate the true mutant gene frequencies

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

18-year-old man with hepatitis b virus infection and Hepatoblastoma

A young man presented with a large liver mass and positive hepatitis B virus markers. This 18-year-old male has developed ascites, jaundice, high serum alpha fetoprotein [AFP] level, liver mass and portal hypertension, without fever or calcification in the mass. All favored the diagnosis of rapidly, progressive hepatocellular carcinoma, however proven hepatoblastoma in liver biopsy. Hepatoblastoma usually manifests prior to the third year of life, but can rarely be seen in older children or adults. Although HCC rarely can be presented in young patients with HBV infection, but in patients without cirrhosis hepato-blastoma should be considered as the first possible diagnosis

Chromosomal abnormality in patients with secondary amenorrhea

Secondary amenorrhea is a condition in which there is cessation of menses after at least one menstruation. It is a symptom of different diseases, such as hormonal disturbances which range from pituitary to ovarian origin, as well as chromosomal abnormalities. Knowledge of the distinct cause of secondary amenorrhea is of tremendous benefit for the management and monitoring of patients. In this study, we determine the chromosomal abnormalities in patients with secondary amenorrhea in Southwest Iran. We selected 94 patients with secondary amenorrhea who referred to our Cytogenetic Ward from 2004 until 2009. For karyotyping, peripheral blood lymphocyte cultures were set up by conventional technique. In this study, 5.3% [n=5] of patients with secondary amenorrhea presented with chromosomal abnormalities, of which all contained an X element. The chromosomal abnormalities were:i]45, X [n=1]; ii]47, XXX [n=1]; iii] 45, X [13]/45, Xi[X]q[17] [n=1]; iv]45, X[12]/46,X,+mar[12] [n=1]; and v] 46,X,del[Xq][q23q28] [n=1]. Our study revealed that some causes of secondary amenorrhea could be due to chromosomal abnormalities. Therefore, cytogenetic studies should be important tests in the evaluation of patients with secondary amenorrhea

Status of estrogen and progesterone receptors in various phases of the menstrual cycle in breast cancer

Expression of hormone receptors is routinely evaluated in predicting tumor response to hormone therapy in breast cancer patients. Normal female genital organs show cyclic changes in the expression of estrogen and progesterone receptors. This study was designed to assess variations in estrogen and progesterone receptor expression rates in breast cancer patients, who were operated in different phases of the menstrual cycle. From 2001 through 2004, 161 premenopausal patients with breast cancer, who were operated on, were enrolled into this study. Immunohistochemistry for the expression of estrogen and progesterone receptors was performed on their tumor paraffin blocks, using antibodies against estrogen and progesterone receptors. Estrogen receptor expression was seen in 24 out of 30 cases [80%] in early luteal phase, which was significantly higher than that of those operated in early follicular [53%], late follicular [51%] and late luteal [49%] phases [P < 0.05]. Progesterone receptor expression also showed a rising trend in the early luteal phase [87%], as compared with the other phases [P = 0.09]. Expression of estrogen/progesterone receptor shows cyclic changes in breast cancer patients, being highest in the early luteal phase of the menstrual cycle. This variation implies that this phase of the cycle could be the golden time for evaluation of estrogen/progesterone receptor status