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1.
Chinese Medical Journal ; (24): 564-572, 2021.
Article in English | WPRIM | ID: wpr-878081

ABSTRACT

BACKGROUND@#The pathogenesis of osteosarcoma (OS) is still unclear, and it is still necessary to find new targets and drugs for anti-OS. This study aimed to investigate the role and mechanism of the anti-OS effects of miR-296-5p.@*METHODS@#We measured the expression of miR-296-5p in human OS cell lines and tissues. The effect of miR-296-5p and its target gene staphylococcal nuclease and tudor domain containing 1 on proliferation, migration, and invasion of human OS lines was examined. The Student's t test was used for statistical analysis.@*RESULTS@#We found that microRNA (miR)-296-5p was significantly downregulated in OS cell lines and tissues (control vs. OS, 1.802 ± 0.313 vs. 0.618 ± 0.235, t = 6.402, P < 0.01). Overexpression of miR-296-5p suppressed proliferation, migration, and invasion of OA cells. SND1 was identified as a target of miR-296-5p by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of SND1 abrogated the effects induced by miR-296-5p upregulation (miRNA-296-5p vs. miRNA-296-5p + SND1, 0.294 ± 0.159 vs. 2.300 ± 0.277, t = 12.68, P = 0.003).@*CONCLUSION@#Our study indicates that miR-296-5p may function as a tumor suppressor by targeting SND1 in OS.


Subject(s)
Bone Neoplasms/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Endonucleases/genetics , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Humans , MicroRNAs/genetics , Osteosarcoma/genetics
2.
Article in Chinese | WPRIM | ID: wpr-880841

ABSTRACT

OBJECTIVE@#To investigate the effects of overexpression of long noncoding RNA (lncRNA) MEG3 on the proliferation and invasion of glioblastoma U251 cells by suppressing the expression of hypoxia inducible factor 1@*METHODS@#The expression of lncRNA MEG3 and HIF1@*RESULTS@#The expression of MEG3 was significantly lower and HIF1@*CONCLUSIONS@#MEG3 overexpression inhibits the proliferation and invasion of U251 cells through suppressing the expression of HIF1


Subject(s)
Apoptosis , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Humans , MicroRNAs , RNA, Long Noncoding/genetics
3.
Chinese Medical Journal ; (24): 2619-2628, 2021.
Article in English | WPRIM | ID: wpr-921210

ABSTRACT

BACKGROUND@#Microribose nucleic acids (miRNAs) are implicated in the progression of lung adenocarcinoma. MicroRNA-345-5p (miR-345-5p) is a recently identified anti-oncogene in some human cancers, but its functional role and possible molecular mechanism in lung adenocarcinoma remain unknown. This study aimed to identify the biological function and underlying mechanism of miR-345-5p in lung adenocarcinoma cells.@*METHODS@#In this study, lung adenocarcinoma tissues and adjacent tissues were collected in the First Affiliated Hospital of Anhui Medical University between April 2016 and February 2017. The expression of miR-345-5p and ras homolog family member A (RhoA) in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines (A549, H1650, PC-9, and H441) was detected by reverse transcription quantitative polymerase chain reaction analysis. Functional assays including colony formation, flow cytometry analysis, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of lung adenocarcinoma cells. In addition, RNA pulldown and luciferase reporter assays were conducted to evaluate the relationship between miR-345-5p and RhoA. Difference between the two groups was analyzed with Student's t test, while that among multiple groups was analyzed with one-way analysis of variance.@*RESULTS@#MiR-345-5p expression displayed lower level in lung adenocarcinoma tissues (0.241 ± 0.095 vs.1.000 ± 0.233, t = 19.247, P < 0.001) and cell lines (F = 56.992, P < 0.001) than control tissues and cells. Functional experiments demonstrated that upregulation of miR-345-5p inhibited the malignant phenotypes of lung adenocarcinoma cells via suppressing cell proliferation, migration, invasion, and facilitating cell apoptosis. Additionally, RhoA was verified to be the downstream target of miR-345-5p. Expression of RhoA was downregulated by overexpression of miR-345-5p in PC-9 (0.321 ± 0.047 vs. 1.000 ± 0.127, t = 8.536, P < 0.001) and H1650 (0.398 ± 0.054 vs. 1.000 ± 0.156, t = 4.429, P = 0.011) cells. Rescue assays revealed that overexpression of RhoA rescued the suppressive effects of miR-345-5p upregulation on proliferation, migration, and invasion of lung adenocarcinoma cells. Further, miR-345-5p was found to regulate the Rho/Rho-associated protein kinase (ROCK) signaling pathway by downregulation of RhoA in lung adenocarcinoma cells.@*CONCLUSIONS@#MiR-345-5p plays a tumor suppressor role in lung adenocarcinoma cells by downregulating RhoA to inactivate the Rho/ROCK pathway.


Subject(s)
Adenocarcinoma of Lung/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/genetics , MicroRNAs/genetics , Up-Regulation/genetics , rho-Associated Kinases/genetics , rhoA GTP-Binding Protein/genetics
4.
Braz. j. med. biol. res ; 54(8): e10940, 2021. graf
Article in English | LILACS | ID: biblio-1285675

ABSTRACT

Recently, an increasing number of studies have reported that dysregulation of circular RNA (circRNA) expression plays critical roles in the progression of several cancers, including colorectal cancer (CRC). However, the detailed molecular mechanisms of circRNAs involvement in CRC remain largely unknown. Here, we confirmed that the level of circEGFR was significantly increased in CRC tissues compared to matched adjacent non-tumor tissues, and a high level of circEGFR was correlated with poor clinicopathological characteristics and poor prognosis in patients with CRC. Moreover, increased circEGFR expression promoted CRC cell proliferation, migration, and invasion in vitro. Mechanistically, circEGFR acted as a ceRNA for miR-106a-5p to relieve the repressive effect of miR-106a-5p on DDX5 mRNA. Moreover, circEGFR enhanced DDX5 expression, thereby upregulating p-AKT levels. Together, these findings showed that circEGFR promoted CRC cell proliferation, migration, and invasion through the miR-106a-5p/DDX5/AKT axis, and may serve as a promising diagnostic marker and therapeutic target for CRC patients.


Subject(s)
Humans , Colorectal Neoplasms/genetics , MicroRNAs/genetics , Gene Expression Regulation, Neoplastic , Cell Movement/genetics , Cell Line, Tumor , Cell Proliferation/genetics , DEAD-box RNA Helicases , RNA, Circular
5.
Braz. j. med. biol. res ; 54(8): e10062, 2021. tab, graf
Article in English | LILACS | ID: biblio-1249323

ABSTRACT

Long non-coding RNA (lncRNA) is an essential regulator of carcinogenesis and cancer progression. In the study, we explored the role of lncRNA DLGAP1-AS1 in gastric cancer (GC). qRT-PCR was carried out to detect DLGAP1-AS1 expression in GC tissues and cell lines. CCK-8 assay, EdU assay, and transwell experiments were employed to detect the malignant biological behaviors of GC cells with DLGAP1-AS1 knockdown or overexpression. Bioinformatics and dual-luciferase report assay were used to confirm the binding relationship between DLGAP1-AS1 and miR-515-5p. MARK4 expression was detected by western blot after DLGAP1-AS1/miR-515-5p was selectively regulated. DLGAP1-AS1 was up-regulated in GC tissues and cell lines, and its high expression was closely associated with larger tumor size, higher TNM stage, and lymph node metastasis. Furthermore, DLGAP1-AS1 overexpression enhanced cell proliferation, migration, and invasion, and miR-515-5p could reverse these effects. DLGAP1-AS1 participated in the regulation of the MARK4 signaling pathway by targeting miR-515-5p. DLGAP1-AS1 promoted GC progression through miR-515-5p/MARK4 signaling pathway.


Subject(s)
Humans , Stomach Neoplasms/genetics , MicroRNAs/genetics , RNA, Long Noncoding/genetics , Gene Expression Regulation, Neoplastic , Cell Movement/genetics , Protein-Serine-Threonine Kinases , Cell Line, Tumor
6.
Braz. j. med. biol. res ; 54(6): e10474, 2021. tab, graf
Article in English | LILACS | ID: biblio-1285667

ABSTRACT

Osteosarcoma is a highly malignant tumor that occurs in the bone. Previous studies have shown that multiple microRNAs (miRNAs) regulate the development of osteosarcoma. This study aimed to explore the role of miR-629-5p and its target gene, caveolin 1 (CAV1), in osteosarcoma development. To analyze the expression of miR-629-5p and CAV1 mRNA in osteosarcoma tissues and cell lines, qRT-PCR analysis was performed. Dual-luciferase reporter experiments were subsequently performed to validate the relationship between CAV1 and miR-629-5p. CCK8 assay was used to measure osteosarcoma cell proliferation, and wound-healing assay was performed to study their migratory phenotype. Our findings revealed that miR-629-5p was overexpressed in osteosarcoma tissues and cells, and thereby enhanced cell proliferation and migration. Further, we validated that miR-629-5p targets CAV1 mRNA directly. CAV1 expression, which was negatively correlated with miR-629-5p expression, was found to be downregulated in osteosarcoma tissue samples. Moreover, our data showed that an increase in CAV1 level led to a decline in osteosarcoma cell proliferation and migration, which could be rescued by miR-629-5p upregulation. Overall, our study confirmed that miR-629-5p promoted osteosarcoma proliferation and migration by directly inhibiting CAV1.


Subject(s)
Humans , Bone Neoplasms/genetics , Osteosarcoma/genetics , MicroRNAs/genetics , Gene Expression Regulation, Neoplastic , Cell Movement/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Caveolin 1/genetics
7.
Braz. j. med. biol. res ; 54(2): e10394, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153512

ABSTRACT

MicroRNAs (miRNAs) have been indicated to be frequently dysregulated in various cancers and promising biomarkers for colon cancer. The present study aimed to assess the prognostic significance and biological function of miR-1273a in colon cancer. The expression levels of miR-1273a was estimated using quantitative real-time polymerase chain reaction. Kaplan-Meier survival curves and Cox regression analysis were used to evaluate the prognostic value of miR-1273a in patients of colon cancer. The effects of miR-1273a on cell proliferation, migration, and invasion were investigated by cell experiments. The expression of miR-1273a was downregulated in colon cancer tissues and tumor cell lines compared with the normal controls (all P<0.001). The aberrant expression of miR-1273a was associated with vascular invasion (P=0.005), differentiation (P=0.023), lymph node metastasis (P=0.021), and TNM stage (P=0.004). The patients with low miR-1273a expression had low overall survival compared with the patients with high miR-1273a expression (log-rank P=0.002). miR-1273a was detected to be an independent prognostic biomarker for patients. Furthermore, the results of cell experiments revealed that miR-1273a downregulation promoted, while miR-1273a upregulation suppressed the cell proliferation, migration, and invasion. In conclusion, all data indicated that a downregulated expression of miR-1273a predicted poor prognosis for colon cancer and enhanced tumor cell proliferation, migration, and invasion. Thus, we suggest that methods to promote miR-1273a expression may serve as novel therapeutic strategies in colon cancer.


Subject(s)
Humans , Male , Female , Middle Aged , Colonic Neoplasms/diagnosis , MicroRNAs/genetics , Biomarkers, Tumor/genetics , Cell Movement/genetics , Colonic Neoplasms/genetics , Cell Proliferation/genetics , Neoplasm Invasiveness
8.
Braz. j. med. biol. res ; 53(12): e9732, 2020. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS | ID: biblio-1132505

ABSTRACT

Preeclampsia (PE) is a complex pregnancy syndrome. Convincing evidence indicates that long non-coding RNAs (lncRNAs) are involved in the pathogenesis of PE. This research mainly investigated the mechanism of family with sequence similarity 99 member A (FAM99A) in PE. The expressions of FAM99A, miR-134-5p, and YAP1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell apoptosis, migration, and invasion were detected by flow cytometry or transwell assay. The interaction between miR-134-5p and FAM99A or YAP1 was confirmed by dual-luciferase reporter assay. The protein expression of YAP1 was determined by western blot assay. FAM99A and YAP1 were significantly up-regulated, and miR-134-5p was significantly down-regulated in PE tissues (n=30). miR-134-5p was verified as a candidate of FAM99A and YAP1. FAM99A promoted cell metastasis, but reduced apoptosis in HTR8/SVneo cells by regulating miR-134-5p. miR-134-5p down-regulated YAP1 expression to suppress cell metastasis, while it induced apoptosis in HTR8/SVneo cells. FAM99A positively modulated YAP1 expression by sponging miR-134-5p. FAM99A modulated YAP1 to accelerate cell migration and invasion, and inhibited cell apoptosis in PE cells by sponging miR-134-5p. The novel regulatory network may shed light on the pathogenesis of PE.


Subject(s)
Humans , Female , Pregnancy , Adult , Pre-Eclampsia/genetics , RNA, Long Noncoding/genetics , Trophoblasts , Cell Movement/genetics , MicroRNAs
9.
Chinese Medical Journal ; (24): 2437-2443, 2020.
Article in English | WPRIM | ID: wpr-877835

ABSTRACT

BACKGROUND@#Epithelial to mesenchymal transition (EMT) is strongly linked with tumor invasion and metastasis, which performs a vital role in carcinogenesis and cancer progression. Emerging evidence suggests that microRNAs (miRNAs) expression are closely associated to EMT by regulating targeted genes. MiR542 has been found to be involved in the EMT program and bound up with various cancers. However, the functions of miR542 and its underlying mechanism in glioblastoma multiforme (GBM) remain largely unknown. In the current study, we investigated the effect of astrocyte elevated gene-1 (AEG-1) on U251 cells aggressiveness, proliferation, apoptosis, and cell cycle.@*METHODS@#The screening of targeted miRNAs was performed, as well as the functional roles and mechanisms of miR542 were explored.@*RESULTS@#MiR542 was selected as the target because of the most significantly differential expression and this high level of expression negatively correlated with cell migration and proliferation, which suggested that miR542 could be a novel tumor suppressor. Moreover, we confirmed that AEG-1 was a direct targeted gene of miR542 by luciferase activity assay, reverse transcription-polymerase chain reaction, and immunoblotting analysis. Furthermore, miR542 suppressed the expression of AEG-1, which upgraded the level of E-cadherin and degraded Vimentin expression contributing to retraining EMT.@*CONCLUSION@#The in vitro findings demonstrated that miR542 inhibited the migration and proliferation of U251 cells and suppressed EMT through targeting AEG-1, indicating that miR542 may be a potential anti-cancer target for GBM.


Subject(s)
Astrocytes , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Humans , MicroRNAs/genetics , Neoplasm Invasiveness/genetics
10.
Article in Chinese | WPRIM | ID: wpr-880799

ABSTRACT

OBJECTIVE@#To explore the inhibitory effects of silencing long non-coding RNA (LncRNA) HIF1A-AS2 on epithelialmesenchymal transition (EMT) and tumor stem cell-like phenotype in cervical cancer cells.@*METHODS@#We designed 3 shRNA constructs for silencing HIF1A-AS2 in CaSki cells, and the shRNA with the strongest interference effect was selected for subsequent experiment. CaSki cells were transfected with shRNA-NC or Sh-HIF1A-AS2, and the changes in cell viability, invasion ability, EMT, expressions of EMT-related proteins, formation of cell spheres and expressions of stem cell markers were detected.@*RESULTS@#Transfection with shRNA-NC and Sh-HIF1A-AS2 did not significantly affected the viability of CaSki cells (@*CONCLUSIONS@#Silencing HIF1A-AS2 can inhibit proliferation, invasion and migration of cervical cancer cells


Subject(s)
Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , RNA, Long Noncoding/genetics , RNA, Small Interfering/genetics , Uterine Cervical Neoplasms/genetics
11.
Chinese Journal of Biotechnology ; (12): 2151-2161, 2020.
Article in Chinese | WPRIM | ID: wpr-878474

ABSTRACT

Kidney is one of the most important organs of the body and the mammalian kidney development is essential for kidney unit formation. The key process of kidney development is metanephric development, where mesenchymal-epithelial transition (MET) plays a crucial role. Here we investigated the biological function of PPP3CA in metanephric mesenchyme (MM) cells. qRT-PCR and Western blotting were used to detect PPP3CA and MET makers expression in mK3, mK4 cells respectively at mRNA and protein level. Subsequently, PPP3CA was stably knocked down via lentivirus infection in mK4 cells. Flow cytometry, EdU/CCK-8 assay, wound healing assay were conducted to clarify the regulation of PPP3CA on cell apoptosis, proliferation and migration respectively. PPP3CA was expressed higher in epithelial-like mK4 cells than mesenchyme-like mK3 cells. Thus, PPP3CA was silenced in mK4 cells and PPP3CA deficiency promoted E-cadherin expression, cell apoptosis. Moreover, PPP3CA knock down attenuated cell proliferation and cell migration in mK4 cell. The underlying mechanism was associated with the dephosphorylation of PPP3CA on ERK1/2. Taken together, our results indicated that PPP3CA mediated MET process and cell behaviors of MM cells, providing new foundation for analyzing potential regulator in kidney development process.


Subject(s)
Animals , Apoptosis/genetics , Cell Line , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Silencing , Mesenchymal Stem Cells/cytology , Mesoderm , Mice
12.
Int. braz. j. urol ; 45(3): 549-559, May-June 2019. tab, graf
Article in English | LILACS | ID: biblio-1012314

ABSTRACT

ABSTRACT Objective: To study the expression patterns of long noncoding RNA (lncRNA) colon cancer-associated transcript 1 (CCAT1) and the changes in cell proliferation, apoptosis, migration and invasion induced by silencing CCAT1 in bladder cancer cells. Materials and Methods: The expression levels of CCAT1 were determined using realtime quantitative polymerase chain reaction in cancerous tissues and paired normal tissues from 34 patients with bladder cancer. The relationship between clinical characteristics and CCAT1 expression was analyzed. And then we conducted cell experiments. Bladder urothelial carcinoma cell lines T24 and 5637 cells were transfected with CCAT1 small interfering RNA (siRNA) or scramble siRNA. Cell proliferation and apoptosis changes were determined using a Cell Counting Kit-8 (CCK-8) assay and a flow cytometry assay. Migration and invasion changes were measured using a wound healing assay and a trans-well assay. microRNAs (miRNAs) were predicted by Starbase 2.0, and their differential expression levels were studied. Results: CCAT1 was significantly upregulated in bladder cancer (P < 0.05). CCAT1 upregulation was positively related to tumor stage (P = 0.004), tumor grade (P = 0.001) and tumor size (P = 0.042). Cell proliferation, migration and invasion were promoted by abnormally expressed CCAT1. miRNAs miR-181b-5p, miR-152-3p, miR-24-3p, miR-148a-3p and miR-490-3p were potentially related to the aforementioned functions of CCAT1. Conclusion: CCAT1 plays an oncogenic role in urothelial carcinoma of the bladder. In addition, CCAT1 may be a potential therapeutic target in this cancer.


Subject(s)
Humans , Male , Female , Aged , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , RNA, Long Noncoding/analysis , Sincalide/analysis , Time Factors , Wound Healing/genetics , Down-Regulation , Gene Expression , Gene Expression Regulation, Neoplastic , Up-Regulation , Cell Movement/genetics , MicroRNAs/genetics , RNA, Small Interfering , Cell Line, Tumor , Cell Proliferation/genetics , Real-Time Polymerase Chain Reaction , Flow Cytometry
13.
Univ. med ; 59(3)2018. ilus
Article in Spanish | LILACS, COLNAL | ID: biblio-995004

ABSTRACT

El desarrollo neurológico humano requiere una serie de pasos que permitan orientar, regular y diferenciar los diversos componentes cerebrales, para así garantizar, de una manera bastante precisa, la correcta organización y funcionamiento de las estructuras neuronales. La neurogénesis está clásicamente dividida en cuatro etapas consecutivas: proliferación, migración, diferenciación y maduración. En los humanos, estas ocurren desde la tercera semana de gestación hasta la vida adulta y precisan de un complejo grupo de paquetes genéticos, así como de algunos factores asociados, que se han ido descubriendo gracias a los avances en la biología molecular. El artículo es una revisión acerca del desarrollo neuroembriológico humano y los componentes genéticos más relevantes encontrados en la literatura.


The human neuronal development requires a number of concrete steps which lead to orientation, regulation and differentiation of several brain components. They must be done to guarantee, in a very precise way, the correct organization and functioning of the neuronal structures. Neurogenesis is commonly divided into four consecutive stages: proliferation, migration, differentiation and maturation. In humans, those stages take place since the third week of prenatal Iife until the adult Iife. They also require a complex group of genetic packs and associated molecular factors, most of which have been recen tly discovered by the molecular biology technology. A review was made about the human neuronal and embryological development and the most relevant genetic components described by the literature so far.


Subject(s)
Cell Movement/genetics , Neurogenesis/genetics , Embryonic and Fetal Development , Cell Differentiation/genetics
14.
Biol. Res ; 51: 50, 2018. graf
Article in English | LILACS | ID: biblio-1011394

ABSTRACT

BACKGROUND: Accumulating studies have demonstrated that high-mobility group A2 (HMGA2), an oncofetal protein, plays a role in tumor development and progression. However, the molecular role of HMGA2 in ovarian carcinoma is yet to be established. MicroRNAs (miRNAs), a group of small noncoding RNAs, negatively regulate gene expression and their dysregulation has been implicated in tumorigenesis. The aim of this study was to investigate the potential involvement of a specific miRNA, miR-219-5p, in HMGA2-induced ovarian cancer. METHODS: The ovarian cancer cell line, SKOV3, was employed, and miR-219-5p and HMGA2 overexpression vectors constructed. The CCK-8 kit was used to determine cell proliferation and the Transwell® assay used to measure cell invasion and migration. RT-PCR and western blot analyses were applied to analyze the expression of miR-219-5p and HMGA2, and the luciferase reporter assay used to examine the interactions between miR-219-5p and HMGA2. Nude mice were employed to characterize in vivo tumor growth regulation. RESULTS: Expression of miR-219-5p led to suppression of proliferation, invasion and migration of the ovarian cancer cell line, SKOV3, by targeting HMGA2. The inhibitory effects of miR-219-5p were reversed upon overexpression of HMGA2. Data from the luciferase reporter assay showed that miR-219-5p downregulates HMGA2 via direct integration with its 3'-UTR. Consistent with in vitro findings, expression of miR-219-5p led to significant inhibition of tumor growth in vivo. CONCLUSION: Our results collectively suggest that miR-219-5p inhibits tumor growth and metastasis by targeting HMGA2.


Subject(s)
Humans , Animals , Female , Mice , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Gene Expression Regulation, Neoplastic/genetics , HMGA2 Protein/metabolism , MicroRNAs/physiology , Ovarian Neoplasms/genetics , Cell Movement/genetics , HMGA2 Protein/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Neoplasm Invasiveness , Neoplasm Metastasis
15.
Biol. Res ; 49: 1-10, 2016. ilus, graf, tab
Article in English | LILACS | ID: lil-774432

ABSTRACT

BACKGROUND: Reprimo (RPRM), a highly glycosylated protein, is a new downstream effector of p53-induced cell cycle arrest at the G2/M checkpoint, and a putative tumor suppressor gene frequently silenced via methylation of its promoter region in several malignances. The aim of this study was to characterize the epigenetic inactivation and its biological function in BC cell lines. METHODS: The correlation between RPRM methylation and loss of mRNA expression was assessed in six breast cancer cell lines by methylation specific PCR (MSP), 5'-Aza-2'-deoxycytidine treatment and RT-PCR assays. MDA-MB-231 cells were chosen to investigate the phenotypic effect of RPRM in cell proliferation, cell cycle, cell death, cell migration and invasion. RESULTS: In the cancer methylome system (CMS) (web-based system for visualizing and analyzing genome-wide methylation data of human cancers), the CpG island region of RPRM (1.1 kb) was hypermethylated in breast cancer compared to normal breast tissue; more interesting still was that ERa(+) tumors showed higher methylation intensity than ERa(-). Downregulation of RPRM mRNA by methylation was confirmed in MDA-MB-231 and BT-20 cell lines. In addition, overexpression of RPRM in MDA-MB-231 cells resulted in decreased rates of cell migration, wound healing and invasion in vitro. However, RPRM overexpression did not alter cell viability, phosphatidylserine (PS) translocation or G2/M cell cycle transition. CONCLUSION: Taken together, these data suggest that RPRM is involved in decreased cell migration and invasion in vitro, acting as a potential tumor suppressor gene in the MDA-MB-231 cell line.


Subject(s)
Female , Humans , Breast Neoplasms/pathology , Cell Cycle Proteins/physiology , Cell Movement/physiology , Cell Proliferation/physiology , Glycoproteins/physiology , Analysis of Variance , Blotting, Western , Breast Neoplasms/genetics , Cell Cycle , Cell Line, Tumor , Cell Survival , Cell Cycle Proteins/genetics , Cell Movement/genetics , Cell Proliferation/genetics , DNA Methylation , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Glycoproteins/genetics , Neoplasm Invasiveness , Real-Time Polymerase Chain Reaction , Statistics, Nonparametric
16.
Braz. j. med. biol. res ; 47(1): 24-34, 01/2014. graf
Article in English | LILACS | ID: lil-697676

ABSTRACT

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.


Subject(s)
Humans , Antineoplastic Agents/pharmacology , Cell Movement/genetics , Cell Proliferation/genetics , /genetics , Fanconi Anemia Complementation Group F Protein/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Drug Resistance , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , RNA Interference , RNA, Small Interfering
17.
Arq. bras. cardiol ; 101(2): 141-148, ago. 2013. ilus, tab
Article in Portuguese | LILACS | ID: lil-685389

ABSTRACT

FUNDAMENTO: O fator de crescimento endotelial vascular (VEGF - vascular endothelial growth factor) induz a mobilização de células progenitoras endoteliais (CPEs) com capacidade de proliferação e diferenciação em células endoteliais, contribuindo, dessa forma, para o processo angiogênico. OBJETIVO: Buscamos avaliar o comportamento de CPEs em pacientes com doença cardíaca isquêmica e angina refratária que receberam injeções intramiocardicas de 2000 µg de VEGF165 como terapia única. MÉTODOS: O estudo foi uma subanálise de um ensaio clínico. Pacientes com doença cardíaca isquêmica avançada e angina refratária foram avaliados para inclusão no estudo. Os critérios de inclusão foram: sinais e sintomas de angina e/ou insuficiência cardíaca apesar de tratamento medicamentoso máximo e área de isquemia miocárdica de, no mínimo, 5% conforme avaliado por uma tomografia computadorizada por emissão de fóton único (TCEFU). Os critérios de exclusão foram: idade > 65 anos, fração de ejeção do ventrículo esquerdo < 25% e cancer diagnosticado. Os pacientes cujos níveis de CPE foram avaliados foram incluídos. A intervenção consistiu na administração de 2000 µg de VEGF 165 de plasmídeo injetado no miocárdio isquêmico. A frequência de células CD34+/KDR+ foi analisada por citometria de fluxo antes e 3, 9, e 27 dias após a intervenção. RESULTADOS: Um total de 9 pacientes foram incluídos, 8 homens, média de idade de 59,4 anos, fração de ejeção ventricular esquerda de 59,3%, e classe de angina predominante III. Observou-se um aumento significativo dos níveis de CPEs no terceiro dia após a intervenção. Todavia, 9 e 27 dias após a intervenção, os níveis de CPEs foram similares aos basais. CONCLUSÃO: Identificamos uma mobilização transitória de CPE, com pico no terceiro dia após a intervenção com VEGF 165 em pacientes com angina refratária. Todavia, os níveis de CPEs apresentaram-se semelhantes aos basais 9 e 27 dias após a intervenção.


BACKGROUND: Vascular endothelial growth factor (VEGF) induces mobilization of endothelial progenitor cells (EPCs) with the capacity for proliferation and differentiation into mature endothelial cells, thus contributing to the angiogenic process. OBJECTIVE: We sought to assess the behavior of EPCs in patients with ischemic heart disease and refractory angina who received an intramyocardial injections of 2000 µg of VEGF 165 as the sole therapy. METHODS: The study was a subanalysis of a clinical trial. Patients with advanced ischemic heart disease and refractory angina were assessed for eligibility. Inclusion criteria were as follows: signs and symptoms of angina and/or heart failure despite maximum medical treatment and a myocardial ischemic area of at least 5% as assessed by single-photon emission computed tomography (SPECT). Exclusion criteria were as follows: age > 65 years, left ventricular ejection fraction < 25%, and a diagnosis of cancer. Patients whose EPC levels were assessed were included. The intervention was 2000 µg of VEGF 165 plasmid injected into the ischemic myocardium. The frequency of CD34+/KDR+ cells was analyzed by flow cytometry before and 3, 9, and 27 days after the intervention. RESULTS: A total of 9 patients were included, 8 males, mean age 59.4 years, mean left ventricular ejection fraction of 59.3% and predominant class III angina. The number of EPCs on day 3 was significantly higher than that at baseline (p = 0.03); however, that on days 9 and 27 was comparable to that at baseline. CONCLUSION: We identified a transient mobilization of EPCs, which peaked on the 3th day after VEGF 165 gene therapy in patients with refractory angina and returned to near baseline levels on days 9 and 27.


Subject(s)
Female , Humans , Male , Middle Aged , Angina Pectoris/therapy , Cell Movement/genetics , Endothelial Cells/physiology , Genetic Therapy/methods , Multipotent Stem Cells/physiology , Vascular Endothelial Growth Factor A/genetics , Cell Movement/physiology , Multipotent Stem Cells/cytology , Myocardial Ischemia/therapy , Neovascularization, Physiologic/genetics , Plasmids/genetics , Time Factors , Treatment Outcome
18.
Arq. bras. endocrinol. metab ; 55(8): 501-511, nov. 2011. ilus
Article in Portuguese | LILACS | ID: lil-610449

ABSTRACT

O hipogonadismo hipogonadotrófico isolado (HHI) congênito caracteriza-se pela falta completa ou parcial de desenvolvimento puberal em decorrência de defeitos na migração, síntese, secreção ou ação do hormônio liberador de gonadotrofinas (GnRH). Baixas concentrações de esteroides sexuais e valores reduzidos ou inapropriadamente normais de gonadotrofinas hipofisárias (LH e FSH) definem, do ponto de vista laboratorial, essa condição clínica. A secreção dos demais hormônios hipofisários encontra-se normal, bem como a ressonância magnética de região hipotalâmica-hipofisária, demonstrando a ausência de uma causa anatômica. Alterações olfatórias, como anosmia ou hiposmia, podem estar associadas ao HHI, caracterizando a síndrome de Kallmann. Uma lista crescente de genes está envolvida na etiologia do HHI, sugerindo a heterogeneidade e a complexidade da base genética dessa condição. Distúrbios na rota de migração dos neurônios secretores de GnRH e dos neurônios olfatórios formam a base clínico-patológica da síndrome de Kallmann. Mutações nos genes KAL1, FGFR1/FGF8, PROK2/PROKR2, NELF, CHD7, HS6ST1 e WDR11 foram associadas a defeitos de migração neuronal, causando a síndrome de Kallmann. É notável que defeitos nos genes FGFR1, FGF8, PROKR2, CHD7 e WDR11 foram também associados ao HHI sem alterações olfatórias (HHI normósmico), porém em menor frequência. Adicionalmente, defeitos nos KISS1R, TAC3/TACR3 e GNRH1/GNRHR foram descritos exclusivamente em pacientes com HHI normósmico. Neste trabalho, revisaremos as características clínicas, hormonais e genéticas do HHI.


Congenital isolated hypogonadotropic hypogonadism (IHH) is characterized by partial or complete lack of pubertal development due to defects in migration, synthesis, secretion or action of gonadotropin-releasing hormone (GnRH). Laboratory diagnosis is based on the presence of low levels of sex steroids, associated with low or inappropriately normal levels of pituitary gonadotropins (LH and FSH). Secretion of other pituitary hormones is normal, as well magnetic resonance imaging of the hypothalamohypophyseal tract, which shows absence of an anatomical defects. When IHH is associated with olfactory abnormalities (anosmia or hyposmia), it characterizes Kallmann syndrome. A growing list of genes is involved in the etiology of IHH, suggesting the heterogeneity and complexity of the genetic bases of this condition. Defects in olfactory and GnRH neuron migration are the etiopathogenic basis of Kallmann syndrome. Mutations in KAL1, FGFR1/FGF8, PROK2/PROKR2, NELF, CHD7, HS6ST1 and WDR11 are associated with defects in neuronal migration, leading to Kallmann syndrome. Notably, defects in FGFR1, FGF8, PROKR2, CHD7 and WDR11 are also associated with IHH, without olfactory abnormalities (normosmic IHH), although in a lower frequency. Mutations in KISS1R, TAC3/TACR3 and GNRH1/GNRHR are described exclusively in patients with normosmic IHH. In this paper, we reviewed the clinical, hormonal and genetic aspects of IHH.


Subject(s)
Humans , Gonadotropin-Releasing Hormone/genetics , Hypogonadism/genetics , Kallmann Syndrome/genetics , Mutation/genetics , Cell Movement/genetics , Gonadotropin-Releasing Hormone , Hypogonadism/congenital , Neurons
19.
Säo Paulo; s.n; 2000. 147 p. ilus, tab.
Thesis in Portuguese | LILACS | ID: lil-280793

ABSTRACT

A lisencefalia clássica (LC) é uma malformação cerebral caracterizada por uma superfície cerebral lisa devido a um distúrbio na migração neuronal. A LC tem sido observada em associação com a Síndrome de Miller-Dieker (SMD) e a Seqüência da Lisencefalia Isolada (SLI). A literatura mostra que deleções visíveis ou submicroscópicas ocorrem no mesmo loco entre 10 a 40 por cento dos pacientes com SLI. Um gene conhecido como LIS1 foi mapeado em l7pl3.3 e suas alterações têm sido responsáveis pela maioria dos casos de LC. Um outro gene foi identificado em Xq22-q23 (XLIS) e está relacionado à LC ligada ao X e aos quadros de Heterotopia com Banda Sucortical. O presente estudo aborda os aspectos clínicos, radiológicos e citogenéticos em 10 pacientes com SLI. À avaliação clínica e radiológica, os pacientes foram categorizados como portadores da SLI. A abordagem citogenética foi realizada pelas técnicas de citogenética clássica e pela metolodogia da hibridação ín situ com fluorescência (FISH) com a sonda do marcador Dl7S379 (Oncor©). Todos pacientes mostravam um cariótipo normal e uma paciente (caso 2) revelou uma microdeleção pelo método da FISH. A deleção era de novo e foi procedido uma amplificação de um fragmento de Dl7S379, através da PCR, com o objetivo de identificar a origem parental da deleção. Para tanto, foram utilizadas seqüência iniciadoras que flanqueiam uma região que contém um polimorfismo de repetição CA. Contudo, a família estudada não foi informativa ara esta abordagem


Subject(s)
Cell Movement/genetics , Cerebrum/abnormalities , Cerebral Cortex/abnormalities , Cytogenetics , In Situ Hybridization, Fluorescence
20.
Rev. colomb. obstet. ginecol ; 45(1): 65-8, ene.-mar. 1994. tab
Article in Spanish | LILACS | ID: lil-293276

ABSTRACT

OBJETIVO: estudiar el comportamiento del espermatozoide "Y" en espermograma normal, moco cervical y endometrio en mujeres en edad reproductiva. MATERIAL Y ETODOS: Se estudiaron 36 mujeres con ligadura de trompas provenientes de un centro de Salud de la ciudad de Cali. Con control de temperatura se orientó la prueba post-coito y se determinó la concentración de espermatozoide "Y" en moco cervical y endometrio por técnica de Fluorescencia con quinacrina. Como prueba de significa se utilizó el coeficiente de correlación. RESULTADOS: El promedio de espermatozoides "Y" en espermograma normal fue de 43 por ciento, en moco cervical 34.48 por ciento y en endometrio de 34.6 por ciento. No hubo diferencias significativas a lo largo del ciclo mestrual. CONCLUSION: El espermatozide "Y" en este estudio disminuye su concentración en moco y endometrio sin encontrarse diferencias significativas a lo largo del ciclo, dato que confirma los hallazgos de otros autores pero contradictorio para otros


Subject(s)
Humans , Male , Female , Adult , Cell Movement/genetics , Cell Movement/immunology , Cell Movement/physiology , Sperm Motility/physiology
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