Effects of microRNA-424-5p on proliferation, migration and invasion of colorectal cancer cells by regulating OTX1 / 国际肿瘤学杂志

Objective To investigate the correlation of the expression of microRNA-424-5p (miR-424-5p) and orthodenticle homeobox 1 (OTX1) gene in colorectal cancer (CRC) tissue,and the effects of miR-424-5p on the proliferation,migration and invasion of CRC cells HCT116.Methods A total of 60 patients with CRC were collected from June 2017 to June 2018 in Department of Colorectal Surgery of Third Affiliated Hospital of Kunming Medical University.Real time quantitative PCR (RT-qPCR) was used to detect the expression of miR-424-5p and OTX1 mRNA in 60 cases of CRC tissues,para-carcinoma tissues and CRC cell lines.The correlation between the expression of miR-424-5p and OTX1 gene was further analyzed.miR-NC (miR-NC group) and miR-424-5p-mimic (miR-424-5p-mimic group) were transfected into HCT116 cells.CCK-8 assay,scratch assay and Transwell assay were used to detect the effects of miR-424-5p on the proliferation,migration and invasion of HCT116 cells.The effect of miR-424-5p on the expression of OTX1 protein was detected by Western blotting.The luciferase report assay was used to detect the influence of miR-424-5p on the luciferase activity of OTX1-3'UTR vector.Results The relative expressions of OTX1 mRNA in CRC tissues and para-carcinoma tissues were 1.049 ±0.446 and 0.639 ±0.178 (t =-6.583,P ＜0.001);and the relative expression of miR-424-5p in CRC tissues and para-carcinoma tissues were 0.865 ± 0.261 and 1.329 ± 0.387 (t =7.705,P ＜ 0.001),with statistically significant differences.Negative correlation was found between the expression of miR-424-5p and OTX1 mRNA in CRC tissues (r =-0.439,P =0.015).The absorbance values of HCT116 cells transfected with miR-424-5p-mimic were 0.813 ± 0.064,0.960 ± 0.098,1.287 ± 0.192 on 72,96 and 120 hours respectively,and those of HCT116 cells transfected with miR-NC were 1.163 ±0.158,1.645 ±0.117 and 2.043 ±0.236.The proliferation ability of miR-424-5p-mimic group was lower than that of miR-NC group and the differences between the two groups were statistically significant (t =3.538,P =0.024;t =7.778,P =0.001;t =4.257,P =0.013).The scratch assay showed that the migration of HCT116 cells in miR-424-5p-mimic group was inhibited as compared with miR-NC group.The numbers of cells permeating septum of miR-NC and miR-424-5p-mimic group were 177.104 ± 17.834 and 35.667 ± 13.634,and the difference was statistically significant (t =15.246,P ＜ 0.001).The relative expressions of OTX1 protein in miR-NC and miR-424-5p-mimic group were 0.862 ±0.121 and 0.342 ±0.103 respectively,and the difference was statistically significant (t =16.286,P ＜ 0.001).Luciferase report assay showed that the luciferase activities of wt-OTX1-3'UTR and mut-OTX1-3'UTR vector were 0.305 ± 0.095 and 0.898 ± 0.080 after over expression of miR-424-5p,and the difference was statistically significant (P ＜ 0.001).Conclusion The expressions of miR-424-5p and OTX1 mRNA are negatively correlated in CRC tissue.miR-424-5p can inhibit the proliferation,migration and invasion of CRC cells by down-regulating the expression of OTX1.

OTX1 Contributes to Hepatocellular Carcinoma Progression by Regulation of ERK/MAPK Pathway

Orthodenticlehomeobox 1 (OTX1) overexpression had previously been associated with the progression of several tumors. The present study aimed to determine the expression and role of OTX1 in human hepatocellular carcinoma (HCC). The expression level of OTX1 was examined by quantitative real-time PCR (qRT-PCR) in 10 samples of HCC and paired adjacent non-cancerous tissues, and by immunohistochemistry (IHC) analysis in 128 HCC samples and matched controls. The relationship between OTX1 expression and the clinicopathological features werealso analyzed. Furthermore, the effects of OTX1 knockdown on cell proliferation and migration were determined in HCC cell lines. Axenograft mouse model was also established to investigate the role of OTX1 in HCC tumor growth. TheqRT-PCR and IHC analyses revealed that OTX1 was significantly elevated in HCC tissues compared with the paired non-cancerous controls. Expression of OTX1 was positively correlated with nodal metastasis status (P = 0.009) and TNM staging (P = 0.001) in HCC tissues. In addition, knockdown of OTX1 by shRNA significantly inhibited the proliferation and migration, and induced cell cycle arrest in S phase in vitro. Tumor growth was markedly inhibited by OTX1 silencing in the xenograft. Moreover, OTX1 silencing was causable for the decreased phosphorylation level of ERK/MAPK signaling. In conclusion, OTX1 contributes to HCC progression possibly by regulation of ERK/MAPK pathway. OTX1 may be a novel target for molecular therapy towards HCC.

Análise molecular dos genes OTX1 e OTX2 em meduloblastomas / Molecular analysis of OTX1 and OTX2 genes in medulloblastoma

INTRODUÇÃO: O meduloblastoma, tumor maligno do Sistema Nervoso Central mais comum em crianças, foi inicialmente descrito de forma uniforme em 1925 por Bailey e Harvey Cushing. A despeito do avanço diagnóstico e terapêutico, os índices de morbimortalidade persistem altos. Grupos epidemiologicamente semelhantes podem ter desfechos diferentes, e evoluções desfavoráveis ocorrem em pacientes com marcadores de bom prognóstico. Os avanços nas pesquisas em biologia molecular procuram explicar os diferentes comportamentos da doença, e de forma sistemática, buscam identificar genes que sirvam como alvos terapêuticos, já que o tratamento disponível atualmente ainda é bastante insatisfatório e com muitos efeitos colaterais. Simeone e colaboradores identificaram os genes OTX1 e OTX2, presentes em humanos, e cuja função é organizar, compartimentalizar e hierarquizar a formação do sistema nervoso central, especialmente o cerebelo. Os genes OTX1 e OTX2 são expressos no tecido cerebelar em humanos até a nona semana de vida extra-uterina, exclusivamente. Os mesmos autores também identificaram que os mesmos genes são alvo terapêutico do ácido transretinóico, que inibe a expressão gênica. Estudos prévios demonstraram a expressão dos genes OTX1 e OTX2 em meduloblastomas, o que torna o ácido uma potencial terapêutica para estes tumores, assim como os genes OTX1 e OTX2 potenciais alvos para desenvolvimento de novas drogas terapêuticas. OBJETIVOS: Estudar a prevalência dos genes OTX1 e OTX2 em uma amostra de 60 pacientes, e estabelecer correlações entre a expressão gênica e aspectos clínicos, patológicos e de evolução. CASUÍSTICA E MÉTODO: Realizada análise retrospectiva de 60 pacientes com diagnóstico meduloblastoma, operados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, e no Hospital do Câncer de Barretos. Organizado um banco de dados de 60 pacientes contendo dados da expressão gênica dos genes OTX1 e OTX2 (obtida através da técnica de PCR...

INTRODUCTION: Medulloblastoma, the most common malignant tumor of the central nervous system in children, was first uniformly described in 1925 by Bailey and Harvey Cushing. Despite the diagnostic and therapeutic advances, the morbidity and mortality rates remain high. Epidemiologically similar groups may have different outcomes, and adverse developments occur in patients with markers of good prognosis. Advances in molecular biology research seeks to explain the different behaviors of the disease, and consistently seek to identify genes that serve as drug targets, since the treatment currently available is still unsatisfactory and with many side effects. Simeone and colleagues identified genes OTX1 and OTX2 in humans, and whose function is to organize, prioritize and compartmentalize the formation of the central nervous system, especially the cerebellum. OTX1 and OTX2 genes are expressed in cerebellar tissue in humans until the ninth week of extra uterine life, exclusively. The same authors also found that the same genes are therapeutic target of trans-retinoic acid, which inhibits gene expression. Previous studies have demonstrated the expression of OTX1 and OTX2 genes in medulloblastomas, which makes the acid a potential therapy for these tumors, as well as the genes OTX2 and OTX1 potential targets for developing new therapeutic drugs. OBJECTIVES: To study the prevalence of OTX1 and OTX2 genes in a sample of 60 patients, and to establish correlations between gene expression and clinical, pathological and follow up aspects. CASUISTICS AND METHODS: A retrospective analysis of 60 patients diagnosed with medulloblastoma, assisted at Hospital of the Faculty of Medicine, University of São Paulo, and the Cancer Hospital of Barretos. Organized a database of 60 patients which contains the gene expression of OTX1 and OTX2 genes (obtained through the technique of real-time PCR) and clinical and epidemiological data. Performed statistical tests to establish a...