miR-21 regulates the proliferation, invasion and radiosensitivity of cervical cancer HeLa cells by targeting RECK / 中华放射肿瘤学杂志

Objective:To explore the effect of miR-21 on cell proliferation, apoptosis, invasion and radiosensitivity of cervical cancer HeLa cells and unravel the underlying mechanism.Methods:RT-qPCR assay was used to detect the expression levels of miR-21 in cervical cancer tissues and adjacent non-tumor tissues, normal cervical epithelial cells (H8) and cervical cancer cell lines (HeLa, SiHa, ME180). HeLa cell line with inhibition of miR-21 or knockdown of RECK were constructed. CCK-8, Caspase3/7 live cell apoptosis detection, wound healing test, Transwell invasion, clone formation assay, Western blot and immunofluorescence were performed to detect cell viability, apoptosis, migration, invasion, radiosensitivity and related proteins. The dual luciferase assay verified whether miR-21 targeted RECK.Results:MiR-21 level in the cervical cancer tissues was significantly higher than that in its corresponding adjacent non-tumor tissues ( P<0.05). The expression levels of miR-21 in cervical cancer cell lines HeLa, SiHa and ME180 were significantly up-regulated compared with those in normal cervical epithelial cells H8(all P<0.05). MiR-21 knockdown significantly inhibited HeLa cell viability, promoted cell apoptosis, reduced radiation tolerance, down-regulated the expression of Cyclin D 1,Bcl-2, MMP-2 and MMP-9, and up-regulated the expression P21 and Bax proteins (all P<0.05). miR-21 targeted the 3’-UTR of RECK mRNA and negatively regulated the expression of RECK. Silencing RECK reversed the effects of miR-21 knockdown on HeLa cell apoptosis, migration, invasion and radiosensitivity. Conclusions:Inhibiting the expression of miR-21 significantly decreases cell viability, induces cell apoptosis, weakens cell migration and invasion capabilities, and enhances the radiosensitivity of HeLa cells. The potential mechanism is closely related to the targeted up-regulation of RECK.

Progress in epigenetic regulation of the tumor suppressor gene RECK / 肿瘤研究与临床

The RECK protein is highly expressed in normal cells and tissues,however,down-regulated in transformed cells or tumor tissues.Subsequent functional studies have demonstrated that RECK owns the tumor suppressor activity.Hence,it is urgent and important to unmask the mechanisms involved in regulation of RECK expression.Epigenetic regulation plays a vital role in modulating gene expression,besides,multiple research also suggested the involvement of epigenetic regulation in RECK expression.The epigenetic regulation mechanisms of controlling the gene expression currently known will be reviewed in this paper.

Correlation between RECK gene methylation and the prognosis of laryngeal squamous cell carcinoma / 医学研究生学报

Objective The incidence of laryngeal cancer has characteristic of regional differences, but the etiology is not clear. The aim of this study was to analyze the correlation between RECK gene promoter methylation and prognosis of laryngeal squamous cell carci-noma patients through detecting the RECK gene methylation status of primary laryngeal squamous cell carcinoma and adjacent tissues . Methods Methylation specific PCR assay was used to detect the RECK gene promoter methylation status of 70 laryngeal squamous cell carci-noma specimens in our hospital from July 2006 to Dcember 2007, and the differences of methylation status with different pathological parame-ters were compared.The correlation between RECK gene promoter methylation and prognosis of 64 patients completed five-year follow-up was analyzed. Results The RECK gene methylation rate (86.67%) of patients with poor differentiation in tumor cells was much higher than that of the patients with a moderate and better tumor cell differentiation (43.64%) (P<0.05).In 29 pairs of laryngeal cancer-adjacent tis-sues specimens matches, the RECK gene methylation in laryngeal carcinoma (55.12%) was higher than normal tissues (27.59%) ( P=0.029).RECK gene methylation significantly shortened the tumor free survival and overall survival analyzed by Log-rank (P=0.024, P=0.017).Lymph node metastasis and clinical stage in classⅢ-Ⅳsignificantly shortened the tumor free survival and overall survival (P=0.029, P=0.024;P=0.033, P=0.032).Moderate and better tumor cell differentiation significantly shortened the tumor free survival (P=0.024, P=0.049).Lymph node metastasis, clinical stage, and RECK gene methylation were independent risk factors of overall survival. Conclusion RECK gene promoter methylation in human laryngeal squamous cell carcinoma is an early event and may occur in the adjacent normal tissues, predicting a poor prognosis in patients.

Expression of reversion-inducing cysteine-rich protein with Kazal motifs and its relationship with matrix metalloproteinase-9 in prostate carcinoma tissues / 第二军医大学学报

Objective: To investigate the expression of the reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and matrix metalloproteinase-9(MMP-9) in the prostate carcinoma tissues and to evaluate the role of RECK in the tumorigenesis of prostate carcinoma. Methods: Twenty specimens of prostate cancer and 12 specimens of normal prostate were harvested. RT-PCR and real-time RT-PCR were used to determine the expression of RECK mRNA and RT-PCR was used to determine the expression of MMP-9 mRNA in the specimens. Western blotting analysis was used to determine the expression of RECK protein. Results: It was found that the expression of RECK mRNA in the prostate carcinoma tissues was lower than that in the normal prostate tissues (P<0.01); MMP-9 expression in the prostate carcinoma tissues was significantly higher than that of the normal prostate tissues (P<0.01). Western blotting analysis showed that the expression of RECK protein in the carcinoma tissues was lower than that in the normal prostate tissue (P<0.01). Conclusion: RECK gene expression is lower in the prostate carcinoma tissues; RECK may inhibit the progression and metastasis of cancer through inhibiting MMP-9 expression.

Expression of RECK Gene in Placentas from Patients with Preeclampsia and Its Correlation with MMP-2 Activation / 华中科技大学学报(医学版)

Objective To investigate the expression of RECK gene in placentas of patients with preeclampsia and its correlation with MMP-2 activation,and explore the possible roles of RECK gene in the placental trophoblast invasion mechanism.Methods RT-PCR and Western blot were used to detect the expression of RECK mRNA and protein respectively in the placental tissues of normal late pregnant women (normal pregnant group,22 cases) and pre-eclamptic patients(22 mild cases and 20 severe cases).Gelatinase zymography was used to determine MMP-2 activation ratio.Results The expression levels of RECK mRNA and protein from placenta tissues in mild,severe pre-eclamptic group were both significantly higher than those in nomal pregnant group.Moreover,the expression levels of RECK mRNA and protein in severe pre-eclamptic group were obviously increased as compared with those in mild pre-eclamptic group.There was significant difference among the three groups (all P<0.01).MMP-2 activation ratio in mild,severe pre-eclamptic group was significantly lower than that in normal pregnant group.MMP-2 activation ratio in severe pre-eclamptie groups was obviously reduced as compared with mild pre-eclamptic group.There was significant difference among the three groups(all P<0.01).The expression leVels of RECK mRNA and protein were significantly negatively correlated with MMP-2 activation ratio (both P<0.01).Conclusion The abnormal high expression of RECK and inhibition of MMP-2 activation in placentas of pre-eclamptic patients may participate in the process of placental trophoblast shallow invasion.

Analysis of RECK (a novel MMP inhibitor) Expression and its Roles as a Biomarker and Evaluation of RECK as a Potential Therapeutic Agent for Osteosarcoma / 대한정형외과연구학회지

PURPOSE: The purpose of this study is to analyze the mechanism of RECK gene (a novel MMP inhibitor) in human osteosarcoma and evaluation of RECK as a prognostic factor and therapeutic target. MATERIALS AND METHODS: Osteosarcoma cell lines were established from tumor samples of 23 patients who had been treated from March 2003 to April 2004 and 4 standard cell lines (HOS, MG-63, SaOS-2, U-2OS). We isolated the RNA from 27 cell lines and evaluated the expression level of RECK gene using quantitative real time-PCR method. MMP-2 and MMP-9 expression were evaluated by gelatin zymography. Five cell lines were selected which had a statistical significance between RECK gene up-regulation and MMP expression (p=0.01). Then 5 cell lines and 3 standard cell lines were transfected by RECK gene. We compared RECK gene expression with MMP down-regulation between transfected cell lines and non-transfected cell lines. Invasion of transfected cell lines were evaluated by invasion assay using matrigel. RESULTS: RECK genes were expressed in all cell lines and 1 cell line showed especially high expression. In zymography, pro-MMP-2 was expressed in almost cell lines whereas pro-MMP-9 was rarely expressed. RECK gene expressions were increasingly high and MMP expressions were low in transfected cell lines via zymography. Transfected HOS cells decreased invasiveness in matrigel invasion assay and showed small number of migrated cells. It had a statistical significance (p<0.01). CONCLUSION: It is expected that down-regulation of MMP by RECK gene expression can be used as a biologic marker. It can be a new therapeutic strategies and valuable prognostic factors in treating osteosarcoma.