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BACKGROUND: Breast cancer, the most common cancer in women worldwide, causes the vast majority of cancer-related deaths. Undoubtedly, tumor metastasis and recurrence are responsible for more than 90 percent of these deaths. MicroRNAs are endogenous noncoding RNAs that have been integrated into almost all the physiological and pathological processes, including metastasis. In the present study, the role of miR-128 in breast cancer was investigated. RESULTS: Compared to the corresponding adjacent normal tissue, the expression of miR-128 was significantly suppressed in human breast cancer specimens. More importantly, its expression level was reversely correlated to histological grade of the cancer. Ectopic expression of miR-128 in the aggressive breast cancer cell line MDA-MB-231 could inhibit cell motility and invasive capacity remarkably. Afterwards, Metadherin (MTDH), also known as AEG-1 (Astrocyte Elevated Gene 1) and Lyric that implicated in various aspects of cancer progression and metastasis, was further identified as a direct target gene of miR-128 and its expression level was up-regulated in clinical samples as expected. Moreover, knockdown of MTDH in MDA-MB-231 cells obviously impaired the migration and invasion capabilities, whereas re-expression of MTDH abrogated the suppressive effect caused by miR-128. CONCLUSIONS: Overall, these findings demonstrate that miR-128 could serve as a novel biomarker for breast cancer metastasis and a potent target for treatment in the future.
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Humans , Female , Breast Neoplasms/genetics , MicroRNAs/physiology , MicroRNAs/genetics , Neoplasm Invasiveness/genetics , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Gene Expression Regulation, Neoplastic , RNA-Binding Proteins , Cell Line, Tumor , Membrane Proteins , Neoplasm Recurrence, LocalABSTRACT
OBJECTIVE To detect the reversal effect of Guizhi Fuling Wan on cisplatin-resistant ovarian cancer SKOV3/DDP cells and its relationship with protein expression of phosphatase and tensin homolog (PTEN) and metadherin (MTDH). METHODS Guizhi Fuling Wan (GFW) concentrated solution was prepared according to the Chinese Pharmacopoeia 2015 edition, Wistar rats were given GFW viagavage at 4 g·kg-1·d-1,8 g·kg-1·d-1,16 g·kg-1·d-1,or given saline as blank control for 5 days.Blood samples were taken and the corresponding drug-containing low-dose sera, medium-dose sear, high-dose sera and blank sera were prepared.The XCELLigence RTCA S16 real-time label-free cell analyzer was used to detect the reversal effect by the sera combined with cisplatin or paclitaxel in SKOV3/DDP cells. Annexin V-FITC and PI double-staining were used to detect the apoptosis-inducing effect of the sera in the cells. RT-qPCR and western blot were used to detect the mRNA and protein expression of PTEN and MTDH after the cells treated with the sera. RESULTS The inhibition rate of low-dose sera against SKOV3/DDP cells was less than 5%.After the low-dose sera combined with cisplatin or pacli-taxel, the IC50 of SKOV3/DDP cells against cisplatin and paclitaxel decreased by 3.01 and 1.79-fold, respectively.The total apoptosis rates induced by the low-dose sera,medium-dose sear,high-dose sera and blank sera in SKOV3/DDP cells were 11.08±0.13,19.42±0.30,24.23±0.31,and 3.21±0.24,respec-tively; there was a significant difference between the groups (P<0.01). RT-qPCR results showed that, compared with the blank serum, the sera can up-regulate the expression of PTEN mRNA and down-regulate the expression of MTDH mRNA in a dose-dependent manner. Western blot results showed that the induction effect to PTEN protein and the inhibition effect to MTDH protein by the sera were gradually enhanced with thesera dose increasement. CONCLUSION The resistance reversal effect of Guizhi Fuling Wan on ovarian cancer SKOV3/DDP cells may be related to the inhibition of MTDH, up-regulation of PTEN and induction of apoptosis, providing with an experiment basis for the applica-tion of Guizhi Fuling Wan as a reversal agent for chemotherapy resistance of ovarian cancer.
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@#AIM:To investigate the expression of MTDH and β-catenin in ocular adnexal lymphoma lesions and its clinical significance. <p>METHODS: Resected specimens were collected from patients suffering from B-cell non-Hodgkin lymphoma(NHL)(<i>n</i>=40)and lymphadenosis(<i>n</i>=20)of ocular adnexal in Affiliated Hospital of Qingdao University from June 1995 to December 2015. Lymphadenosis of ocular adnexal was acted as the control group. PCR and immunohistochemistry were employed to examine the MTDH and β-catenin mRNA and protein expression respectively. The relationship between the MTDH and β-catenin protein expression level and the clinical pathological characteristics were analyzed. <p>RESULTS:The expression of mRNA and protein of MTDH and β-catenin in ocular adnexal lymphoma lesions were higher than that in control group(<i>P</i><0.05). The expression of MTDH and β-catenin proteins were related to pathologic type of tumors, but not related to age, gender or pathogenic site. With the increase of pathologic grade, MTDH and β-catenin labeling frequency increased gradually. And there was a positive correlation between MTDH and β-catenin(<i>r</i>=0.389, <i>P=</i>0.036). <p>CONCLUSION: Over expression of MTDH and β-catenin may play a significant role in the ocular adnexal lymphoma. The expression of MTDH and β-catenin has a positive relationship.
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Objective This study aimed at observing expression and clinical significance of metadherin in gastric adenocarcinoma and exploring the potentially regulating mechanism of metadherin in invation and migration of gastric cancer.Methods Expressions of metadherin and E-cadherin in primay lesion of gastric cancer were detected by immunohistochemistry and their correlation to clinicopathologic characteristics and prognosis were analyzed by Chi-square tests.Transwell assay and wound healing assay were applied for the ability of invasion and metastasis in gastric cancer cells.Then,the down-regulatied metadherin expression in MKN45 cells by RNA interference (siRNA) was carried out and furthermore,the regulation role of metadherin in epithelial-mesenchymal transition was analyzed also in invasion and migration of gastric cancer cells.Results The positive expression of metadherin was correlated to invading depth (P =0.029),lymph node metastasis (P =0.001),TNM stage (P =0.014) and inhibiting E-cadherin expression (P =0.001).The patients with positive metabherin shared poorer prognosis.Furthermore,the down-regulated metabherin in MKN45 cells would result in the increasing expression of E-cadherin,as well as decreasing expression of N-eadherin,Slug and Snail.At the same time,the abilities of invasion (P =0.027) and migration (P =0.008) of MKN45 cells was decreased.Conclusion metabherin induces EMT in metastasis of gastric adenocarcinoma via activating either Slug or Snail but not twist,which would result in the poorer prognosis.
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Objective To investigate the effects of RNA interference technique silencing MTDH on proliferation,metastasis and invasion of human breast cancer cell MDA-MB-231.Methods The MTDH miRNA was transfected into Human breast cancer cell MDA-MB-231 by means of lipofectamine 2000.The silencing efficiency of MTDH was examined by RT-PCR and western blot.The influence of MDA-MB-231 cell proliferation,metastasis and invasion ability from inhibition MTDH gene was conducted by using MTT assay,scratch experiments,and Transwell assay.Results MTDH miRNA effectively inhibited MTDH mRNA and protein levels.The best inhibition rate were 79.41％ and 80.56％ (P ＜ 0.05).With miRNA interferring cells,the ability of cells proliferation was decreased (P ＜ 0.05),and cells invasive and metastasis were depressed (P ＜ 0.05).Conclusion MTDH expression is obviously suppressed after transfected by MTDH miRNA in MDA-MB-231 cells.The inhibition of MTDH expression from microRNA leads to significant suppression of proliferation,invasion and metastasis of breast cancer cell MDA-MB-231.
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Objective:To study MTDH and PDCD1O level in colorectal cancer and its clinical significance.Methods:Between December 2014 and December 2015,55 cases of colorectal cancer and 55 intestinal polyps were selected as the research object.Real-time fluorescence quantitative technology(RT-PCR) was used to detect MTDH and PDCD10.Results:By RT-PCR detection,Colorectal cancer tissue MTDH,PDCD10 mRNA was higher than tissue adjacent to carcinoma,colon polyps group,and the difference was statistically significant (P＜0.05),and MTDH,PDCD10 mRNA level comparison of tissue adjacent to carcinoma,intestinal polyp tissue,there was no statistically significant difference (P＞0.05).MTDH,PDCD10 mRNA level of colorectal cancer tissues showed no significant difference between the patients with different age,gender (P＞0.05),and with the increase of patients with malignant degree,MTDH,PDCD10 mRNA level was rising,MTDH,PDCD10 expression in colorectal cancer tissues was associated with liver metastasis,TNM staging,lymph node metastasis.MTDH,PDCD10 expression in the organization of Ⅲ-Ⅳ,with liver metastasis and lymph node metastasis of colorectal cancer patients,was higher than Ⅰ-Ⅱ,no liver metastasis and lymph node metastasis of colorectal cancer patients (P＜0.05).MTDH expression in colorectal cancer tissues positively correlated with the expression of PDCD10 (r=0.409,P=0.409).Conclusions:Expressions of MTDH,PDCD10 in colorectal cancer tissues were higher than normal tissue and intestinal polyp tissues.MTDH,PDCD10 of colorectal cancer tissues associated with TNM stages,differentiation degree,distant metastasis,liver metastasis.
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Objective To explore the relationship between gene MTDH expression and its role in promo-ting gastric carcinoma metastasis .Methods We collected clinical specimens and cultured gastric carcinoma cell lines.By Western blotting and Real -time PCR methods,protein and mRNA levels in tissues and MTDH relation-ship with EMT were detected .Results There was 86%of patients who expressed MTDH positively and 13%of normal gastric mucosa was positive expression .The results showed that the expressive level of MTDH gene in gas-tric carcinoma was higher than in the normal tissues .The expression of MTDH was correlated with TNM stage、mi-crovascular invasion、recurrence and metastasis .The expressive level of MTDH was correlated with two epithelial mesenchymal transition markers ( E-cadherin and N-cadherin ) .Conclusion MTDH may promote gastric car-cinoma metastasis through the induction of EMT process and may be a candidate biomarker for therapeutic target .
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Objective:To investigate the expression and clinical significance of microRNA-30a in renal cell carcinoma (RCC). Methods:The miRNA-30a expression in renal tissues and cell lines was detected using quantitative real-time polymerase chain reac-tion (qRT-PCR), and the relationship between miRNA-30a expression and the clinicopathological features of RCC was analyzed. The effect of miRNA-30a on cancer cells was evaluated using methyl thiazolyl tetrazolium (MTT) assay. In addition, a bioinformatics algo-rithm was adopted to predict the potential targets of miRNA-30a. Results:miRNA-30a was downregulated both in RCC tissues and cell lines. Patients with higher miRNA-30a expression exhibited better prognosis than those with lower miRNA-30a expression. Bioin-formatics algorithm and qRT-PCR both indicated that metadherin (MTDH) was a target of miRNA-30a. Moreover, MTT results showed that miRNA-30a could inhibit cell proliferation. Conclusion:miRNA-30a was inhibited in renal cancer, and low miRNA-30a expres-sion was associated with poor prognosis. In addition, miRNA-30a could inhibit the growth of cells through MTDH.
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PURPOSE: Astrocyte elevated gene-1 (AEG-1) plays important roles in tumorigenesis such as proliferation, invasion, metastasis, angiogenesis, and chemoresistance. We examined the expression of AEG-1 in patients with hepatocellular carcinoma (HCC). METHODS: Eighty-five samples were collected from patients with HCC who underwent surgery and were histopathologically confirmed to have HCC. Two independent pathologists, experienced in evaluating immunohistochemistry and blinded to the clinical outcomes of the patients, reviewed all samples. They determined AEG-1 expression semiquantitatively by assessing the percentage of positively stained immunoreactive cells and staining intensity. Clinicopathological data were analyzed in association with prognosis. RESULTS: The association was estimated by univariate and multivariate analyses with Cox regression. Tumor size (hazard ratio [HR], 2.285; 95% confidence interval [CI], 1.175-4.447; P = 0.015), microvascular invasion (HR, 6.754; 95% CI, 1.631-27.965; P = 0.008), and AEG-1 expression (HR, 4.756; 95% CI, 1.697-13.329; P = 0.003) were independent prognostic factors for overall survival. Those for disease-free survival rate were tumor size (HR, 2.245; 95% CI, 1.282-3.933; P = 0.005) and AEG-1 expression (HR, 1.916; 95% CI, 1.035-3.545; P = 0.038). The cumulative 5-year survival and recurrence rates were 89.2% and 50.0% in the low-expressing group and 24.5% and 82.4% in the high-expressing group, respectively. CONCLUSION: The results suggest that AEG-1 overexpression could serve as a valuable prognostic marker in patients with HCC.
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Humans , Astrocytes , Carcinogenesis , Carcinoma, Hepatocellular , Disease-Free Survival , Immunohistochemistry , Multivariate Analysis , Neoplasm Metastasis , Prognosis , RecurrenceABSTRACT
Background and purpose:miR-22 has been reported to be down-regulated in gastric cancer, lung cancer, colorectal cancer, and breast cancer. However, its expression in glioma was still poorly known. This study aimed to explicit whether miR-22 suppresses cell proliferation by targeting MTDH, thus to reveal molecular mechanism that miR-22 functions as a tumor suppressor in glioma. Methods:Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted for detecting the expression of miR-22 in gliomas and normal brain tissues. MTDH 3’UTR-luciferase vector was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-22 on luciferase activity. U251 cells were transfected with miR-22 mimics, and MTDH siRNA as for postive control, then Western blot was performed to detect the expressions of MTDH protein. The proliferation ability of U251 cells was evaluated by MTT assay. Results:miR-22 was down-regulated in glioma tissues. Glioma patients with relatively high expression of miR-22 showed lower mortality compared with low expression of miR-22 by using Kaplan-Meier survival curves. We demonstrated miR-22 could bind to the 3’ untranslated region (UTR) of MTDH and inhibited the luciferase activity. Western blot showed that the expression of MTDH protein was inhibited by restored miR-22 or siR MTDH in U251 cells. Overexpression of miR-22 or siR MTDH inhibited the proliferation of U251 cells. Conclusion:miR-22 suppresses cell proliferation by targeting MTDH in glioma.
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Objective To investigate if down regulation of MTDH could inhibit proliferation and induce apoptosis in breast cancer SK-BR-3 cells.Methods RNA interference was employed to reduce MTDH expression in human breast cancer SK-BR-3 cells.Western blot assay was applied to measure the down regulation of MTDH.MTT assay was performed to assess the proliferation of SK-BR-3 cell.Flow cytometry was employed to detect cell cycle and apoptosis.Western blot assay was applied to detect the molecular alterations that was associated with cell proliferation,cell cycle and apoptosis.Results MTDH down regulation significantly inhibited cell proliferation.48 hours and 72 hours after trasnfection,the absorbance value(A value)in blank control,negative control and treatment group was (2.0 ± 0.1) vs (1.9 ± 0.3) vs (0.9 ± 0.1) (P =0.02) and (2.7 ± 0.2) vs (2.5 ± 0.4) vs (1.3 ± 0.2) (P =0.008).MTDH down regulation resulted in accumulation of the G0/G1 phase cells and reduction of S and G2/M phase cells.Moreover,MTDH down regulation significantly induced cell apoptosis.The cell apoptosis rate in blank control,negative control and trial group was (1.3 ± 0.2) ％,(1.4 ± 0.3) ％ and (19.6 ± 2.7) ％ (P =0.012).MTDH down regulation resulted in a decreased expression of cyclinD1 and Bcl-2,an increased expression of P21 and the activation of caspase-3.Conclusions Reduced MTDH expression in SKBR-3 cells can inhibit proliferation and induce apoptosis,which may be associated with decreased expression of cyclinD1 and Bcl-2,an increased expression of P21 and the activation of caspase-3.
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Objective To establish a quantitative SYBR GreenⅠreal-time PCR method for detection of Metadherin (MTDH) gene expression level in breast cancer, and to investigate the relationship between MTDH mRNA expression level and the clinicopathological characters. Methods Real-time PCR was employed to determine the expression level of MTDH mRNA in peripheral blood of 80 specimens of breast cancer patients and normal females. Results In the 80 specimens, MTDH mRNA was positively expressed in the peripheral blood of 61 cases of breast cancer patients while negatively expressed in the 19 cases of normal females. Among the 61 breast cancer patients, MTDH mRNA showed high expression in 34 cases, accounting for 55.7%, and showed low expression in 27 cases, accounting for 44.3%. Both of the differences in expression rate has statistic significance (Ratio = 2.02 ± 0.81, P<0.05). MTDH expression relative to GAPDH expression in the peripheral blood of breast cancer patients was 1.15 ± 0.36. MTDH mRNA expression has no connection with age, estrogen and progesterone receptors , as well as HER-2.(P＞0.05). There is statistical difference for MTDH mRNA expression level between the group with lymph node metastasis and the group without lymph node metastasis (Ratio=2.02 ± 0.81,P<0.05). MTDH mRNA expression level changed between clinicopathological stage I, II and III, IV. Conclusion The established SYBR Green Ⅰ quantitative real-time PCR method can successfully detect the expression level of MTDH mRNA, which may be closely related to the occurrence and development of breast cancer.