肺癌A549细胞上皮-间质转化及其对微小RNA表达的影响

目的 探讨肺癌A549细胞上皮-间质转化(EMT)对微小RNA(miRNA)表达的影响.方法 采用不同浓度的转化生长因子β1(TGF-β1)诱导肺癌A549细胞发生EMT,应用相差显微镜观察A549细胞形态学变化,采用Western blot法检测EMT相关标记蛋白表达的变化,应用miRNA芯片检测EMT前后miRNA表达的变化,应用荧光定量逆转录聚合酶链反应(RT-PCT)验证芯片结果的可靠性.结果 肺癌A549细胞发生EMT后,细胞形态拉长,细胞间连接变得疏松.肺癌A549细胞发生EMT后,上皮标记蛋白E-钙黏附素(E-cadherin)表达降低,而间质标记波形蛋白(Vimentin)和纤维连接蛋白(Fibronectin)表达上调.miRNA芯片获得51个miRNA在诱导前后有统计学意义(P＜0.05),且表达差异在2倍以上.18个表达上调,33个表达下调,其中mir-33a和mir-193a-3p的表达经诱导后分别下调了92.8%和86.5%;荧光定量RT-PCR检测mir-33a和mir-193a-3p的表达经诱导后分别下调了73.1%和56.6%.结论 EMT可影响肺癌A549细胞中miRNA的表达变化,miRNA可能通过EMT调节肺癌侵袭转移.

Abstract：

Objective To investigate the effect of epithelial-mesenchymal transition (EMT) on the expression of microRNAs (miRNAs) in lung cancer A549 cells. Methods Transforming growth factor beta1 (TGF-β1) in different concentrations was used to induce EMT in lung cancer A549 cells. The morphological changes were observed under phase-contrnst microscope. The changes of EMT-related proteins were analyzed by Western blot. The changes of miRNAs expression after EMT were detected by microRNA (miRNA) array. Real time quantitative RT-PCR was applied to verify the reliability of miRNA array results.Results The lung cancer A549 cells became elongated and the cell-cell junction became loose after EMT.The epithelial protein marker E-cadherin was down-regulated and the mesenchymal protein markers vimentin and fibronectin up-regulated. There were 51 miRNAs showing statistically significunt changes of expression more than double (P ＜ 0. 05 ) after EMT. Among them 18 were up-regulated and 33 down-regulated. Of them, mir-33a was down-regulated by 92.8% and mir-193a-3p by 86.5%. Real time quantitative RT-PCR showed that mir-33a was down-regulated by 73.1% and mir-193a-3p by 56.6%. Conclusion Epithelial-mesenchymal trnsition has effects on the expression of miRNAs, and miRNAs may regulate the invasion and metastasis of lung cancer cells via EMT.     

Evaluate the expression of uPA, PAI-1 in human gastric cancer and its correlation with the angiogenesis by the application of tissue microarray

OBJECTIVE To investigate the expression of urokinase-type plasminogen （uPA）, its inhibitor-1 （PAI-1） mRNA and its protein in human gastric cancer and to find out the relationship among the tumor differentiation, angiogenesis, and other clinical pathologic factors. METHODS In situ hybridization （ISH） was used to get the uPA, PAI-lmRNA in 110 cases with human gastric cancer in 2-tissue microarray （TMA）. Immunohistochemical staining （S-P method） for uPA, PAI-1 protein and CD34 were performed in the 110 cases in 2 TMA. RESULTS The expression of the uPA, PAI-lmRNA and their protein happened in the cytoplasm of gastric cancer cells were induced by the poor differentiation of the GC, and the expression of uPA had an increasing trend while the expression of the PAI-1 had a decreasing trend. The microvessel density （MVD） had a positive correlation with the clinical stages and the significant relationship with the lymph node metastasis （P 〈 0.05）. The MVD in uPA positive group was significantly higher than those in uPA negative group （P 〈 0.05）. The expression of PAI-1 has no correlation neither with the clinical stages nor the lymph node metastasis. CONCLUSION The uPA play an important role in invasion and metastasis of GC through promoting angiogenesis. Interdicting the secretion and function of the uPA may allow the target therapy against the tumor invasion. As a new high-throughput technology, the tissue microarray is a valuable way to be used in clinical treatment.     

Expression of TGF-β1, Snail, E-cadherin and N-cadherin in Gastric Cancer and Its Significance

OBJECTIVE To investigate the expression of TGF-β1,Snail,E-cad-herin and N-cadherin in gastric cancer(GC),and to examine its relationship to malignant features of the tumors. METHODS The expression of TGF-β1,Snail,E-cadherin and N-cadherin proteins was detected in GC and adjacent tissues by immunohistochemical staining,and compared with the clinico-pathological data. RESULTS Positive rates of expression for TGF-β1,Snail,E-cadherin and N-cadherin were 63.5%,83.3%,37.5%and 44.8%in GC,and 28.8%, 41.3%,100%,11.3%in adjacent tissues,respectively.The expression of all four proteins showed a significant difference between the GCs and adjacent tissues(P<0.05).The positive rate of TGF-β1,Snail and N-cadherin,or the negative rate of E-cadherin expression was significantly related to the differentiated degree,histological type,invasion and metastasis of GC.In addition,the expression of N-cadherin was positively related to that of TGF-β1, but negatively related to that of E-cadherin.There was negative correlation between expression of E-cadherin and TGF-β1 and Snail in GC(P<0.05). CONCLUSION The over-expression of TGF-β1 and Snail and decreased expression of E-cadherin and the abnormal expression of N-cad-herin were involved in the process of invasion and metastasis of GC.The data showed that E-cadherin might switch to N-cadherin.TGF-β1 and Snail might play a fundamental role in the process.     

Antisense Oligonucleotide Targeting TGF-β1 Abrogates Tumorigenicity of Rhabdomyosarcoma in vivo

OBJECTIVE Over-expression of transforming growth factor β1 (TGF-β1) has been observed in many advanced cancers.The present study was aimed at developing potential antisense oligonucleotides (ASONs) to repress TGF-β1 expression in rhabdomyosarcoma (RMS) RD cells, and to examine their effect on tumorigenicity of RD cells in vivo.METHODS ASONs targeting the region surrounding the start codon of TGF-β1 were synthesized and transferred into cells in the form of complexes with Lipofectamine 2000. The TGF-β1 protein was determined by immunofluorescence and ELISA.The cell viability and cell cycle were examined by MTT and flow cytometry. The RD cells, with or without TGF-β1ASON, in 50 μl of serum-free EMDM medium were injected subcutaneously into the right flank of nude mice. The tumors were then measured and weighed.RESULTS The ASON sequence targeting the first start site at bases 841-855 of the human TGF-β1 gene had the greatest effect on attenuating the expression of TGF-β1 (P<0.05). The ASONs induced a decrease in OD values after 6 d (P<0.05). Analysis of the cell cycle revealed that the ASON induced a significant decrease in cells in the S phase and an increase in cells in the G1 phase (P<0.05). In the nude mice model, the mean tumor volume, after 2 weeks of treatment with Lipofectamine or ASON,decreased to 88.5% or 55% respectively, compared to the control tumor size, resulting in a significant difference (P<0.01).CONCLUSION The sequence of the ASON, which targeted the start condon at the bases 841-855 of the human TGF-β1 gene, was demonstrated to be a useful agent for studying the regulation of TGF-β1 over-expression in RD cells, and has important therapeutic potential for suppressing the tumorigenicity of human RMS in vivo.     

MTA1基因表达与人骨肉瘤细胞浸润和转移的关系

Objective： To compare the expression level of metastasis associated-1 （MTA1） in the higher and lower metastasis sublines of human osteosarcoma cells （MG63）, and to investigate the relationship between the expression level of MTA1-EGFP and in vitro invasion and metastasis of human osteosarcoma cells. Methods： The expression level of MTA1 in two sublines of MG63 cells was detected by semi-quantitative RT-PCR, and cell invasion assay and cell proliferation assay were used to evaluate the invasive capacity in vitro in two sublines. The lower metastasis line of MG-63 cells were transfected with MTA1-EGFP full-length cDNA expression plasmid by lipofectamine. The changes of the MTA1-EGFP expression and in vitro invasion potential were measured after transfection. Results： M8 subline expressed significantly higher level of MTA1 than that of M6 subline by RT-PCR. The invasive potentials of low metastasis MG63 cell line were increased after MTA1 gene transfection. Conclusion： There may be a relationship between MTA1 and invasive potentials of human osteosarcoma cells, and MTA1 may play a role in the molecular mechanism of tumor metastases and be a potential target for gene therapy of osteosarcoma. Further studies of MTA1 in human ostersarcoma cell metastasis are needed.     

Biological Significance and the Related Molecular Mechanism of Ets1 mRNA Expression in Lung Cancer by Tissue Microarray(TMA)

Objective: To investigate the expressions and molecular mechanism of Ets-1 mRNA, and TGFβ1 and c-Met proteins in the pathogenesis, progression of lung cancer by tissue microarray (TMA) method. Methods: The expressions of Ets-1 mRNA, and TGFβ1 and c-Met proteins were detected in 89 primary lung cancers, 12 lung cancer with lymph-node metastasis and 12 precancerous lesions by FISH(fluorescence in situ hybridization) and immunohistochemical method, and 10 normal lung tissues were used as controls. Results: The expressions of Ets-1 mRNA, and TGFβ1 and c-Met proteins were significantly higher in 89 primary lung cancer than in the control group (P<0.05). The expressions of Ets-1 mRNA, and TGFβ1 and c-Met proteins were related to lymph node metastasis and clinical stages. There was a positive correlation between the Ets-1 mRNA expression and TGFβ1 and c-Met proteins (P<0.05). Conclusion: Ets-1 mRNA, TGFβ1 and c-Met proteins may be related to the pathogenesis, progression and malignant behavior of lung cancer. They may play an important role in prognosis assessment of lung cancer.     

Integrinβ1 asODN-inhibition of Cell Attachment and Invasion of the Human Gastric Cancer Cell Line SGC7901

OBJECTIVE To study the inhibition of adhesion and invasion of SGC7901cells into the ECM by integrinβ1 antisense oligodeoxynucleotide asODN.METHODS asODN and control ODN were transfected into SGC7901 cells using liposomes as vectors. The distribution of the ODN was followed by immunochemistry and changes in the expression of integrinβ1 mRNA and protein were determined by RT-PCR and FCM, respectively. The adhesion and invasion into the ECM were measured by the MTT and Boyden chamber methods, respectively.RESULTS Integrinβ1 asODN which was transfected into SGC7901 cells distributed evenly in the cytoplasm and nucleus. PCR and FCM revealed a weakened band at 489bp and a left-shift curve, respectively. Adhesion and invasion assays showed decreased activity with an inhibition rate of 54% and 76%. The extent of decrease induced by integrinβ1 asODN was larger than that caused by random control ODN (P＜0.001).CONCLUSION Transfection of integrinβ1 asODN into SGC7901 cells induced a decrease in the expression of integrinβ1 mRNA and protein,resulting in a decrease in adhesion and invasion into the ECM, with a greater effect than random control ODN.     

MicroRNAs:regulators of cancer metastasis and epithelial-mesenchymal transition (EMT)

Tumor metastasis is the main cause of death in patients with solid tumors. The epithelial-mesenchymal transition(EMT) process, in which epithelial cells are converted into mesenchymal cells, is frequently activated during cancer invasion and metastasis. MicroRNAs(miRNAs) are small, non-coding RNAs that provide widespread expressional control by repressing mRNA translation and inducing mRNA degradation. The fundamental roles of miRNAs in tumor growth and metastasis have been increasingly well recognized. A growing number of miRNAs are reported to regulate tumor invasion/metastasis through EMT-related and/or non-EMT–related mechanisms. In this review, we discuss the functional role and molecular mechanism of miRNAs in regulating cancer metastasis and EMT.     

Analysis of miR-205 and miR-155 expression in the blood of breast cancer patients

The purpose of this study was to identify and validate circulating microRNAs (miRNAs) in human plasma for use as breast cancer (BC) biomarkers and to analyze their relationship to clinicopathologic features and its preliminary biological function. Genome-wide expression profiling of miRNAs in BC was investigated by microarray analysis. miR-155 was up-regulated greater than two-fold in BC compared with Normal Adjacent Tissue (NAT), whereas let-7b, miR-381, miR-10b, miR-125a-5p, miR-335, miR-205 and miR-145 were down- regulated greater than two-fold. Our hypothesis was that circulating miRNAs are also present and differentially expressed in the serum of BC patients compared to controls. Using real-time PCR (RT-PCR), we analyzed miR-205 and miR-155 in archived serum from 30 participants, 20 with breast cancer and 10 healthy people. miR-205 was down-regulated in BC patient serum while miR-155 was up-regulated. Furthermore, we analyzed the relationship between the expression levels of these two miRNAs and the clinicopathologic parameters of BC patients. High expression of miR155 was associated with clinical stage, molecular type, Ki-67 and p53 in BC patients (P<0.05). By contrast, we found no significant correlation between miR-205 and BC patient clinicopathologic parameters. Functional analysis showed that ectopic expression of miR-205 significantly inhibits cell proliferation and promotes apoptosis. miR-205 was down-regulated and miR-155 was up-regulated in BC patient serum. miR-155 was positive correlated with clinical stage and ki-67 and negatively correlated with p53 status.     

Differential expression of serum miR-126,miR-141 and miR-21 as novel biomarkers for early detection of liver metastasis in colorectal cancer简

Objective：MicroRNAs （miRNAs） have potential as diagnostic biomarkers in cancer.Evaluation of the association between miRNA expression patterns and early detection of liver metastasis in colorectal cancer （CRC） has not been reported.Methods：We investigated the expression of metastasis-associated miRs-31,335,206,141,126,200b,200c,21,Let7a,Let7b and Let7c in localized,liver-metastatic and other organ-metastatic CRC （OM-CRC）.Expressions of target miRNAs in serum were evaluated in 116 consecutive localized CRC （L-CRC）,72 synchronous liver-metastatic CRC （SLM-CRC） and 36 other OM-CRC by quantitative real-time PCR.Results：Seven of 11 tested miRNAs could be detected from serum.Four miRNAs,miR-126,Let-7a,miR141 and miR-21 were identified as metastasis-associated miRNAs.Compared with L-CRC,significant upregulated expression was observed for miR-141 and miR-21 in SLM-CRC and OM-CRC,down-regulated expression was observed for miR-126 in SLM-CRC and OM-CRC,and up-regulated expression of Let-7a in OM-CRC.The receiver operating characteristic （ROC） curve showed serum miR-126 had a cut-off [log10 relative quantity （log10RQ）=--0.2005] with 77.78％ sensitivity and 68.97％ specificity with an area under curve （AUC） of 0.7564,miR-141 had a cut-off （1og10RQ=-0.2285） with 86.11％ sensitivity and 76.11％ specificity with an AUC of 0.8279,and miR-21 had a cut-off （log10RQ=-0.1310） with 73.61％ sensitivity and 66.38％ specificity with an AUC of 0.7479.Conclusions：We identified liver metastasis-associated miRNAs,suggesting serum miR-126,miR-141 and miR-21 may be novel biomarkers for clinical diagnosis of early stage liver-metastatic CRC.     

Overexpression of HOXB9 promotes metastasis and indicates poor prognosis in colon cancer简

Objective：Homeobox B9 （HOXB9） is proposed to be involved in tumor angiogenesis and metastasis.We investigated the role of HOXB9 in the progression of colon cancer.Methods：HOXB9 expression was investigated by immunohistochemically and Western blotting in 128 colon cancer patients and the results were analyzed statistically associated with clinicopathological data and survival of the patients.The effect of HOXB9 on cell invasion and metastases abilities were analyzed in vitro and in vivo.Results：HOXB9 is overexpressed in colon cancer tissues and significandy correlated with metastasis and poor survival of patients （P＜0.05,respectively）.Additionally,high levels of expression of HOXB9 were observed in metastatic lymph nodes.The down-regulation of HOXB9 expression can inhibit the migration and invasive ability of colon cancer cells,while exogenous expression of HOXB9 in colon cancer cells enhanced cell migration and invasiveness.Moreover,stable knockdown of HOXB9 reduced the liver and lung metastasis of colon cancer in vivo.Conclusions：HOXB9 may play an important role in the invasion and metastasis of colon cancer cells and may be a useful biomarker for metastasis and prognostic of colon cancer.     

Expression of miR-125b in the new, highly invasive glioma stem cell and progenitor cell line SU3

MicroRNA (miR)-125b has been shown to play a potential role in the development of glioma stem cells.However,the relationship between miRNA and glioma stem cells is still elusive.This study was designed to elucidate this potential relationship.We established a highly invasive glioma stem cell and progenitor (GSCP) cell line SU3.SU3 cell suspensions were injected into nude mice brains in situ,and the invasiveness of graft tumors was analyzed using hematoxylin and eosin staining as well as immunohistochemistry.Real-time polymerase chain reaction (PCR) was used to measure the expression levels of miR-125b in SU3 and other cells.In vitro,SU3 cells expressed CD133 and nestin as well as differentiation markers glial fibrillary acidic protein (GFAP) and β-tubulin III,which were consistent with the characteristics of glioma stem cells.Scratch assays indicated that the migration ability of SU3 cells was stronger than that of U251 stem cells (U251s).In vivo,SU3 cells invaded into each part of the mouse brain from the caudate nucleus in a diffuse pattern and highly expressed invasive and proliferative cell markers matrix metalloprotease 2 (MMP2),MMP9,and Ki-67.Real-time PCR results revealed that the levels of miR-125b and MMP9 were significantly higher in SU3 and SU2,also a highly invasive GSCP cell line we established before,than in U251s.High expression of miR-125b both in newly established GSCPs,SU3,and long-term cultured GSCPs,SU2 suggests that miR-125b exhibits oncogene-like behavior.This behavior should be considered in further studies of miR-125b in cancer stem cells.Furthermore,MMP9,which plays a role in cancer stem cell invasion,may be a target gene of miR-125b.     

The expression of TGF-β1, ADAM12 and HB-EGF in primary hepatic carcinoma

Objective: To detect the expression and location of TGF-β1, ADAM12 and HB-EGF in primary hepatic carcinoma and study their effect on the growth and metastasis of hepatoma carcinoma cell. Methods: TGF-β1, ADAM12 and HB-EGF were detected by RT-PCR and immunohistochemistry in 30 cases of hepatic carcinoma tissues, 30 cases of adjacent carci-noma tissues and 5 cases of normal hepatic tissues. Results: RT-PCR analyses showed that the mRNA expression of TGF-β1, ADAM12 and HB-EGF were markedly increased in each hepatic carcinoma tissue compared with its adjacent tissue (P < 0.01), but no signal was detected in normal hepatic tissue, tmmunohistochemistry showed the same outcome on the expression of above three factors in hepatic tissues as RT-PCR. Proteins location analyses showed the proteins of TGF-β1, ADAM12 and HB-EGF all distributed in the stroma of hepatic carcinoma tissues. The positive correlation was found between TGF-β1 and ADAM12 (r=0.6137, P < 0.05), as well as ADAM12 and HB-EGF (r=0.5763, P < 0.05). The protein expression of TGF-β1, ADAM12 and HB-EGF were correlated with the size of tumors, degree of differentiation of hepatoma carcinoma cells, portal vein thrombus and the metastasis of absorbent glands, especially with hepatic cirrhosis caused by hepatitis B virus. Conclu-sion: TGF-β1, ADAM12 and HB-EGF possibly play an important role in the process of growth, invasion and metastasis of hepatoma carcinoma cell, meanwhile, the above three factors may collectively participate in the transition from hepatic cirrhosis caused by hepatitis B virus to hepatocellular carcinoma.     

Expression of 6 microRNAs in prostate cancer and its significance

OBJECTIVE Numerous microRNAs （miRNAs） are deregulated in human cancers. The experimental evidence supports that miRNAs plays a role in the initiation and progression of human malignancies.The present study was undertaken to evaluate the differential expression of 6 miRNAs as biomarker for early detection of prostate cancer, and then to determine whether the expression profiling of these miRNAs could predict the prognosis of prostate cancer. METHODS The expression profilings of these 6 miRNAs were investigated using the method of locked nucleic acid （LNA）- modified oligonucleotide in situ hybridization （ISH）. And the technology of tissue microarray （TMA） was employed using the formalin-fixed, paraffin-embedd （FFPE） specimens taken from 52 patients with prostate carcinoma （PCa） and 38 patients with benign prostatic hyperplasia （BPH）. RESULTS The rates of positive expression for 6 miRNAs （miR- 15b, miR-16, let-7g, miR- 96,miR-182 and miR-183） were 26.92%, 15.38%, 15.38%, 67.31%, 61.54% and 71.15% in the specimens of prostate cancer, and 57.89%, 76.32%, 68.42%, 44.74%, 31.58%, 47.37% in the tissues of benign prostatic hyperplasia, respectively. The expressions of all 6 miRNAs between the prostate cancer and benign prostatic hyperplasia tissues were significantly different （P 〈 0.05）. The positive rate of these 6 miRNAs was significantly related to the Gleason Grading of prostate cancer （P 〈 0.01）. There was no significant correlation between the expression of these miRNAs and age and the concentration of serum PSA of the patient （P 〉 0.05）. We also found that the expression of miR-15b, miR-96 and miR-182 correlated with clinical stages of tumor （P 〈 0.05）. The expression of miR-96 correlated with lobus prostatae of tumor invasion （P 〈 0.01）, but the expressions of the remaining five miRNAs were not correlated with that （P 〉 0.05）. In addition, the expression of miR-15b was negatively related to that of miR-96, miR-182 and miR-183, respectively （P 〈 0.01, r 〈 0.00）.There was a positive correlation among the expressions of miR-96, miR-182 and miR-183 in prostate cancer （P 〈 0.01, r 〉 0.00）. The expression of miR-16 was positively related to that of miR-let-7g （P 〈 0.01, r 〉 0.00）. CONCLUSION The results suggest that miRNA expression profiling could have relevance to the biological and clinical behavior of prostate cancer, and they might be important biomarkers for early detection and prognostic assessment of prostate cancer.