MicroRNA let-7f inhibits tumor invasion and metastasis by targeting MYH9 in human gastric cancer

Background

MicroRNAs (miRNAs) are important regulators that play key roles in tumorigenesis and tumor progression. A previous report has shown that let-7 family members can act as tumor suppressors in many cancers. Through miRNA array, we found that let-7f was downregulated in the highly metastatic potential gastric cancer cell lines GC9811-P and SGC7901-M, when compared with their parental cell lines, GC9811 and SGC7901-NM; however, the mechanism was not clear. In this study, we investigate whether let-7f acts as a tumor suppressor to inhibit invasion and metastasis in gastric cancers.

Methodology/Principal

Real-time PCR showed decreased levels of let-7f expression in metastatic gastric cancer tissues and cell lines that are potentially highly metastatic. Cell invasion and migration were significantly impaired in GC9811-P and SGC7901-M cell lines after transfection with let-7f-mimics. Nude mice with xenograft models of gastric cancer confirmed that let-7f could inhibit gastric cancer metastasis in vivo after transfection by the lentivirus pGCsil-GFP- let-7f. Luciferase reporter assays demonstrated that let-7f directly binds to the 3′UTR of MYH9, which codes for myosin IIA, and real-time PCR and Western blotting further indicated that let-7f downregulated the expression of myosin IIA at the mRNA and protein levels.

Conclusions/Significance

Our study demonstrated that overexpression of let-7f in gastric cancer could inhibit invasion and migration of gastric cancer cells through directly targeting the tumor metastasis-associated gene MYH9. These data suggest that let-7f may be a novel therapeutic candidate for gastric cancer, given its ability to reduce cell invasion and metastasis.     

用基因芯片结合荧光差异显示—PCR筛选和识别胃腺癌转移相关的基因

使用来源于同一个胃腺癌病人的原发灶RF－1（ATCC编号：CRL－1864）和转移灶RF－48细胞系（ATCC编号,CRL－1863）作为研究肿瘤转移分子机制的模型,RF－1（实验组）和RF－48（对照组）的mRNA通过逆转录方法,将Cy3和Cy5两种荧光染料分别标记到两种细胞的cDNA上,制备成cDNA探针,并与表达谱芯片（双点4096条基因）进行杂交与扫描,重复2次实验,利用计算机数据处理判断基因是否在上述两种细胞中有表达差异,共筛选出差异表达的基因共138条,其中81条在RF－48细胞中表达明显上调,57条在RF－48细胞中表达显著下调,同时也通过荧光差异显示－PCR（FDD－PCR）技术,克服了45个涉及胃腺癌转移相关基因,包括未被发现的基因3个,在两种筛选方法中都存在差异表达的基因共有7条,对部分可能与肿瘤志移机制有关的差异表达基因的作用进行了分析和讨论,基因芯片技术可高通量,大规模地研究基因表达水平,FDD－PCR技术可克隆出未发现的新基因,二者结合,初步筛选出与转移相关的基因,有助于揭示胃腺癌转移的分子机制。     

nm23-H1基因转染L9981肺癌细胞前后基因表达谱的变化

应用基因芯片检测L9981细胞转染nm23-H1基因前后细胞基因表达谱的改变.提取L9981细胞转染nm23-H1基因前后细胞的总RNA,纯化为mRNA后再转录为cDNA.cDNA经限制性内切酶Sau3AI消化后,cDNA片段分别用cy3和cy5标记,与定制的包含14000个基因芯片杂交.杂交结果经扫描和软件分析,nm23-H1基因转染L9981细胞后发现1156(8.26%,1156/14000)个基因表达上调,而642(4.59%,642/14000)个基因表达下调.涉及基因包括信号传导、癌基因与抑癌基因、转移相关基因、细胞周期与凋亡、细胞外基质与细胞骨架相关基因,以及细胞因子和转录因子等.nm23-H1基因是通过对转移相关基因的调节来发挥其抑制肺癌细胞株L9981侵袭和转移作用的.     

Genomic and proteomic expression patterns in HPV-16 E6 gene transfected stable human carcinoma cell lines

cDNA microarray and proteomics studies were performed to analyze the genomic and proteomic expression patterns in HPV-16 E6 gene transfected stable human carcinoma cell lines. Among 1024 known genes and ESTs tested by cDNA microarray, we found 50 upregulated and 35 downregulated genes in RC10.1 HPV-16 E6 transfected human colon adenocarcinoma cells compared to RKO cells, and 27 upregulated and 43 downregulated genes in A549E6 HPV-16 E6 transfected human lung adenocarcinoma cells compared to A549 cells. Employing two dimensional gel electrophoresis and MALDI-TOF-MS, the global pattern of protein expressions in RC10.1 human colon adenocarcinoma and A549E6 human lung adenocarcinoma cell lines stably expressing the HPV 16-E6 gene were compared with those of RKO and A549 cell lines to generate a differential protein expression catalog. We found 13 upregulated and 13 downregulated proteins in RC10.1 (E6-expressing RKO) cells compared to RKO cells and 12 upregulated and 14 downregulated proteins in A549E6 (E6-expressing A549) cells compared to A549 cells. The identified genes and proteins were classified into several groups according to the subcellular function. Expressing pattern of three genes and proteins (CDK5, Bak, and I-TRAF) were matched in both analyses of cDNA microarray and proteomics. These powerful approaches using cDNA microarray and proteomics could provide in-depth information on the impact of HPV-16 E6-related genes and proteins. Differential gene and protein expression patterns by transfection of HPV-16 E6 will provide the nucleus of valuable resource for investigation of the biochemical basis of cervical carcinogenesis. Further understanding of this data base may provide valuable resources for developing novel diagnostic markers and therapeutic targets of cervical cancer.     

Isolation of diallyl trisulfide inducible differentially expressed genes in human gastric cancer cells by modified cDNA representational difference analysis

Extensive epidemiologic studies indicated protective effects of consumption of garlic on reducing human gastric cancer (HGC) incidence. Diallyl trisulfide (DATS), a critical organic allyl sulfur component of garlic, was reported to have chemopreventive effects in inhibiting tumor process. We used DATS to treat HGC cell line BGC823 cells, and showed that DATS induces G1/S arrest and apoptosis in BGC823 cells demonstrated by a flow cytometric analysis. To further isolate DATS inducible differentially expressed genes in BGC823 cells, we combined a highly specific subtractive hybridization of cDNA representational difference analysis (cDNA RDA) with a sensitive bidirectional radioactive detection of mRNA differential display (mRNA DD) to develop a subtractive hybridization differential display (SHDD) method. This modified method adopted a first round of bidirectional subtractive hybridization between two sample cDNAs and a second round of bidirectional subtractive hybridization between the two resultant first-round difference products. Bidirectional subtractive hybridizations magnified the differences between the two sample cDNAs and favored isolating mRNA species with very small expression differences. We employed the SHDD method to detect DATS inducible differentially expressed genes in BGC823 cells. A total of 14 cDNA fragments (11 upregulated and 3 downregulated by DATS treatment) were isolated and confirmed by reverse Northern blot analysis. Our data show that SHDD is a powerful technique for identifying differentially expressed mRNA species between two sample cDNAs and provide useful cellular and molecular information for understanding the effects of garlic against human gastric cancer.     

Long noncoding RNA NNT-AS1 promotes gastric cancer proliferation and invasion by regulating microRNA-363 expression

Increasing studies showed that long noncoding RNAs (lncRNAs) had crucial regulatory roles in various tumors, including gastric cancer (GC). Recent studies demonstrated that lncRNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) played an important role in several tumors. However, the role and expression of NNT-AS1 in GC progression remain unknown. In our study, we indicated that NNT-AS1 expression was upregulated in GC samples compared with the nontumor tissues. We also showed that NNT-AS1 expression was upregulated in the GC cell lines. Ectopic expression of NNT-AS1 promoted GC cell line HGC-27 cell proliferation, cell cycle progression, and invasion. In addition, we showed that NNT-AS1 acted as a sponge competing endogenous RNA for microRNA-363 (miR-363), which was downregulated in the GC samples and cell lines. miR-363 expression was negatively related with NNT-AS1 expression in GC samples. Upregulated expression of miR-363 suppressed GC cell growth, cycle, and invasion. Furthermore, we reported that elevated expression of NNT-AS1 promoted GC cell proliferation, cycle, and invasion partly by suppressing miR-363 expression. These results indicated that lncRNA NNT-AS1 acted as an oncogene in the development of GC partly by inhibiting miR-363 expression.     

Proteomics-based strategy to delineate the molecular mechanisms of RhoGDI2-induced metastasis and drug resistance in gastric cancer

Rho GDP dissociation inhibitor 2 (RhoGDI2) was initially identified as a regulator of the Rho family of GTPases. Our recent works suggest that RhoGDI2 promotes tumor growth and malignant progression, as well as enhances chemoresistance in gastric cancer. Here, we delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance using two-dimensional gel electrophoresis (2-DE) on proteins derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. Differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 47 differential protein spots were identified; 33 were upregulated, and 14 were downregulated by RhoGDI2 overexpression. Upregulation of SAE1, Cathepsin D, Cofilin1, CIAPIN1, and PAK2 proteins was validated by Western blot analysis. Loss-of-function analysis using small interference RNA (siRNA) directed against candidate genes reveals the need for CIAPIN1 and PAK2 in RhoGDI2-induced cancer cell invasion and Cathepsin D and PAK2 in RhoGDI2-mediated chemoresistance in gastric cancer cells. These data extend our understanding of the genes that act downstream of RhoGDI2 during the progression of gastric cancer and the acquisition of chemoresistance.     

肿瘤相关基因表达检测寡核苷酸芯片的制备及其初步应用

为研制肿瘤相关寡核苷酸芯片,并实现其在抗肿瘤反义核酸“癌泰得”作用机理研究方面的初步应用,制备了包含近450种肿瘤相关基因特异寡核苷酸探针的寡核苷酸芯片,建立了相应的质控标准.“癌泰得”用脂质体转染HepG2肿瘤细胞,提取细胞总RNA反转录并荧光标记cDNA,用制备的寡核苷酸芯片检测肝癌细胞HepG2的肿瘤相关基因表达水平,用软件分析获得其差异基因表达谱.0.4 μmol/L的反义核酸“癌泰得”作用于HepG2细胞15 h后,MDNCF、DHS等基因mRNA表达下调,MUC2、MPP11、LAT、HRIF-B、JNK3A1等mRNA基因表达上调,初步检测到了“癌泰得”的抗肿瘤作用可能的相关基因,为进一步的分子作用机理的探讨奠定基础.结果表明,制备的肿瘤相关芯片敏感度高、特异性高、重复性均较好,可用于检测肿瘤相关基因的表达谱,为临床诊断和基础研究提供了技术平台.     

Perilaldehyde activates AMP-activated protein kinase to suppress the growth of gastric cancer via induction of autophagy

Background and Aim: Perillaldehyde (PAH), one of the major oil components in Perilla frutescens, is very critical to health maintenance, for a wide range of human chronic diseases, including cancers. AMP-activated protein kinase (AMPK) has been implicated in the activation of autophagy in distinct tissues. This study was designed to explore whether PAH prevents gastric cancer growth and to investigate the molecular mechanism. Methods and Results: In cultured mouse gastric cancer cell line MFCs and human gastric cancer cell lines GC9811-P, PAH activated AMPK by increasing the Thr172 phosphorylation and activity in a time-/concentration-dependent manner. Furthermore, incubation of MFCs with PAH also increased autophagy as determined by monodansylcadaverine (MDC) staining, which was reversed by AMPK inhibitor compound C. PAH further decreased MFCs cell survival, which was abolished by compound C or autophagy inhibitor 3-Methyladenine (3-MA). In vivo studies indicated that 4-week administration of PAH (100 mg/kg/day) suppressed the growth of gastric cancer and increased the levels of autophagy-related proteins, including beclin-1, LC3-II, cathepsin, caspase-3, p53, and cathepsin in tumors isolated from the xenograft model of gastric cancer in mice. Moreover, these anticancer effects produced by PAH were abolished by coadministration of compound C or 3-MA in vivo. Conclusions: PAH increases AMPK phosphorylation and activity to induce gastric cancer cell autophagy to inhibit the growth of gastric cancer. In perspective, therapy of PAH should be applied to treat patients with gastric cancer.     

Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury, the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS. The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorporation of different fluorescence (Cy3, Cy5) labeled dUTP as the hybridization probes. The mixed probes were hybridized to the cDNA microarray chips. The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0. Among the 14112 target genes, 293 genes were found to be significantly differentially expressed, in which 188 genes were up-regulated and 105 genes were down-regulated. Based on the analysis of biological functions of those differentially expressed genes, it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reactions, cell synthesis, metabolism, apoptosis and transportation in liver cell, which might be quite important for elucidating the regulatory network of gene expression associated with the liver damage, also important for finally discovering the pathogenic mechanisms of immunological liver damage.     

Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury,the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS.The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorpora-tion of different fluorescence(Cy3,Cy5) labeled dUTP as the hybridization probes.The mixed probes were hybridized to the cDNA microarray chips.The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0.Among the 14112 target genes,293 genes were found to be significantly differentially expressed,in which 188 genes were up-regulated and 105 genes were down-regulated.Based on the analysis of biological functions of those differentially expressed genes,it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reac-tions,cell synthesis,metabolism,apoptosis and transportation in liver cell,which might be quite im-portant for elucidating the regulatory network of gene expression associated with the liver damage,also important for finally discovering the pathogenic mechanisms of immunological liver damage.     

Profiling highly conserved microrna expression in recombinant IgG‐producing and parental Chinese hamster ovary cells

MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY? miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT‐PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44‐derived CHO lines producing a recombinant human IgG. We observed that miR‐221 and miR‐222 were significantly downregulated in all IgG‐producing cell lines when compared with parental DG44, whereas miR‐125b was significantly downregulated in one IgG‐producing line. In another IgG‐producing line, miR‐19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let‐7b and miR‐221 were significantly downregulated. In parental CHO K1, let‐7b, miR‐15b, and miR‐17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Differential expression of metastasis-associated cell surface glycoproteins and mRNA in a murine large cell lymphoma

A metastatic variant cell subline of the Abelson virus-transformed murine large lymphoma/lymphosarcoma RAW117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117-H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117-P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus-associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117-P and -H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one-half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.     

Analysis of co-expression networks for circular RNAs and mRNAs reveals that circular RNAs hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 are candidate oncogenes in gastric cancer

Accumulating evidence has suggested that circular RNAs (circRNAs) play important roles in oncogenesis and tumor progression. However, our knowledge of circRNAs in gastric cancer (GC) remains limited. To investigate circRNAs involved in GC oncogenesis, we examined differentially-expressed circRNAs and mRNAs in GC tissues and paired noncancerous mucosa tissues using circRNA and mRNA microarrays. Next, we built gene co-expression networks according to the degree of correlation to predict the critical circRNAs in GC. Through bioinformatics analysis, we observed three newly identified circRNAs that are substantially upregulated in GC: hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15. Additionally, hsa_circ_0047905 and hsa_circ_0138960 positively correlated with their parental gene mRNA. Knockdown of hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 in GC cells, resulted in downregulation of parental gene expression. Functional assays suggested that inhibition of these three circular RNAs suppresses GC cell proliferation and invasion in vitro. Those findings suggest that hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 might act as tumor promoters in the pathogenesis of gastric cancer.     

N-myc Downstream Regulated Gene 1 (NDRG1) Promotes Metastasis of Human Scirrhous Gastric Cancer Cells through Epithelial Mesenchymal Transition

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.     

Establishment and molecular characterization of decitabine‐resistant K562 cells

The clinical activity of decitabine (5‐aza‐2‐deoxycytidine, DAC), a hypomethylating agent, has been demonstrated in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. However, secondary resistance to this agent often occurs during treatment and leads to treatment failure. It is important to clarify the mechanisms underlying the resistance for improving the efficacy. In this study, by gradually increasing concentration after a continuous induction of DAC, we established the DAC‐resistant K562 cell line (K562/DAC) from its parental cell line K562. The proliferation and survival rate of K562/DAC was significantly increased, whereas the apoptosis rate was remarkably decreased than that of K562 after DAC treatment. In K562/DAC, a total of 108 genes were upregulated and 118 genes were downregulated by RNA‐Seq. In addition, we also observed aberrant expression of DDX43/H19/miR‐186 axis (increased DDX43/H19 and decreased miR‐186) in K562/DAC cells. Ectopic expression of DDX43 in parental K562 cells rendered cells resistant to the DAC. Taken together, we successfully established DAC‐resistant K562 cell line which can serve as a good model for investigating DAC resistance mechanisms, and DDX43/H19/miR‐186 may be involved in DAC resistance in K562.     

Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression.

We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.