LncRNA LY6E-DT and its encoded metastatic-related protein play oncogenic roles via different pathways and promote breast cancer progression.

Abnormal long noncoding RNA (lncRNA) expression plays an important role in tumor invasion and metastasis. Here, we show that lncRNA LY6E divergent transcript (LY6E-DT) levels are increased in breast cancer (BC) tissues. Transcription factor SP3 binds directly to the LY6E-DT promoter, activating its transcription. Moreover, LY6E-DT N6-methyladenosine modification by methyltransferase-like protein 14 (METTL14) promotes its expression, dependent on the "reader" insulin-like growth factor 2 mRNA binding protein 1(IGF2BP1)-dependent pathway. Notably, we discovered that the lncRNA LY6E-DT encodes a conserved 153-aa protein, "Metastatic-Related Protein" (MRP). Both LY6E-DT and MRP promote BC invasion and metastasis, and MRP expression could distinguish BC patients with lymph node metastasis from those without. Mechanistically, MRP binds heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC), enhancing the interaction between HNRNPC and epidermal growth factor receptor (EGFR) mRNA, increasing EGFR mRNA stability and protein expression and subsequently activating the phosphatidylinositol 3­kinase/protein kinase B signaling (PI3K) pathway. LncRNA LY6E-DT promotes the interaction between Y box binding protein 1 (YBX1) and importin α1 and increases YBX1 protein entry into the nucleus, where it transcriptionally activates zinc finger E-box-binding homeobox 1(ZEB1). Our findings uncover a novel regulatory mechanism underlying BC invasion orchestrated by LY6E-DT and its encoded MRP.

HRCT1, negatively regulated by miR-124-3p, promotes tumor metastasis and the growth of gastric cancer by activating the ERBB2-MAPK pathway.

BACKGROUND: Gastric cancer is the fourth leading cause of cancer-related deaths worldwide. And patient outcomes are poor due to tumor relapse and metastasis. To develop new therapeutic strategies, it is of great importance to explore the mechanism underlying the progression of gastric cancer. METHODS: Primary gastric cancer samples with lymph node metastases (LNM) and without LNM were subjected to mRNA microarray assay. The differentially expressed genes were confirmed by RT-qPCR. HRCT1 protein expression was further detected using an immunohistochemistry (IHC) assay. In vitro and in vivo assays were performed to investigate the role of HRCT1 in tumor invasion, metastasis, and proliferation. The expressions of the downstream target genes of HRCT1 were detected by microarray, RT-qPCR and Western blot assays. Dual-luciferase reporter and Western blot assays were carried out to identify miRNAs target to HRCT1. RESULTS: HRCT1 was upregulated in gastric cancer, and high expression of HRCT1 was associated with poor overall survival (OS) and disease-free survival (DFS). Moreover, HRCT1protein expression was an independent predictor for poor OS and DFS. HRCT1 could promote gastric cancer cells' migration, invasion, and proliferation in vitro as well as tumor metastasis and growth in vivo. Notably, our data showed that HRCT1 promoted gastric cancer progression by activating the ERBB2-MAPK signaling pathway. At least partially, the expression of HRCT1 could be negatively regulated by miR-124-3p. CONCLUSIONS: The upregulated expression of HRCT1 predicts poor survival for patients with gastric cancer. HRCT1 promotes tumor progression by activating the ERBB2-MAPK pathway. HRCT1, negatively regulated by miR-124-3p, may be a potential therapeutic target for patients with gastric cancer.

Elevated ZBTB7A expression in the tumor invasive front correlates with more tumor budding formation in gastric adenocarcinoma.

PURPOSE: Tumor budding (TB) is reported to predict nodal involvement and recurrence in multiple human malignancies. However, it is not clear how TB forms. The purpose of this study is to find markers related to TB formation in gastric cancer and to investigate the underlying mechanisms. METHODS: TB was scored on hematoxylin-eosin staining slides in 122 gastric cancer cases. Immunostaining score of CREB1, GAGE12I, CTNND1, KIF26B and ZBTB7A both at the invasive front and in the center of the tumor were assigned to each case. Spearman's correlation with the TB score was performed to find the TB-related markers. In vitro study and RNA-seq using gastric cancer cell lines were done to unveil the mechanisms. RESULTS: TB could predict lymph node metastasis and is negatively associated with overall survival of the patients. The expression of ZBTB7A in the invasive front, rather than the other four markers, was much higher than that in the tumor center and was positively correlated with TB score. ZBTB7A could enhance migration and invasion of gastric cancer cells in vitro. RNA-seq data followed by RT-qPCR and western blot verification demonstrated the activation of EGFR-MAPK-ERK and PI3K-AKT-mTOR pathways and increased expression of EMT related markers upon ZBTB7A over-expression. CONCLUSION: Higher ZBTB7A expression in the tumor margin may contribute to the dissociation of tumor cells from the tumor mass to form TB by initiating EMT via EGFR-MEK-ERK and PI3K-AKT-mTOR pathway.

TIPRL, a Novel Tumor Suppressor, Suppresses Cell Migration, and Invasion Through Regulating AMPK/mTOR Signaling Pathway in Gastric Cancer.

Invasion and metastasis of gastric cancer after curative resection remain the most common lethal outcomes. However, our current understanding of the molecular mechanism underlying gastric cancer metastasis is far from complete. Herein, we identified TOR signaling pathway regulator (TIPRL) as a novel metastasis suppressor in gastric cancer through genome-wide gene expression profiling analysis using mRNA microarray. Decreased TIPRL expression was detected in clinical gastric cancer specimens, and low TIPRL expression was correlated with more-advanced TNM stage, distant metastasis, and poor clinical outcome. Moreover, TIPRL was identified as a direct target of miR-216a-5p and miR-383-5p. Functional study revealed that re-expression of TIPRL in gastric cancer cell lines suppressed their migratory and invasive capacities, whereas inverse effects were observed in TIPRL-deficient models. Mechanistically, TIPRL downstream effectors and signaling pathways were investigated using mRNA microarray. Gene expression profiling revealed that TIPRL could not modulate the downstream genes at transcriptional levels, thereby implying that the regulation might occur at the post-transcriptional levels. We further demonstrated that TIPRL induced phosphorylation/activation of AMPK, which in turn attenuated phosphorylation of mTOR, p70S6K, and 4E-BP1, thereby leading to inactivation of mTOR signaling and subsequent suppression of cell migration/invasion in gastric cancer. Taken together, TIPRL acts as a novel metastasis suppressor in gastric cancer, at least in part, through regulating AMPK/mTOR signaling, likely representing a promising target for new therapies in gastric cancer.

LncRNA-HNF1A-AS1 functions as a competing endogenous RNA to activate PI3K/AKT signalling pathway by sponging miR-30b-3p in gastric cancer.

BACKGROUND: Accumulating evidence demonstrated that long noncoding RNAs (lncRNAs) played important regulatory roles in many cancer types. However, the role of lncRNAs in gastric cancer (GC) progression remains unclear. METHODS: RT-qPCR assay was performed to detect the expression of HNF1A-AS1 in gastric cancer tissues and the non-tumourous gastric mucosa. Overexpression and RNA interference approaches were used to investigate the effects of HNF1A-AS1 on GC cells. Insight into competitive endogenous RNA (ceRNA) mechanisms was gained via bioinformatics analysis, luciferase assays and an RNA-binding protein immunoprecipitation (RIP) assay, RNA-FISH co-localisation analysis combined with microRNA (miRNA)-pulldown assay. RESULTS: This study displayed that revealed expression of HNF1A-AS1 was associated with positive lymph node metastasis in GC. Moreover, HNF1A-AS1 significantly promoted gastric cancer invasion, metastasis, angiogenesis and lymphangiogenesis in vitro and in vivo. In addition, HNF1A-AS1 was demonstrated to function as a ceRNA for miR-30b-3p. HNF1A-AS1 abolished the function of the miRNA-30b-3p and resulted in the derepression of its target, PIK3CD, which is a core oncogene involved in the progression of GC. CONCLUSION: This study demonstrated that HNF1A-AS1 worked as a ceRNA and promoted PI3K/AKT signalling pathway-mediated GC metastasis by sponging miR-30b-3p, offering novel insights of the metastasis mechanism in GC.

The Olfactory Receptor Family 2, Subfamily T, Member 6 (OR2T6) Is Involved in Breast Cancer Progression via Initiating Epithelial-Mesenchymal Transition and MAPK/ERK Pathway.

Breast cancer is the most common female malignancy worldwide, however its molecular pathogenesis still needs in-depth investigation. Here we first revealed that the olfactory receptor family 2, subfamily T, member 6 (OR2T6) was significantly over-expressed in breast cancer tissues compared with normal breast tissues. OR2T6 expression was tightly correlated with higher TNM staging, positive lymph node metastasis, and associated with poorer patients' overall and disease-free survival. And OR2T6 enhanced the proliferation, invasion, and migration ability of breast cancer cell lines in vitro (MCF-7 and MDA-MD-231). Mechanically, it promoted the expression of mesenchymal markers (Vimentin, N-cadherin, and ß-catenin) while inhibited E-cadherin expression, suggesting that OR2T6 played a key role in the regulation of epithelial-mesenchymal transition (EMT) process. Moreover, the human gene expression microarray clarified that MAPK/ERK pathway could be initiated by OR2T6 at mRNA level, which was further confirmed at protein level by western blot analysis. Thus, we concluded that OR2T6, as a novel oncogene, contributed to the progression of breast carcinoma by the initiation of EMT and MAPK/ERK pathway.

GAGE7B promotes tumor metastasis and growth via activating the p38δ/pMAPKAPK2/pHSP27 pathway in gastric cancer.

BACKGROUND: Gastric cancer is the second most common cause of cancer-related mortality; thus, the mechanisms underlying tumor metastasis and growth in gastric cancer need to be extensively explored. METHODS: Differentially expressed genes were examined in gastric cancer samples with lymph node metastasis (LNM) and without LNM using mRNA microarray and RT-qPCR. The effects of G antigen 7B (GAGE7B) on the metastasis, growth, and angiogenesis of gastric cancer were investigated in vitro and in vivo. GAGE7B protein expression was detected by immunohistochemical (IHC) analysis. Microarray, RT-qPCR, and western blot assays were performed to detect downstream target genes of GAGE7B. Dual-luciferase reporter and western blot assays were used to identify miRNAs that could negatively regulate GAGE7B. RESULTS: GAGE7B was significantly overexpressed in samples with LNM. High expression levels of GAGE7B were associated with advanced clinical stage and poor patient survival. GAGE7B dramatically enhanced the metastasis, growth, and angiogenesis ability of gastric cancer. GAGE7B was further demonstrated to promote the progression of gastric cancer by activating the p38δ/pMAPKAPK2/pHSP27 pathway. However, the GAGE7B-induced p38δ/pMAPKAPK2/pHSP27 pathway was inactivated by miR-30c, as the expression levels of both GAGE7B and p38δ were found to be directly suppressed by miR-30c. Intriguingly, GAGE7B was found to be a ceRNA for p38δ, as it activated the p38δ/pMAPKAPK2/pHSP27 pathway by competitively binding miR-30c. CONCLUSIONS: GAGE7B may serve as a prognostic indicator in gastric cancer. GAGE7B significantly promotes gastric cancer progression by upregulating the p38δ/pMAPKAPK2/pHSP27 pathway, but it is negatively regulated by miR-30c. GAGE7B and miR-30c may be potential therapeutic targets in gastric cancer.

Human Epidermal Growth Factor Receptor-2 Promotes Invasion and Metastasis in Gastric Cancer by Activating Mitogen-activated Protein Kinase Signaling.

Increasing evidence supports an important role for the human epidermal growth factor receptor-2 (HER2) gene and mitogen-activated protein kinase (MAPK) signaling pathways in the progression of human cancers by enhancing cancer cell metastasis and proliferation. However, the relationship between HER2 and MAPK signaling pathways in gastric cancer (GC) remains unclear. In the present study, dual in situ hybridization was performed to detect HER2 gene amplification and reverse transcription-quantitative polymerase chain reaction was used to investigate the mRNA expression of members of the MAPK signaling pathway, including rapidly accelerated fibrosarcoma (RAF), extracellular regulated signal-activated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), in 112 primary GC tissue samples. The results revealed that 19/112 (17%) of tissue samples showed positive amplification of HER2, which was correlated with tumor invasion and metastasis. Upregulation of RAF, ERK, p38, and JNK was also observed in samples associated with metastasis. Moreover, the expression levels of RAF and ERK in samples with HER2 gene amplification were significantly increased compared with those without HER2 amplification. However, the expression levels of both p38 and JNK were not significantly correlated with HER2 gene amplification. Our results simultaneously showed the association between HER2 gene amplification and the expression levels of MAPK signaling pathway proteins and clinicopathologic characteristics in GC. These findings provide the basis for investigating the regulation of MAPK signaling pathways by HER2 and potential therapeutic targets for inhibiting metastasis and invasion in GC.

Overexpression of TMPRSS4 promotes tumor proliferation and aggressiveness in breast cancer.

Transmembrane protease serine 4 (TMPRSS4) is a novel type II transmembrane serine protease that is overexpressed in various types of human cancers and has an important function in cancer progression. However, there is a paucity of data available regarding the biological effects of TMPRSS4 on breast cancer (BC) cells and the underlying mechanisms. In this study, expression of TMPRSS4 in BC tissues was detected by immunohistochemistry. The relationship between TMPRSS4 expression and clinicopathological characteristics as well as prognosis was evaluated. The effects of TMPRSS4 on cell proliferation, migration and invasion were investigated in BC cell lines in vitro. Additionally, RT-qPCR and western blot analysis were used to determine the expressions of epithelial-mesenchymal transition (EMT) biomarkers and TMPRSS4 in BC cell lines. We found that TMPRSS4 was overexpressed in BC tissues and its expression level was closely correlated with tumor size, histological grade, lymph node metastasis, clinical stage as well as poor survival (all P<0.05) and could be recognized as an independent prognostic factor for BC patients. Overexpression of TMPRSS4 promoted the proliferation, migration and invasion of BC cells in vitro. Moreover, TMPRSS4 knockdown significantly enhanced the expression of E-cadherin and claudin-1 and inhibited the expression of vimentin and Slug, indicating suppression of EMT. Our results suggest that TMPRSS4 plays a crucial role in the progression of BC. Moreover, TMPRSS4 overexpression promoted the proliferation, invasion and migration of BC cells by possibly inducing EMT. To conclude, TMPRSS4 may be a potential therapeutic target for cancer treatment.

Prognostic Value of microRNA Signature in Patients with Gastric Cancers.

The occurrence of lymph node metastases (LNM) after endoscopic submucosal dissection (ESD) in patients with gastric cancer (GC) leads to poor prognosis. However, few biomarkers are available to predict LNM in GC patients. Thus, we measured expression of 6 cancer-related miRNAs using real-time RT-PCR in 102 GC samples that were randomized into a training set and a testing set (each, 51 cases). Using logistic regression, we identified 4-miRNA (miR-27b, miR-128, miR-100 and miR-214) signatures for predicting LNM in GC patients. Patients with high-risk scores for the 4-miRNA signature tended to have higher LNM than those with low-risk scores. Meanwhile, the ROC curve of the 4-miRNA signature was better for predicting LNM in GC patients. In addition, Cox regression analysis indicated that a 2-miRNA signature (miR-27b and miR-214) or a miR-214/N stage signature was predictive of survival for GC patients. This work describes a previously unrecognized 4-miRNA signature involved in LNM and a 2-miRNA signature or miR-214/N stage signature related to GC patients' survival.

Suppression of SPIN1-mediated PI3K-Akt pathway by miR-489 increases chemosensitivity in breast cancer.

Drug resistance is one of the major obstacles for improving the prognosis of breast cancer patients. Increasing evidence has linked the association of aberrantly expressed microRNAs (miRNAs) with tumour development and progression as well as chemoresistance. Despite recent advances, there is still little known about the potential role and mechanism of miRNAs in breast cancer chemoresistance. Here we describe that 16 miRNAs were found to be significantly down-regulated and 11 up-regulated in drug-resistant breast cancer tissues compared with drug-sensitive tissues, using a miRNA microarray. The results also showed miR-489 to be one of the most down-regulated miRNAs in drug-resistant tissues and cell lines, as confirmed by miRNA microarray screening and real-time quantitative PCR. A decrease in miR-489 expression was associated with chemoresistance as well as lymph node metastasis, increased tumour size, advanced pTNM stage and poor prognosis in breast cancer. Functional analysis revealed that miR-489 increased breast cancer chemosensitivity and inhibited cell proliferation, migration and invasion, both in vitro and in vivo. Furthermore, SPIN1, VAV3, BCL2 and AKT3 were found to be direct targets of miR-489. SPIN1 was significantly elevated in drug-resistant and metastatic breast cancer tissues and inversely correlated with miR-489 expression. High expression of SPIN1 was associated with higher histological grade, lymph node metastasis, advanced pTNM stage and positive progesterone receptor (PR) status. Increased SPIN1 expression enhanced cell migration and invasion, inhibited apoptosis and partially antagonized the effects of miR-489 in breast cancer. PIK3CA, AKT, CREB1 and BCL2 in the PI3K-Akt signalling pathway, demonstrated to be elevated in drug-resistant breast cancer tissues, were identified as downstream effectors of SPIN1. It was further found that either inhibition of SPIN1 or overexpression of miR-489 suppressed the PI3K-Akt signalling pathway. These data indicate that miR-489 could reverse the chemoresistance of breast cancer via the PI3K-Akt pathway by targeting SPIN1. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MicroRNA-27b, microRNA-101 and microRNA-128 inhibit angiogenesis by down-regulating vascular endothelial growth factor C expression in gastric cancers.

Vascular Endothelial Growth Factor C (VEGF-C) has critical roles in angiogenesis in human cancers; however, the underlying mechanisms regulating VEGF-C expression remain largely unknown. In the present study, VEGF-C protein expression and the density of blood vessels or lymphatic vessels were determined by immunohistochemistry in 103 cases of gastric cancer tissues. Suppression of VEGF-C by miR-27b, miR-101 and miR-128 was investigated by luciferase assays, Western blot and ELISA. The miRNAs expression levels were detected in human gastric cancers by real-time quantitative PCR. Cell proliferation, migration and invasion assays were performed to assess the effect of miRNAs on gastric cancer cells and human umbilical vascular endothelial cells (HUVECs). Our data showed that high VEGF-C expression was significantly associated with increased tumor size, advanced TNM classification and clinical stage, higher microvessel density (MVD) and lymphatic density (LVD), as well as poor survival in patients with gastric cancer. Furthermore, VEGF-C was found to be a direct target gene of miR-27b, miR-101, and miR-128. The expression levels of the three miRNAs were inversely correlated with MVD. Overexpression of miR-27b, miR-101, or miR-128 suppressed migration, proliferation activity, and tube formation in HUVECs by repressing VEGF-C secretion in gastric cancer cells. We conclude that miR-27b, miR-101 and miR-128 inhibit angiogenesis by down-regulating VEGF-C expression in gastric cancers.

C/EBPα-induced miR-100 expression suppresses tumor metastasis and growth by targeting ZBTB7A in gastric cancer.

MicroRNAs have been reported to play key roles in various human cancers, including gastric cancer. However, understanding of the expression of miR-100 and its regulatory mechanisms in human gastric cancer remains elusive. In this study, we reveal that miR-100 is downregulated in gastric cancer samples and gastric cancer cell lines. Furthermore, lower miR-100 expression was found in primary gastric cancer samples with lymphatic metastasis compared to those without lymphatic metastasis. Overexpression of miR-100 suppressed tumor growth in vivo and inhibited gastric cancer invasion and metastasis in vitro and in vivo. Furthermore, we demonstrated that miR-100 reduced gastric cancer aggressiveness by directly targeting ZBTB7A. Knockdown of ZBTB7A by siRNA disrupted gastric cancer progression by impairing tumor invasion and metastasis. High expression of ZBTB7A was significantly correlated with poorer prognosis in gastric cancer patients. Our results also showed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) could induce the expression of miR-100 by binding to the putative promoter region of miR-100. This study demonstrated that miR-100 could be induced by C/EBPα and may act as a tumor suppressor gene by inhibiting ZBTB7A.

Catenin-δ1, negatively regulated by miR-145, promotes tumour aggressiveness in gastric cancer.

Increasing evidence supports the association of catenin-δ1 (CTNND1, p120ctn) with tumour development and progression. However, the mechanism and clinical significance of CTNND1 deregulation in gastric cancer remain unknown. The expression level and cellular localization of CTNND1 were determined by immunohistochemistry in 126 human gastric cancer and 50 non-tumourous tissues. The cellular localization of CTNND1 and epithelial cadherin (E-cadherin) were detected by immunofluorescence. Cell proliferation, apoptosis, migration and invasion assays were performed to assess the effect of CTNND1 cDNA or CTNND1 siRNA transfection on gastric cancer cells. Luciferase assay, western blot analysis and in vivo assays were used to determine whether CTNND1 could be regulated by miR-145. The results demonstrate that the cytoplasmic localization of CTNND1 protein, rather than expression level, was indicative of higher clinical stage, positive lymph node metastasis and poorer prognosis in gastric cancers. CTNND1 could promote gastric cancer cell migration and invasion with little effect on cellular proliferation and apoptosis. CTNND1 was proved to be a direct target gene for miR-145. Besides suppressing cytoplasmic CTNND1 expression, miR-145 could recover the membranous localization of CTNND1 and E-cadherin. We conclude that cytoplasmic CTNND1 can serve as an independent prognostic factor for patients with gastric cancers. MiR-145 inhibits invasion of gastric cancer cells not only by down-regulating cytoplasmic CTNND1 expression but also by inducing the translocation of CTNND1 and E-cadherin from the cytoplasm to the cell membrane through down-regulating N-cadherin.

Aberrant expression of CD227 is correlated with tumor characteristics and invasiveness of breast carcinoma.

PURPOSE: Increasing evidences demonstrate that CD227 plays a crucial role in the development and progression of breast cancer. However, the function of CD227 in breast carcinoma was still controversial and the investigation on CD227 in Asian race was scarce. METHODS: To investigate the relationship between CD227 and tumor characteristics of breast carcinoma, CD227, estrogen receptor (ER), progesterone receptor (PR), Her2/neu and Ki-67 were detected by immunohistochemistry in a series of 227 patients. The Kaplan-Meier method and log-rank tests were used to estimate the correlation between CD227 expression and patients' prognosis. Furthermore, in vitro invasion assay was performed to examine the effect of CD227 on the invasiveness of breast carcinoma cells after transfection with CD227 cDNA or antisense phosphorothioate oligodeoxynucleotides (ASODN) against CD227 mRNA. RESULTS: Our data demonstrate that the cytoplasm staining and high expression of CD227 were positively related to the aggressiveness of breast cancer. Both circumferential membrane staining and cytoplasm staining were associated with lymph node metastasis. Moreover, the cytoplasm staining and overexpression of CD227 were found to be related to Her-2/neu positivity, higher Ki-67 positivity and poorer survival of patients. We further demonstrated that the invasion ability of breast carcinoma cells could be enhanced or inhibited by CD227 cDNA or ASODN, respectively. CONCLUSIONS: We conclude that the aberrant expression of CD227, especially cytoplasm staining could be predictive for tumor aggressiveness, lymph node metastasis, poorer outcome of patients with breast cancers. And CD227 could promote the invasion ability of breast cancer cells, suggesting a potential role of CD227 as an oncogene in breast carcinoma.

Clinicopathological significance of microRNA-214 in gastric cancer and its effect on cell biological behaviour.

Accumulating evidence indicates that numerous microRNAs are involved in the tumorigenesis and progression of gastric cancer, while the clinical significance of microRNA-214 in gastric cancer is poorly understood and the exact role of microRNA-214 in gastric cancer remains obscure. In the present study, expression levels of microRNA-214 in 80 gastric carcinoma tissues, 18 nontumourous gastric tissues, and 4 types of gastric cancer cell lines were quantified by reverse transcription followed by real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between microRNA-214 expression and cliniopathological characteristics including prognosis was explored. To investigate the potential role of microRNA-214 in gastric cancer cell biological behaviour, we performed cell proliferation, apoptosis, migration and invasion assays in four gastric cancer cell lines and an immortalized gastric cell line in vitro. Our results showed that microRNA-214 was dramatically downregulated in gastric cancer tissues and gastric cancer cell lines, compared with nontumourous gastric tissues. Stepwise downregulation of microRNA-214 expression was observed among nontumourous gastric mucosa, nonmetastasis gastric cancer tissues, and metastasis gastric cancer tissues. The expression of microRNA-214 was significantly inversely correlated with lymph node metastasis and tumour size but had no correlation with the patient's prognosis. Ectopic expression of microRNA-214 could inhibit cell migration and invasion ability in SGC7901 and MKN45 gastric cancer cells. And knockdown of microRNA-214 significantly facilitated cell proliferation, migration and invasion in a cell-specific manner in MKN28, BGC823 and GES-1 cells. Colony stimulating factor 1 (CSF1) was identified as a target gene of microRNA-214. In summary, our data demonstrated that microRNA-214 is a promising novel biomarker for lymph node metastasis in patients with gastric cancer. And we identified that downregulation of microRNA-214 may regulate the proliferation, invasion and migration of gastric cancer cells by directly targeting CSF1.

Deregulated expression of miR-145 in manifold human cancer cells.

MicroRNAs play important roles in the processes of tumor initiation and progression. The expression level of miR-145 in gastric, liver, and cervical cancers has been rarely investigated. Whether miR-145 may function as a common tumor suppressor in the generation of tumor phenotype needs to be clarified. miR-145 expression was determined by RT-qPCR in various human cancer tissues including those of gastric, liver, colon, and cervical cancers. Cancer cell lines were transfected with miR-145 precursor, anti-miR-145 inhibitor, or negative control, and cells' proliferation, migration, and invasion activities were analyzed. The gene target of miR-145 was confirmed by luciferase assay and Western blot. The miR-145 expression level was lower by 37.68-, 2.64-, 2.69- and 2.39-fold in gastric, liver, colon, and cervical cancer tissues, respectively, compared to corresponding nontumorous controls. Moreover, miR-145 levels were significantly downregulated in various cancer cell lines. We further demonstrated that miR-145 could suppress anchorage-independent growth and cell motility in both the liver cancer cell line Hep-G2 and the gastric cancer cell line MKN-45, and inhibited cell proliferation in a cell type-specific manner. Insulin receptor substrate-1 (IRS1) was identified as a target gene of miR-145, by which miR-145 was able to suppress cell proliferation. miR-145 suppresses cell proliferation, anchorage-independent growth, cell motility, and may serve as a tumor suppressor.

[Expression of microRNA-100 in human gastric cancer].

OBJECTIVE: To investigate the expression of microRNA-100 (miR-100) in human gastric cancer cells and its role of miR-100 in migration, invasion and proliferation in gastric cancer cell line SGC7901. METHODS: Total RNAs were extracted from formalin-fixed and paraffin-embedded tissue samples of SGC7901 cells, gastric cancer (50 cases), non-tumor (18 cases) and lymph nodes with metastases (18 cases). The expression of miR-100 was examined by reverse transcription (RT)-qPCR. Additionally, SGC7901 cells were transfected with Pre-miR-100 and negative control constructs, and then their ability of migration, invasion and proliferation in vitro was documented after 48 hours. RESULTS: RT-qPCR showed that although miR-100 expressed in all samples, compared to non-tumour tissues, the expression was lower both in SGC7901 cells and gastric cancer tissues (P = 0.0077, P < 0.01). SGC7901 cells and primary gastric cancer tissues with lymph nodes metastasis had lower miR-100 expression than those of without lymph node metastasis (P = 0.0361, P = 0.0356). The migration ability and invasion of SGC7901 cells transfeced with pre-miR-100 decreased as compared with control cells (P = 0.0025, P = 0.0028 respectively). However, miR-100 expression had no significant effects on the cell proliferation. CONCLUSIONS: Expression of miR-100 inhibits the migration and invasion of gastric cancer cells without significant alteration of proliferation. Therefore, miR-100 may play an inhibitory role in the progression of gastric carcinoma.

Restoration of chemosensitivity in cancer cells with MDR phenotype by deoxyribozyme, compared with ribozyme.

One of the main mechanisms for multidrug resistance (MDR) involves multidrug resistance gene 1 (MDR1) which encodes P-glycoprotein (Pgp). Pgp acts as a drug efflux pump and exports chemotherapeutic agents from cancer cells. Specific inhibition of Pgp expression by gene therapy is considered a well-respective strategy having less innate toxicities. At present, the investigation of DRz in reversal MDR is scarce. In the study, phosphorothioate DRz that targets to the translation initiation codon AUG was synthesized and transfected into breast cancer cells and leukemia cells with MDR phenotype. ASODN (antisense oligonucleotide) and ribozyme targets to the same region were also synthesized for comparison analysis. Alterations in MDR1 mRNA and Pgp were determined by RT-PCR, Northern blot, flow cytometry and Rh123 retention tests. Chemosensitivity of the treated cells was determined by MTT assay. The results showed that DRz could significantly suppress expression of MDR1 mRNA and inhibit synthesis of Pgp. The efflux activity of Pgp was inhibited accordingly. Chemosensitivity assay showed that a 21-fold reduction in drug resistance for Adriamycin and a 45-fold reduction in drug resistance for Vinblastine were found in the treated cells 36h after transfection. These data suggest that DRz targeted to the translation initiation codon AUG can reverse MDR phenotype in cancer cells and restore their chemosensitivity. Moreover, the reversal efficiency of DRz is better than that of ribozyme and ASODN targets to the same region of MDR1 mRNA.