The MiR-495/Annexin A3/P53 Axis Inhibits the Invasion and EMT of Colorectal Cancer Cells.

More and more reports have shown that the dysregulation of miRNAs can contribute to the progression and metastasis of human cancers. Many studies have shown that the down-regulation of the miR-495 level occurs in a variety of cancers, including colorectal cancer (CRC). However, the precise molecular mechanisms of miR-495 in CRC have not been well clarified. In the current study, we investigated the biological functions and molecular mechanisms of miR-495 in CRC cell lines. qRT-PCR was used to determine the level of miR-495 in CRC cell lines and tissues. A miR-495 mimic and inhibitor were transfected into CRC cells, and the effects of miR-495 on the invasion and EMT were explored by qRT-PCR as well as transwell and Western blot assays. Meanwhile, luciferase assays were performed to validate Annexin A3 as a miR-495 target in CRC cells. In our study, we found that miR-495 is down-regulated in CRC tissues and cell lines. Moreover, the low level of miR-495 was associated with increased expression of Annexin A3 in CRC tissues and cell lines. The invasion and EMT of CRC cells were suppressed by the overexpression of miR-495. However, the down-regulation of miR-495 promoted the invasion and EMT of CRC cells. Bioinformatics analysis predicted that Annexin A3 was a potential target gene of miR-495. Next, the luciferase reporter assay confirmed that miR-495 could directly target Annexin A3. Consistent with the effect of miR-495, the down-regulation of Annexin A3 by siRNA inhibited the invasion and EMT of CRC cells through the up-regulation of p53. The introduction of Annexin A3 in CRC cells partially blocked the effects of the miR-495 mimic. The introduction of miR-495 directly targeted Annexin A3 to inhibit the invasion and EMT of CRC cells by up-regulating p53, and the down-regulation of Annexin A3 was essential for inhibiting the invasion and EMT of CRC cells by overexpressing miR-495. Overall, the re-activation of the miR-495/Annexin A3/ p53 axis may represent a new strategy for overcoming metastasis of CRC.

Bai Z ，Wang J ，Wang T ，Li Y ，Zhao X ，Wu G ，Yang Y ，Deng W ，Zhang Z ... -  《-》

The Effect of LncRNA H19/miR-194-5p Axis on the Epithelial-Mesenchymal Transition of Colorectal Adenocarcinoma.

Since the combined actions of lncRNAs and miRNAs have been considered to be involved in the occurrence and development of various neoplasms, the main purpose of this study was to discover whether and how lncRNA H19 and miR-194 influenced the epithelial-mesenchymal transition (EMT) process of colorectal adenocarcinoma (CRA). Totally 214 pairs of CRA and adjacent normal tissues were collected, and 5 human CRA cell lines (i.e. HCT116, HT-29, RKO SW280 and Lovo) were purchased. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was adopted to quantify the H19 and miR-194-5p expressions in cells and tissues. The expressions of FoxM1, E-cadherin, vimentin, N-cadherin were determined using western blot. On the side, si-H19, si-NC, miR-194-5p mimic, miR-194-5p inhibitor and negative control (NC) were transfected into CRA cell lines. Meanwhile, the invasive, migratory and proliferative conditions of the cells were assessed through transwell, wound healing and colony-forming experiments, with final verification of the relationship between H19 and miR-194-5p employing dual-luciferase reporter gene assay. Highly-expressed H19, lowly-expressed miR-194-5p, low-grade differentiation and lymph node metastasis appeared as the independent predictors of unfavorable prognosis in CRA patients' (all P< 0.05). It indicated that FoxM1 expression displayed positive correlations with H19 expression, yet negative associations with miR-194-5p expression within CRA tissues (P< 0.05). In addition, transfection of H19-siRNA and miR-145-5p mimic triggered a conspicuous increase in E-cadherin expression, as well as an evidently down-regulation in vimentin and N-cadherin expressions within HT29 and RKO cells (P< 0.05). On the other hand, the invasive and migratory capacities of CRA cells were significantly hindered (P< 0.05). Moreover, the luciferase reporter gene assay confirmed that H19 modified miR-194-5p expression through directly targeting at it (P< 0.05). Ultimately, FoxM1 could reverse the role of miR-194-5p in inhibiting invasion, migration and EMT of CRA cells (P< 0.05). LncRNA H19/miR-194/FoxM1 axis could serve as a profound target for the diagnosis and treatment of CRA.

Li CF ，Li YC ，Wang Y ，Sun LB ... -  《-》

MicroRNA-340 Induces Apoptosis and Inhibits Metastasis of Ovarian Cancer Cells by Inactivation of NF-x03BA;B1.

Aberrant expression of microRNA-340 (miR-340) has been frequently reported in some cancers excluding ovarian cancer (OC). The role and its molecular mechanism of miR-340 in OC have not been reported. Real-time PCR was performed to detect the expression of miR-340 in OC cell lines. MiR-340 mimic and negative control were transfected into OC cells and the effects of miR-340 on the cell proliferation, cell cycle, apoptosis and metastasis were investigated by Brdu-ELISA assay, flow cytometry, qRT-PCR, Transwell and ELISA assays. Furthermore, protein level of NF-x03BA;B1 was measured by Western blotting. Meanwhile, luciferase assays were performed to validate NF-x03BA;B1 as miR-340 target in OC cells. In this study, we explored the effects of miR-340 overexpression on apoptosis, invasion and EMT in OC cells. The mRNA level of miR-340 in OC cell lines and tissues was evidently reduced. The miR-340 mimic was transiently transfected into OC cells using Lipofectamine™ 2000 reagent. Subsequently, the Brdu-ELISA results showed that introduction of miR-340 inhibited cell proliferation. Our data also demonstrated that miR-340 mimic arrested cell cycle progression and promoted apoptosis of OC cells. In addition, miR-340 overexpression could also inhibit invasion and EMT of OC cells. qRT-PCR were used to determined the expressions of matrix metalloproteinase-2 and -9 (MMP-2 and -9) in OC cells. Next, we found that NF-x03BA;B1 expression was evidently reduced by up-regulation of miR-340. Bioinformatics analysis predicted that the NF-x03BA;B1 was a potential target gene of miR-340. Luciferase reporter assay further confirmed that miR-340 could directly target the 3' UTR of NF-x03BA;B1. Moreover, overexpression of NF-x03BA;B1 in OC cells transfected with miR-340 mimic partially reversed the inhibitory of miR-340 mimic. miR-340 induced cell apoptosis and inhibited metastasis in OC cells by down-regulation of NF-x03BA;B1.

Li P ，Sun Y ，Liu Q 《-》

The regulatory effects of metformin on the [SNAIL/miR-34]:[ZEB/miR-200] system in the epithelial-mesenchymal transition(EMT) for colorectal cancer(CRC).

The epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression, metastasis and drug resistance. The transcription factor(TF) and microRNA (miR) chimeric [SNAIL/miR-34]:[ZEB/miR-200] unit is the core regulatory system for the EMT process. Here, we proposed to assess the anti-EMT abilities and explore the inherent pharmacological mechanisms of the classic hypoglycaemic agent metformin for colorectal cancer(CRC). For the EMT model, the TGF-β-induced CRC cell lines SW480 and HCT116 were treated with metformin. The viability, migration and invasion abilities of the cells were evaluated with the Cell Counting Kit-8, wound-healing and trans-well assay. The alterations of the [SNAIL/miR-34]:[ZEB/miR-200] system and the EMT markers E-cadherin and vimentin were detected by western blot, qPCR and immunofluorescent staining. Metformin exhibited inhibitory effects on the proliferation, migration and invasion of the CRC SW480 cells. The up-regulation of E-cadherin and the down-regulation of vimentin for both SW480 and HCT116 cells revealed the anti-EMT abilities of metformin. For the [SNAIL/miR-34]:[ZEB/miR-200] system, metformin increased miR-200a, miR-200c and miR-429 levels and decreased miR-34a, SNAIL1 and ZEB1 levels in the TGF-β-induced EMT. From immunofluorescence, we observed increased E-cadherin and ZEB1 co-expression in metformin-treated cells. Metformin may perform bidirectional regulations of the [SNAIL/miR-34]:[ZEB/miR-200] system in the EMT process for colorectal cancer. Such regulation is expressed as the inhibition of EMT in general as well as an increased higher proportion of E/M hybrid cells in the total population.

Wang Y ，Wu Z ，Hu L 《-》

MicroRNA-183 Acts as a Tumor Suppressor in Human Non-Small Cell Lung Cancer by Down-Regulating MTA1.

MicroRNAs (miRs) often contribute to the progression of non-small cell lung cancer (NSCLC) via regulation of mRNAs that are involved in lung homeostasis. We conducted a study aimed at exploring the roles of miR-183 in the proliferation, epithelial-mesenchymal transition (EMT), invasion and migration of human NSCLC cells via targeting MTA1. NSCLC and adjacent normal tissues were collected from 194 patients with NSCLC. Positive expression of MTA1 protein was detected by immunohistochemistry. The highest levels of expression of miR-183 were detected using RT-qPCR in SPC-A-1 cells, which were selected and assigned to the following groups: blank, negative control (NC), miR-183 mimic, miR-183 inhibitor, siRNA-MTA1, and miR-183 inhibitor + siRNA-MTA1. The expression of miR-183 and the mRNA and protein expression of MTA1, E-cadherin, Vimentin, Snail, PCNA, Bax and Bcl-2 in tissues and transfected cells were measured using RT-qPCR and western blot analysis. Cell proliferation, apoptosis, migration and invasion were evaluated by CCK-8, flow cytometry, scratch tests and Transwell assays. Tumor xenografts were conducted in nude mice to determine tumor growth. SPC-A-1 cells with the highest levels of miR-183 expression were selected. Compared with adjacent normal tissues, the expression of miR-183 and the mRNA and protein expression of E-cadherin and Bax were decreased in NSCLC tissues, while mRNA and protein expression of MTA1, Vimentin, snail, PCNA and Bcl-2 were increased. MiR-183 was over-expressed in the miR-183 mimic group and under-expressed in the miR-183 inhibitor and miR-183 inhibitor + siRNA-MTA1 groups. In the miR-183 mimic and siRNA-MTA1 groups, the mRNA and protein expression of E-cadherin and Bax, as well as cell apoptosis, were enhanced, while the expression levels of MTA1, Vimentin, snail, PCNA and Bcl-2 mRNA and protein, cell proliferation, migration, invasion and tumor growth were reduced relative to the blank and NC groups. The miR-183 inhibitor group exhibited an opposite trend. Our study indicates that miR-183 down-regulates MTA1 to inhibit the proliferation, EMT, migration and invasion of human NSCLC cells.

Yang CL ，Zheng XL ，Ye K ，Ge H ，Sun YN ，Lu YF ，Fan QX ... -  《-》