miR-506通过调控MCL-1抑制耐阿立替尼非小细胞肺癌A549细胞上皮间质转化的作用机制

目的:探究miR-506通过调控MCL-1对耐阿立替尼非小细胞肺癌A549细胞上皮间质转化（epithelial mesenchymal transformation,EMT）及侵袭转移的影响和作用机制。方法:收集2017年12月至2018年12月我院肿瘤科收治的经PET-CT结合组织病理活检及药敏试验确诊为耐阿立替尼的74例非小细胞肺癌患者的癌及癌旁组织以及人非小细胞肺癌A549细胞为研究对象,分别采用细胞转染、免疫组化染色（IHC）、qRT-PCR和Western Blot法检测上述临床组织和细胞样本中miR-506、MCL-1、BAX/Bcl-2凋亡信号途径及EMT标志蛋白表达水平;此外,采用Transwell细胞实验观察miR-506过表达和MCL-1敲减对A549细胞迁移和侵袭能力的影响。结果:免疫组化（IHC）结果显示肺癌患者癌组织中浸润性坏死性病理损伤较癌旁组织明显加重,且癌组织中MCL-1的阳性表达率为94.64%,明显高于癌旁组织的23.27%（P＜0.05）。qRT-PCR和Western Blot结果显示,肺癌组织中miR-506、BAX和E-cadherin的表达明显低于癌旁组织,而MCL-1、Bcl-2和N-cadherin的表达显著高于癌旁组织（P＜0.05）。细胞实验结果表明miR-506过表达和MCL-1敲减能够明显上调BAX和E-cadherin的表达,同时抑制Bcl-2和N-cadherin的表达（P＜0.05）;此外,miR-506过表达和MCL-1敲减均能显著抑制肺癌A549细胞的迁移和侵袭能力（P＜0.05）。结论:miR-506可能通过抑制BAX/Bcl-2/MCL-1凋亡途径发挥抑制耐阿立替尼非小细胞肺癌A549细胞EMT及诱导细胞凋亡作用,有望为临床抗肺癌转移及凋亡抑制靶向治疗提供新分子和靶点。     

MiR-21 regulates epithelial-mesenchymal transition phenotype and hypoxia-inducible factor-1α expression in third-sphere forming breast cancer stem cell-like cells

Cancer stem cells (CSCs) are predicted to be critical drivers of tumor progression due to their "stemness", but the molecular mechanism of CSCs in regulating metastasis remains to be elucidated. Epithelial-mesenchymal transition (EMT), hypoxia-inducible factor (HIF)-1α, and miR-21, all of which contribute to cell migration for metastasis, are interrelated with CSCs. In the present study, third-sphere forming (3-S) CSC-like cells, which showed elevated CSC surface markers (ALDH1(+) and CD44(+)/CD24(-/low)) and sphereforming capacity as well as migration and invasion capacities, were cultured and isolated from breast cancer MCF-7 parental cells, to evaluate the role of miR-21 in regulating the CSC-like cell biological features, especially EMT. EMT, which was assessed by overexpression of mesenchymal cell markers (N-cadherin, Vimentin, alpha-smooth muscle actin [α-SMA]) and suppression of epithelial cell marker (E-cadherin), was induced in 3-S CSC-like cells. Moreover, both of HIF-1α and miR-21 were upregulated in the CSC-like cells. Interestingly, antagonism of miR-21 by antagomir led to reversal of EMT, downexpression of HIF-1α, as well as suppression of invasion and migration, which indicates a key role of miR-21 involved in regulate CSC-associated features. In conclusion, we demonstrated that the formation of CSC-like cells undergoing process of EMT-like associated with overexpression of HIF-1α, both of which are regulated by miR-21.     

Down-regulation of MiR-206 promotes proliferation and invasion of laryngeal cancer by regulating VEGF expression

MicroRNAs (miRNA) are a class of small noncoding RNAs that regulate the expression of their target genes. The aim of the present study was to explore the effects of miR-206 on laryngeal suamous cell carcinoma (LSCC) cells. The expression level of miR-206 was quantified by qRT-PCR in primary LSCC tissues and corresponding adjacent non-neoplastic tissues. MTT, Matrigel invasion assays and flow cytometry methods were used to test the proliferation, invasion and apoptosis of MiR-206 transfection LSCC cells and a mouse model was used to investigate tumorigenesis. MiR-206 was significantly down regulated in the LSCC tissues. Inverse correlation of miR-206 expression was found with the T grade, nodal metastasis and clinical stage of LSCC. Cell proliferation, migration, invasion and tumorigenesis in the LSCC cells were significantly inhibited and apoptotic cells were also increased after miR-206 tansfection. Furthermore, miR-206 transfection down-regulated the expression of vascular endothelial growth factor (VEGF) in the LSCC cells. The loss of miR-206 may play an important role in the progress of LSCC and miR-206 may function as a novel tumor suppressed miRNA.     

Disclosure of a stem cell phenotype in an oral squamous cell carcinoma cell line induced by BMP-4 via an epithelial-mesenchymal transition

A small subset of cells within a malignant neoplasm, named cancer stem cells (CSCs) or tumour initiating cells, are thought to be capable of initiating the neoplasm itself, and of driving its growth and recurrence after treatment. It is unclear whether CSCs can be identified and experimentally induced within oral squamous cell carcinoma (OSCC), although this has been reported for a number of other tumour types. In this study, we aimed to determine whether BMP-4 (bone morphogenetic protein-4) could induce epithelial-mesenchymal transition (EMT) with acquisition of stem cell-like phenotypes in a cell-culture model. Furthermore, the differential expression of ABCG2, a putative CSC marker, was determined in human normal oral mucosa and OSCC tissues at mRNA and protein level. The results showed that after treatment with BMP-4, most Tca8113 cells (a human tongue OSCC cell line) changed their morphology from slabstone to spindle-shaped, and demonstrated enhanced expression of ABCG2 compared with non-treated cells. Expression of Oct-4 was induced in cell nuclei with up-regulation of EMT markers (Snail, Slug and vimentin), and down-regulation of E-cadherin. Interestingly, the expression of hTERT, CD44 and Bmi-1 (generally accepted as markers of CSCs) were up-regulated, but this was not synchronous with the expression of EMT markers. Tumour spheres were formed after stimulation with BMP-4, with high expression of CD44 and ABCG2. In human tissues, ABCG2 was strongly expressed in OSCC, but not in normal mucosa. This study suggests that BMP-4-mediated EMT constitutes one possible pathway for the development of CSCs in oral cancer, implying a transient therapeutic opportunity if EMT can be interrupted early in the evolution of such a neoplasm.     

MicroRNA-34a affects the occurrence of laryngeal squamous cell carcinoma by targeting the antiapoptotic gene survivin

Survivin has been shown to be an ideal target for cancer gene therapy because of its strong antiapoptotic effect. MicroRNA-34a (miR-34a) can function as a tumor suppressor in some cancers through negative regulation of gene expression. However, the relationship between miR-34a and survivin in larynx squamous cell carcinoma (LSCC) has not been explored. The abundance of survivin mRNA and miR-34a in LSCC tissues were measured using quantitative real-time polymerase chain reaction. Their expression levels were analyzed and correlated with tumor differentiation, lymphatic metastasis, clinical stages, and survival rates. MiR-34a mimic was transfected using liposomes to increase its level in LSCC cancer cell line, Hep-2. The effects of miR-34a on survivin protein expression were tested using western blot analysis. Cell cycle analyses were performed using flow cytometry. The results showed that transfection of miR-34a mimic significantly suppressed cell proliferation with decreased survivin protein expression, but did not affect mRNA expression level. The results from LSCC tissue samples showed that miR-34a was downregulated, while survivin expression was upregulated. The miR-34a levels were negatively correlated with histologic differentiation and were positively correlated with survival rate. MiR-34a significantly suppressed cell proliferation by arresting cells at G(0)/G(1) phase in Hep-2 cells. These results indicated that miR-34a may affect the occurrence of LSCC by targeting survivin.     

miR-203 Inhibits the Invasion and EMT of Gastric Cancer Cells by Directly Targeting Annexin A4

Many studies have shown that downregulated miR-203 level is in a variety of cancers including gastric cancer (GC). However, the precise molecule mechanisms of miR-203 in GC have not been well clarified. In the current study, we investigated the biological functions and molecular mechanisms of miR-203 in GC cell lines. We found that miR-203 is downregulated in GC tissues and cell lines. Moreover, the low level of miR-203 was associated with increased expression of annexin A4 in GC tissues and cell lines. The invasion and EMT of GC cells were suppressed by overexpression of miR-203. However, downregulation of miR-203 promoted invasion and EMT of GC cells. Bioinformatics analysis predicted that annexin A4 was a potential target gene of miR-203. Next, luciferase reporter assay confirmed that miR-203 could directly target annexin A4. Consistent with the effect of miR-203, downregulation of annexin A4 by siRNA inhibited the invasion and EMT of GC cells. Introduction of annexin A4 in GC cells partially blocked the effects of miR-203 mimic. Introduction of miR-203 directly targeted annexin A4 to inhibit the invasion and EMT of GC cells. Overall, reactivation of the miR-203/annexin A4 axis may represent a new strategy for overcoming metastasis of GC.     

MiR-203 suppresses tumor growth and invasion and down-regulates MiR-21 expression through repressing Ran in esophageal cancer

The expression of miR-203 has been reported to be significantly down-regulated in esophageal cancer. We showed here that overexpression of miR-203 in esophageal cancer cells dramatically increased cell apoptosis and inhibited cell proliferation, migration and invasion as well as tumor growth and down-regulated miR-21 expression. We subsequently identified that small GTPase Ran was a target gene of miR-203. Furthermore, Ran restoration partially counteracted the tumor suppressive effects of miR-203 and increased miR-21 expression. Taken together, our findings suggest that miR-203 may act as novel tumor suppressor in esophageal cancer through down-regulating the expression of Ran and miR-21.     

Tumor suppressor TSLC1 is implicated in cell proliferation, invasion and apoptosis in laryngeal squamous cell carcinoma by regulating Akt signaling pathway

Overwhelming evidence has demonstrated that TSLC1 (tumor suppressor in lung cancer 1), a novel tumor suppressor, is crucially implicated in various biological processes including progression, proliferation and apoptosis during tumorigenesis. However, the exact functions and molecular details of TSLC1 in laryngeal cancer remain ill-defined. Here, the expression of TSLC1 in laryngeal squamous cell carcinoma (LSCC) tissues and cells was detected, and the biological roles of TSLC1 in LSCC cells were investigated. The results showed that expressions of TSLC1 mRNA and protein were significantly reduced in LSCC tissues with low expression in 18 of 85 (21.18?%) and 16 of 85 (18.82?%), respectively. Additionally, statistical analysis revealed a significant correlation of TSLC1 expression with TNM staging and lymph node metastases (P?<?0.05), but not related to age, gender and tumor differentiation (P?>?0.05). Elevation of TSLC1 level inhibited cell proliferation, reduced cell invasion in vitro and induced cell apoptosis in Hep-2 cells, most importantly, TSLC1 upregulation decreased the level of pAkt, but not changed the level of total Akt in Hep-2 cells. Stepwise investigations demonstrated that overexpression of TSLC1 in Hep-2 cells increased caspase-3 activity and expressions of bax and p21 proteins but decreased the levels of bcl-2, MMP-2 and MMP-9 proteins. These data suggest that TSLC1 may exert essential roles in the progression and development of LSCC, and thus TSLC1 may be a potential molecular target for LSCC treatment.     

miR-24 functions as a tumor suppressor in Hep2 laryngeal carcinoma cells partly through down-regulation of the S100A8 protein

microRNAs, a family of small non-coding RNAs, regulating approximately 30% of all human genes are deeply involved in the pathogenesis of several types of cancers, including laryngeal squamous cell carcinoma (LSCC). Here, we demonstrate that miR-24 is down-regulated in human LSCC tissues. Ectopic expression of miR-24 in Hep2 cells significantly induced cell morphology changes and inhibited cell proliferation and invasion ability in vitro by targeting S100A8 at the translational level. Meanwhile, miR-24 could significantly inhibit Hep2 cell invasion after S100A8 protein blockade. In conclusion, our results suggest that miR-24 may function as a tumor suppressor in LSCC through down-regulation of S100A8, which suggests that miR-24 could serve as a novel potential maker for LSCC therapy.     

lncRNA HOTAIR靶向miR-126激活RhoA/ROCK通路促进喉癌Hep-2细胞增殖、侵袭与EMT

目的:探讨长链非编码RNA-Hox转录反义RNA(long non-coding RNA Hox antisense intergenicRNA,lncRNA HOTAIR)对喉癌Hep-2细胞增殖、侵袭及其上皮间质转化(epithelial-mesenchymal transition,EMT)的影响及机制.方法:利...     

MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2

Epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration, invasion, and drug resistance in several types of cancer. In this study, our aim was to clarify microRNAs (miRNAs)-related mechanisms underlying EMT followed by acquired resistance to chemotherapy in glioblastoma (GBM). We used multiple methods to achieve our goal including microarray analysis, qRT-PCR, western blotting analysis, loss/gain-of-function analysis, luciferase assays, drug sensitivity assays, wound-healing assay and invasion assay. We found that miR-203 expression was significantly lower in imatinib-resistant GBM cells (U251AR, U87AR) that underwent EMT than in their parental cells (U251, U87). Ectopic expression of miR-203 with miRNA mimics effectively reversed EMT in U251AR and U87AR cells, and sensitized them to chemotherapy, whereas inhibition of miR-203 in the sensitive lines with antisense oligonucleotides induced EMT and conferred chemoresistance. SNAI2 was identified as a direct target gene of miR-203. The knockdown of SNAI2 by short hairpin RNA (shRNA) inhibited EMT and drug resistance. In GBM patients, miR-203 expression was inversely related to SNAI2 expression, and those tumors with low expression of miR-203 experienced poorer clinical outcomes. Our findings indicate that re-expression of miR-203 or targeting SNAI2 might serve as potential therapeutic approaches to overcome chemotherapy resistance in GBM.     

lncRNA DNM3OS在喉鳞状细胞癌组织和细胞中的表达及其临床和生物学意义

目的:检测lncRNA DNM3OS在喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)组织和LSCC细胞株中的表达及其临床意义,探讨其对LSCC TU177细胞体外增殖、迁移及侵袭的影响,并分析DNM3OS与EMT的关系.方法:从河北医科大学第四医院生物标本库选取2014年3...     

miR-126 functions as a tumour suppressor in human gastric cancer

MicroRNAs have emerged as important gene regulators and are recognised as key players in carcinogenesis. In the present study, we show that miR-126 was significantly down-regulated in gastric cancer tissues compared with matched normal tissues and was associated with clinicopathological features, including tumour size, lymph node metastasis, local invasion and tumour-node-metastasis (TNM) stage. Ectopic expression of miR-126 in SGC-7901 gastric cancer cells potently inhibited cell growth by inducing cell cycle arrest in G0/G1 phase, migration and invasion in vitro as well as tumorigenicity and metastasis in vivo. Mechanistically, we identified the adaptor protein Crk as a target of miR-126. Taken together, our results suggest that miR-126 may function as a tumour suppressor in gastric cancer, with Crk as a direct target.     

The SOX17/miR-371-5p/SOX2 axis inhibits EMT,stem cell properties and metastasis in colorectal cancer

Cancer stem cells (CSCs) and EMT-type cells, which share molecular characteristics with CSCs, have been believed to play critical roles in tumor metastasis. Although much progress has been garnered in elucidating the molecular pathways that trigger EMT, stemness and metastasis, a number of key mechanistic gaps remain elusive. In the study, miR-371-5p was obviously down-regulated in primary CRC tissues compared with matched adjacent normal mucosa and correlated significantly with differentiation, tumor size, lymphatic and liver metastases. MiR-371-5p could attenuate proliferation, invasion in vitro and metastasis in vivo in CRC cells. It also suppressed EMT by regulating Wnt/β-catenin signaling and strongly decreased the CRC stemness phenotypes. Moreover, demethylation of SOX17 induced miR-371-5p expression and consequently suppressed its direct target SOX2 in CRC cells. MiR-371-5p was necessary for SOX17 mediated cancer-related traits and SOX2 was a functional target of miR-371-5p. A positive relationship between SOX17 and miR-371-5p expression and a negative one between miR-371-5p and SOX2 expression were observed in CRC cell lines and tissues. In conclusion, we identified miR-371-5p as an important “oncosuppressor” in CRC progression and elucidated a novel mechanism of the SOX17/miR-371-5p/SOX2 axis in the regulation of EMT, stemness and metastasis, which may be a potential therapeutic target.     

Cyr61/CCN1 Overexpression Induces Epithelial-Mesenchymal Transition Leading to Laryngeal Tumor Invasion and Metastasis and Poor Prognosis

Background: To examine the expression of cysteine-rich 61 (Cyr61/CCN1) protein in laryngeal squamouscellcarcinoma (LSCC) tissues, and its relationship with the tumor epithelial-mesenchymal transition (EMT),invasion, metastasis, and prognosis. Materials and Methods: Immunohistochemistry was used to detect theexpressions of Cyr61, Vimentin (Vim), and E-cadherin (E-cad) in 88 cases of LSCC tissues and 30 cases oftumor-adjacent normal tissues. Vim and E-cad were used as mesenchymal and epithelial markers, respectively,to determine the relationship between Cyr61 expression and the EMT of LSCC cells. In addition, clinical andhistopathological data were combined to analyze the relationship between the positive-expression rates of Cyr61,Vim and E-cad and LSCC invasion, metastasis and prognosis. Results: In LSCC tissues, Vim expression ratewas significantly higher than that of the tumor-adjacent tissues, whereas E-cad expression rate was significantlylower than that of the tumor-adjacent tissues. The Vim expression rate was significantly higher in stages T3and T4 than in stages T1 and T2 LSCC tissues, whereas E-cad expression rate was significantly lower in stagesT3 and T4 than in stages T1 and T2 LSCC tissues. Compared to the group without lymph node metastasis, theVim expression rate was significantly higher and the E-cad expression rate was significantly lower in the groupwith lymph node metastasis. The expression rate of Cyr61 was significantly higher in LSCC tissues than in thetumor-adjacent normal tissues. In addition, the Cyr61 expression rate was higher in stages T3 and T4 than instages T1 and T2 LSCC, and higher in the group with lymph node metastasis than in the group without lymphnode metastasis. The Vim expression rate was significantly higher in the Cyr61 positive group than in the Cyr61negative group, whereas the E-cad expression rate was significantly higher in the Cyr61 negative group thanin the Cyr61 positive group. Survival analysis indicated that survival rates of Cyr61 positive, Vim positive andE-cad negative groups were significantly lower than that of Cyr61 negative, Vim negative and E-cad positivegroups, respectively. Conclusions: Cyr61 expression is closely associated with LSCC invasion and lymph nodemetastasis. Overexpression of Cyr61 may induce EMT and therefore leads to LSCC invasion and metastasisand poor prognosis. Cyr61 may become a new maker for clinical prediction of LSCC invasion and metastasisand a new target for LSCC treatment.     

MicroRNA与DNA甲基化相互调节机制在肝细胞癌中的研究进展

肝细胞癌是原发性肝癌的主要类型,也是人类恶性程度较高的肿瘤之一,其发病机制至今尚未完全阐明.表观遗传学机制在肿瘤的发生、发展中起重要作用,DNA甲基化和微小RNA (microRNA,miRNA)的调控机制属于表观遗传学的研究范畴.研究表明,DNA甲基化及miRNA在肝细胞癌的形成中分别或协同发挥着重要作用,miRNA是一类在转录后水平调节基因表达的非编码短链RNA.研究表明,DNA甲基化和组蛋白修饰不仅可以调节蛋白编码基因的表达,而且可以调节miRNA的表达.在肝细胞癌中,一些异常表达的miRNAs(如miR-125b、miR-1-1、miR-124、miR-203和miR-191)是通过表观遗传学机制调控的.另外,在肝细胞癌中还发现了一类miRNAs通过调控表观遗传学通路中关键分子来改变整个基因组的表观遗传学状态.本文就DNA甲基化和miRNA之间复杂的相互调节机制在肝细胞癌发生和发展中的研究进展进行综述.     

miR-30通过调节CD90表达抑制肝细胞肝癌

  目的  miRNA能通过转录后调节靶基因的表达量,并在致癌过程中发挥抑癌或促癌作用。研究发现miR-30家族在人类肿瘤中起着重要作用,并参与维持干细胞特性的上皮细胞间质化过程,本研究主要识别miR-30家族与肝细胞肝癌的关系。  方法  应用qRT-PCR实验方法检测93例肝细胞肝癌患者癌组织和癌旁正常组织中miR-30c,miR-30b和miR-30e表达水平,另对121例肝细胞肝癌患者癌组织进行miR-30家族的表达及CD90免疫组织化学表达检测,分析miR-30家族与CD90在肝细胞肝癌中的相关性。  结果  本研究发现miR-30c(P < 0.001)、miR-30b(P=0.004)和miR-30e(P < 0.001)与癌旁正常组织相比,癌组织中表达显著下调。CD90蛋白在癌组织中的表达水平显著高于癌旁正常组织(P=0.007)。MiR-30c(P=0.032)和miR-30e(P=0.015)在CD90阴性表达的患者中表达水平显著高于CD90阳性表达的患者。  结论  miR-30家族在肝细胞肝癌中可作为抑癌miRNA,并通过调节CD90蛋白来发挥这一作用。