MiR-200c Increases the Radiosensitivity of Non-Small-Cell Lung Cancer Cell Line A549 by Targeting VEGF-VEGFR2 Pathway

MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. VEGF family, an important regulator of angiogenesis, also plays a crucial role in the regulation of cancer cell radiosensitivity. VEGFR2 mediates the major growth and permeability actions of VEGF in a paracrine/autocrine manner. MiR-200c, at the nexus of epithelial-mesenchymal transition (EMT), is predicted to target VEGFR2. The purpose of this study is to test the hypothesis that regulation of VEGFR2 pathway by miR-200c could modulate the radiosensitivity of cancer cells. Bioinformatic analysis, luciferase reporter assays and biochemical assays were carried out to validate VEGFR2 as a direct target of miR-200c. The radiosensitizing effects of miR-200c on A549 cells were determined by clonogenic assays. The downstream regulating mechanism of miR-200c was explored with western blotting assays, FCM, tube formation assays and migration assays. We identified VEGFR2 as a novel target of miR-200c. The ectopic miR-200c increased the radiosensitivity of A549 while miR-200c down-regulation decreased it. Besides, we proved that miR-200c radiosensitized A549 cells by targeting VEGF-VEGFR2 pathway specifically, thus leading to inhibition of its downstream pro-survival signaling transduction and angiogenesis, and serves as a potential target for radiosensitizition research.     

Modulation of NF-κB/miR-21/PTEN Pathway Sensitizes Non-Small Cell Lung Cancer to Cisplatin

Background

Platinum-based chemotherapy is a standard strategy for non-small cell lung cancer (NSCLC), while chemoresistance remains a major therapeutic challenge in current clinical practice. Our present study was aimed to determine whether inhibition of the NF-κB/miR-21/PTEN pathway could increase the sensitivity of NSCLC to cisplatin.

Methods

The expression of miR-21 in NSCLC tissues was determined using in situ hybridization. Next, the effect of miR-21 on the sensitivity of A549 cells to cisplatin was determined in vitro. Whether miR-21 regulated PTEN expression was assessed by luciferase assay. Furthermore, whether NF-κB targeted its binding elements in the miR-21 gene promoter was determined by luciferase and ChIP assay. Finally, we measured the cell viability and apoptosis under cisplatin treatment when NF-κB was inhibited.

Results

An elevated level of miR-21 was observed in NSCLC lung tissues and was related to a short survival time. Exogenous miR-21 promoted cell survival when exposed to cisplatin, while miR-21 inhibition could reverse this process. The RNA and protein levels of PTEN were significantly decreased by exogenous miR-21, and the 3′-untranslated region of PTEN was shown to be a target of miR-21. The expression of miR-21 was regulated by NF-κB binding to its element in the promoter, a finding that was verified by luciferase and ChIP assay. Hence, inhibition of NF-κB by RNA silencing protects cells against cisplatin via decreasing miR-21 expression.

Conclusion

Modulation of the NF-κB/miR-21/PTEN pathway in NSCLC showed that inhibition of this pathway may increase cisplatin sensitivity.     

Downregulation of microRNA-92b-3p suppresses proliferation,migration, and invasion of gastric cancer SGC-7901 cells by targeting Homeobox D10

To investigate the effect and mechanism of microRNA-92b-3p (miR-92b-3p) targeting Homeobox D10 (HOXD10) on proliferation, migration, and invasion of gastric cancer, we detected t he expression of miR-92b-3p and HOXD10 in SGC-7901 cells. The effects of miR-92b-3p or HOXD10 on proliferation, migration, invasion, and matrix metalloproteinase (MMP)-2/9 expression in SGC-7901 cells were measured by the Cell Counting Kit-8 assay, Transwell assay, and Western blot, respectively. The results showed that miR-92b-3p expression was increased, and HOXD10 expression was decreased in SGC-7901 cells, compared with human normal gastric epithelial cells GES-1. Functional experiments demonstrated that cell proliferation, migration, invasion, and expression of MMP-2/9 in SGC-7901 cells were significantly inhibited by miR-92b-3p silencing and HOXD10 overexpression. Moreover, HOXD10 was a potential target gene of miR-92b-3p as evidenced by the TargetScan software and double luciferase reporter assay. In the rescue experiment, knockdown of HOXD10, accompanied by higher expression of MMP-2/9, could significantly eliminate the inhibitory effects of miR-92b-3p silencing on cell proliferation, migration, and invasion. In conclusion, miR-92b-3p is highly expressed in gastric cancer SGC-7901 cells, and interfering with its expression might inhibit SGC-7901 cell proliferation, migration, and invasion via downregulating MMP-2/9 expression and targeting HOXD10.     

RhoE Promotes Metastasis in Gastric Cancer through a Mechanism Dependent on Enhanced Expression of CXCR4

RhoE, a novel member of the Rho protein family, is a key regulator of the cytoskeleton and cell migration. Our group has previously shown that RhoE as a direct target for HIF-1α and mediates hypoxia-induced epithelial to mesenchymal transition in gastric cancer cells. Therefore, we assumed that RhoE might play an important role in gastric cancer metastasis. In the present study, we have explored the role of RhoE expression in gastric cancer, cell invasion and metastasis, and the influence of RhoE on regulating the potential expression of down-stream genes. RhoE expression was elevated in gastric cancer tissues as compared with normal gastric tissues. We also found a close correlation between the histological grade and the diagnosis of the patient. Up-regulation of RhoE significantly enhanced the migratory and invasive abilities of gastric cancer cells both in vitro and in vivo. Moreover, down-regulation of RhoE diminished the metastatic potential of cancer cells. PCR array and subsequent transwell assay showed that the regulation of gastric cancer metastasis by RhoE was partially mediated by CXCR4. This observation suggested that CXCR4 might be a downstream effector for RhoE. In summary, our study identified RhoE as a novel prognostic biomarker and metastatic-promoting gene of gastric cancer.     

MicroRNA-200c is involved in proliferation of gastric cancer by directly repressing p27Kip1

P27^Kip1, also known as Cyclin-dependent kinase inhibitor 1B, is an important check-point protein in the cell cycle. It has been identified that although as a tumor suppressor, P27^Kip1 is expressed in different cancer cell types, which shows the therapeutic potential in tumor genesis. In this study, we examined the upstream regulatory mechanism of P27^Kip1 at the microRNA (miRNA) level in gastric carcinogenesis. We used bioinformatics to predict that microRNA-200c (miR-200c) might be a direct upstream regulator of P27^Kip1. It was also verified in gastric epithelial-derived cell lines that overexpression of miR-200c significantly inhibited the expression levels of P27^Kip1, whereas knockdown of miR-200c promoted P27^Kip1 expression in AGS and BGC-823 cells. Furthermore, we identified the direct binding of miR-200c on the P27^Kip1 3′ -UTR sequence by luciferase assay. MiR-200c could enhance the colony formation of cells by repressing P27^Kip1 expression. In addition, the negative correlation between P27^Kip1 and miR-200c in human gastric cancer tissues and matched normal tissues further supported the tumor-promoting action of miR-200c in vivo. Our finding suggested that miR-200c directly regulates the expression of P27^Kip1 and promotes cell growth in gastric cancer as an oncogene, which may provide new clues to treatment.     

miR-92在膀胱癌患者中的表达与膀胱癌细胞侵袭和耐药性的关系

为了考察miR-92在膀胱癌患者中的表达及与膀胱癌细胞侵袭和耐药性的关系,本研究通过RT-PCR检测了膀胱癌患者癌组织和BIU-87细胞中的miR-92表达,通过对BIU-87细胞转染miR-92抑制剂来敲低miR-92的表达。使用10μg/mL的顺铂处理BIU-87细胞24 h、48 h和72 h,Cell Counting Kit-8试剂盒(CCK-8)检测细胞活力。基质胶侵袭实验检测侵袭能力,Annexin V/PI流式细胞仪检测细胞凋亡。RT-PCR和Western blotting检测GSK3β、细胞核β-catenin、Cyclin D1、c-myc和MMP7的表达。研究显示,膀胱癌组织和细胞中miR-92的表达上调且与TNM分期和淋巴结转移相关。敲低miR-92抑制膀胱癌细胞增殖、侵袭和上皮-间质转化,并降低膀胱癌细胞的顺铂耐药性。敲低miR-92导致Cyclin D1、c-myc、MMP7和细胞核β-catenin的表达水平显著降低,而GSK3β的表达水平显著升高。本研究表明,miR-92在膀胱癌患者中明显上调,敲低miR-92可抑制膀胱癌细胞的增殖、转移和上皮-间质转化,并提高化疗药物敏感性。miR-92对膀胱癌细胞生物学行为的调控作用部分由Wnt信号通路相关分子(如GSK3β等)介导。     

Aberrant hypermethylation of miR-9 genes in gastric cancer

Carcinogenesis of the stomach involves multiple steps including genetic mutation or epigenetic alteration of tumor suppressor genes or oncogenes. Recently, tumor suppressive miRNAs have been shown to be deregulated by aberrant hypermethylation during gastric cancer progression. In this study, we demonstrate that three independent genetic loci encoding for miR-9 (miR-9-1, miR-9-2 and miR-9-3) are simultaneously modified by DNA methylation in gastric cancer cells. Methylation-mediated silencing of these three miR-9 genes can be reactivated in gastric cancer cells through 5-Aza-dC treatment. Subsequent analysis of the expression levels of miR-9 showed that it was significantly down-regulated in gastric cancers compared with adjacent normal tissues (P value < 0.005). A similar tendency toward a tumor-specific DNA methylation pattern was shown for miR-9-1, miR-9-2 and miR-9-3 in 72 primary human gastric cancer specimens. Ectopic expression of miR-9 inhibited cell proliferation, migration and invasion, suggesting its tumor suppressive potential in gastric cancer progression.     

miRNA-29c Suppresses Lung Cancer Cell Adhesion to Extracellular Matrix and Metastasis by Targeting Integrin β1 and Matrix Metalloproteinase2 (MMP2)

Our pilot study using miRNA arrays found that miRNA-29c (miR-29c) is differentially expressed in the paired low-metastatic lung cancer cell line 95C compared to the high-metastatic lung cancer cell line 95D. Bioinformatics analysis shows that integrin β1 and matrix metalloproteinase 2 (MMP2) could be important target genes of miR-29c. Therefore, we hypothesized that miR-29c suppresses lung cancer cell adhesion to extracellular matrix (ECM) and metastasis by targeting integrin β1 and MMP2. The gain-of-function studies that raised miR-29c expression in 95D cells by using its mimics showed reductions in cell proliferation, adhesion to ECM, invasion and migration. In contrasts, loss-of-function studies that reduced miR-29c by using its inhibitor in 95C cells promoted proliferation, adhesion to ECM, invasion and migration. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29c inhibited the expression of the luciferase gene containing the 3′-UTRs of integrin β1 and MMP2 mRNA. Western blotting indicated that miR-29c downregulated the expression of integrin β1 and MMP2 at the protein level. Gelatin zymography analysis further confirmed that miR-29c decreased MMP2 enzyme activity. Nude mice with xenograft models of lung cancer cells confirmed that miR-29c inhibited lung cancer metastasis in vivo, including bone and liver metastasis. Taken together, our results demonstrate that miR-29c serves as a tumor metastasis suppressor, which suppresses lung cancer cell adhesion to ECM and metastasis by directly inhibiting integrin β1 and MMP2 expression and by further reducing MMP2 enzyme activity. The results show that miR-29c may be a novel therapeutic candidate target to slow lung cancer metastasis.     

Deregulation of lncRNA-AC078883.3 and microRNA-19a is involved in the development of chemoresistance to cisplatin via modulating signaling pathway of PTEN/AKT

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer death worldwide. As a platinum-based chemotherapeutic drug, cisplatin has been used in the NSCLC treatment for over 30 years, and its effects are impaired by drug resistance. This study aimed to investigate the potential role of lncRNA-AC078883.3 in the development of chemoresistance against cisplatin. Real-time PCR, Western blot analysis, Immunohistochemistry (IHC) assay, bioinformatic analysis, and luciferase assay were collaboratively used to establish the lncRNA-AC078883.3/miR-19a/PTEN/AKT pathway. Also, the effect of cisplatin on cell proliferation was observed via an MTT assay. Furthermore, Cox regression and Kaplan–Meier analyses were used to study whether lncRNA-AC078883.3 is involved in the survival of NSCLC. Compared with the Cisplatin-Sensitive group, the Cisplatin-Resistance group exhibited lower levels of lncRNA-AC078883.3 and PTEN and higher levels of miR-19a and p-Akt. The growth rate of A549 and H460 cells and the IC ₅₀ of DPP in the Cisplatin-Resistance group were higher than those in the Cisplatin-S group. miR-19a contains a putative binding site of lncRNA-AC078883.3, which enabled the luciferase activity of wild-type lncRNA-AC078883.3 to be reduced by miR-19a. In addition, by directly targeting PTEN 3′-untranslated region (UTR), miR-19a repressed the luciferase activity of wild-type PTEN 3′-UTR. The median OS of patients with reduced lncRNA-AC078883.3 expression was longer than that of patients with higher lncRNA-AC078883.3 expression. Finally, compared with low lncRNA-AC078883.3-expression patients, the high lncRNA-AC078883.3-expression patients were associated with lower miR-19a expression and higher PTEN expression. Therefore, we suggested for the first time that the low expression of lncRNA-AC078883.3 contributed to the development of chemoresistance against cisplatin.     

MiR-130a and MiR-374a Function as Novel Regulators of Cisplatin Resistance in Human Ovarian Cancer A2780 Cells

Chemoresistance remains a major obstacle to effective treatment in patients with ovarian cancer, and recently increasing evidences suggest that miRNAs are involved in drug-resistance. In this study, we investigated the role of miRNAs in regulating cisplatin resistance in ovarian cancer cell line and analyzed their possible mechanisms. We profiled miRNAs differentially expressed in cisplatin-resistant human ovarian cancer cell line A2780/DDP compared with parental A2780 cells using microarray. Four abnormally expressed miRNAs were selected (miR-146a,-130a, -374a and miR-182) for further studies. Their expression were verified by qRT-PCR. MiRNA mimics or inhibitor were transfected into A2780 and A2780/DDP cells and then drug sensitivity was analyzed by MTS array. RT-PCR and Western blot were carried out to examine the alteration of MDR1, PTEN gene expression. A total of 32 miRNAs were found to be differentially expressed in A2780/DDP cells. Among them, miR-146a was down-regulated and miR-130a,-374a,-182 were upregulated in A2780/DDP cells, which was verified by RT-PCR. MiR-130a and miR-374a mimics decreased the sensitivity of A2780 cells to cisplatin, reversely, their inhibitors could resensitize A2780/DDP cells. Furthermore, overexpression of miR-130a could increase the MDR1 mRNA and P-gp levels in A2780 and A2780/DDP cells, whereas knockdown of miR-130a could inhibit MDR1 gene expression and upregulate the PTEN protein expression .In a conclusion, the deregulation of miR-374a and miR-130a may be involved in the development and regulation of cisplatin resistance in ovarian cancer cells. This role of miR-130a may be achieved by regulating the MDR1 and PTEN gene expression.     

Involvement of microRNA-93, a new regulator of PTEN/Akt signaling pathway, in regulation of chemotherapeutic drug cisplatin chemosensitivity in ovarian cancer cells

The mechanisms underlying ovarian cancer cell resistance to cisplatin (CDDP) are not fully understood. MicroRNAs (miRNAs) play important roles in tumorigenesis and drug resistance. In this paper, we utilized microRNA array and real-time PCR to show that miR-93 is significantly up-regulated in cisplatin-resistant ovarian cancer cells. In vitro assays show that over-expression and knock-down of miR-93 regulate apoptotic activity, and thereby cisplatin chemosensitivity, in ovarian cells. Furthermore, we found that miR-93 can directly target PTEN, and participates in the regulation of the AKT signaling pathway. MiR-93 inversely correlates with PTEN expression in CDDP-resistant and sensitive human ovarian cancer tissues. These results may have implications for therapeutic strategies aiming to overcome ovarian cancer cell resistance to cisplatin.     

MiR-221/222 promote chemoresistance to cisplatin in ovarian cancer cells by targeting PTEN/PI3K/AKT signaling pathway

Cisplatin resistance is one of the main limitations in the treatment of ovarian cancer, and its mechanism has not been fully understood. The objectives of this study were to determine the role of miR-221/222 and its underlying mechanism in chemoresistance of ovarian cancer. We demonstrated that miR-221/222 expression levels were higher in A2780/CP cells compared with A2780 S cells. An in vitro cell viability assay showed that downregulation of miR-221/222 sensitized A2780/CP cells to cisplatin-induced cytotoxicity. Moreover, we found that knockdown of miR-221/222 by its specific inhibitors promoted the cisplatin-induced apoptosis in A2780/CP cells. Using bioinformatic analysis and luciferase reporter assay, miR-221/222 were found to directly target PTEN. Moreover, knockdown of miR-221/222 in A2780/CP cells significantly upregulated PTEN and downregulated PI3KCA and p-Akt expression. In conclusion, our results demonstrated that miR-221/222 induced cisplatin resistance by targeting PTEN mediated PI3K/Akt pathway in A2780/CP cells, suggesting that miR-221/222/PTEN/PI3K/Akt may be a promising prognostic and therapeutic target to overcome cisplatin resistance and treat ovarian cancer in the future.     

lncRNA CEBPA-AS1靶向miR-455-3p调控胃癌细胞生物学行为的分子机制研究

摘要 目的：探讨lncRNA CEBPA-AS1对胃癌细胞生物学行为的影响及其可能作用机制。方法：qRT-PCR法检测胃癌组织、癌旁组织与正常人胃上皮GES1细胞和人胃癌SNU-1、AGS、HS-746T细胞系中lncRNA CEBPA-AS1、miR-455-3p的表达量。si-NC、si-lncRNA CEBPA-AS1、miR-NC、miR-455-3p mimics、si-lncRNA CEBPA-AS1与anti-miR-NC、si-lncRNA CEBPA-AS1与anti-miR-455-3p分别转染至SNU-1细胞（分别命名为si-NC组、si-lncRNA CEBPA-AS1组、miR-NC组、miR-455-3p组、si-lncRNA CEBPA-AS1+anti-miR-NC组和si-lncRNA CEBPA-AS1+anti-miR-455-3p组）后,MTT实验与平板克隆形成实验分别检测细胞增殖及克隆形成能力,Transwell小室实验检测细胞迁移及侵袭能力,双荧光素酶报告基因实验与qRT-PCR实验验证lncRNA CEBPA-AS1与miR-455-3p的靶向调控关系,Western blot法检测MMP2、MMP9蛋白表达情况。结果：与癌旁组织比较,胃癌组织中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,差异均有统计学意义（均P＜0.05）。与GES1细胞比较,SNU-1、AGS、HS-746T细胞中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,其中SNU-1细胞的lncRNA CEBPA-AS1表达量最高,差异均有统计学意义（均P＜0.05）。与si-NC组比较,si-lncRNA CEBPA-AS1组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。与miR-NC组比较,miR-455-3p组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。lncRNA CEBPA-AS1可靶向结合miR-455-3p,并可负调控miR-455-3p的表达。与si-lncRNA CEBPA-AS1+anti-miR-NC组比较,si-lncRNA CEBPA-AS1+anti-miR-455-3p组细胞活力升高,细胞克隆形成数、迁移及侵袭细胞数增多,MMP2、MMP9蛋白表达水平升高,差异均有统计学意义（P＜0.05）。结论：干扰lncRNA CEBPA-AS1表达可通过靶向调控miR-455-3p而抑制胃癌细胞增殖、克隆形成、迁移及侵袭。     

Aberrant hypermethylation of miR-9 genes in gastric cancer

Carcinogenesis of the stomach involves multiple steps including genetic mutation or epigenetic alteration of tumor suppressor genes or oncogenes. Recently, tumor suppressive miRNAs have been shown to be deregulated by aberrant hypermethylation during gastric cancer progression. In this study, we demonstrate that three independent genetic loci encoding for miR-9 (miR-9-1, miR-9-2 and miR-9-3) are simultaneously modified by DNA methylation in gastric cancer cells. Methylation-mediated silencing of these three miR-9 genes can be reactivated in gastric cancer cells through 5-Aza-dC treatment. Subsequent analysis of the expression levels of miR-9 showed that it was significantly down-regulated in gastric cancers compared with adjacent normal tissues (P value &lt; 0.005). A similar tendency toward a tumor-specific DNA methylation pattern was shown for miR-9-1, miR-9-2 and miR-9-3 in 72 primary human gastric cancer specimens. Ectopic expression of miR-9 inhibited cell proliferation, migration and invasion, suggesting its tumor suppressive potential in gastric cancer progression.