食管癌细胞抗失巢凋亡基因UBCH7的发现与鉴定

失巢凋亡(Anoikis)是细胞失去与细胞外基质(Extra-cellular matrix,ECM)粘附时发生的特殊形式的凋亡,是机体维持组织稳态的关键机制之一。抗失巢凋亡能力的获得是肿瘤细胞发生远处转移的前提条件之一。为了鉴定与食管癌细胞抗失巢凋亡相关的基因,文章首先构建食管癌细胞系的逆转录病毒文库,感染对失巢凋亡敏感的NIH3T3细胞,利用感染病毒cDNA文库的混合细胞系进行软琼脂集落形成实验,挑取在悬浮条件下仍可生长成为较大集落的细胞单克隆(潜在具有抗失巢凋亡能力的细胞),通过逆转录病毒载体特异的引物PCR扩增失巢凋亡抗性克隆基因组中的插入cDNA片段,以此获得食管癌细胞系cDNA文库中潜在的具有失巢凋亡抗性的基因。经测序发现其中一个失巢凋亡克隆中整合的cDNA片段包括人UBCH7/UBE2L3基因全长的编码序列(开放阅读框)。利用携带pMSCV-UBCH7的逆转录病毒感染NIH3T3细胞进行验证,结果显示细胞失巢凋亡抗性增强,并且在具有高转移潜能的食管癌细胞系MLuC1中降调UBCH7表达可减弱其失巢凋亡抗性。这些结果表明,UBCH7/UBE2L3是一个与食管癌失巢凋亡抗性相关的基因。     

食管癌细胞抗失巢凋亡基因UBCH7的发现与鉴定

失巢凋亡(Anoikis)是细胞失去与细胞外基质(Extra-cellular matrix, ECM)粘附时发生的特殊形式的凋亡, 是机体维持组织稳态的关键机制之一。抗失巢凋亡能力的获得是肿瘤细胞发生远处转移的前提条件之一。为了鉴定与食管癌细胞抗失巢凋亡相关的基因, 文章首先构建食管癌细胞系的逆转录病毒文库, 感染对失巢凋亡敏感的NIH3T3细胞, 利用感染病毒cDNA文库的混合细胞系进行软琼脂集落形成实验, 挑取在悬浮条件下仍可生长成为较大集落的细胞单克隆(潜在具有抗失巢凋亡能力的细胞), 通过逆转录病毒载体特异的引物PCR扩增失巢凋亡抗性克隆基因组中的插入cDNA片段, 以此获得食管癌细胞系cDNA文库中潜在的具有失巢凋亡抗性的基因。经测序发现其中一个失巢凋亡克隆中整合的cDNA片段包括人UBCH7/UBE2L3基因全长的编码序列(开放阅读框)。利用携带pMSCV-UBCH7的逆转录病毒感染NIH3T3细胞进行验证, 结果显示细胞失巢凋亡抗性增强, 并且在具有高转移潜能的食管癌细胞系MLuC1中降调UBCH7表达可减弱其失巢凋亡抗性。这些结果表明, UBCH7/UBE2L3是一个与食管癌失巢凋亡抗性相关的基因。     

Hydrogen peroxide inhibits non-small cell lung cancer cell anoikis through the inhibition of caveolin-1 degradation

Anoikis or detachment-induced apoptosis plays an essential role in the regulation of cancer cell metastasis. Caveolin-1 (Cav-1) is a key protein involved in tumor metastasis, but its role in anoikis and its regulation during cell detachment are unclear. We report here that Cav-1 plays a key role as a negative regulator of anoikis through a reactive oxygen species (ROS)-dependent mechanism in human lung carcinoma H460 cells. During cell detachment, Cav-1 is downregulated, whereas ROS generation is upregulated. Hydrogen peroxide and hydroxyl radical are two key ROS produced by cells during detachment. Treatment of the cells with hydrogen peroxide scavengers, catalase and N-acetylcysteine, promoted Cav-1 downregulation and anoikis during cell detachment, indicating that produced hydrogen peroxide plays a primary role in preventing anoikis by stabilizing Cav-1 protein. Catalase and N-acetylcysteine promoted ubiquitination and proteasomal degradation of Cav-1, which is a major pathway of its downregulation during cell anoikis. Furthermore, addition of hydrogen peroxide exogenously to the cells inhibited Cav-1 downregulation by preventing the formation of Cav-1-ubiquitin complex, supporting the inhibitory role of endogenous hydrogen peroxide in Cav-1 degradation during cell detachment. Together, these results indicate a novel role of hydrogen peroxide as an endogenous suppressor of cell anoikis through its stabilizing effect on Cav-1.     

MiR-26b reverses temozolomide resistance via targeting Wee1 in glioma cells

Emerging evidence has demonstrated that microRNAs (miRNA) play a critical role in chemotherapy-induced epithelial-mesenchymal transition (EMT) in glioma. However, the underlying mechanism of chemotherapy-triggered EMT has not been fully understood. In the current study, we determined the role of miR-26b in regulation of EMT in stable temozolomide (TMZ)-resistant (TR) glioma cells, which have displayed mesenchymal features. Our results illustrated that miR-26b was significantly downregulated in TR cells. Moreover, ectopic expression of miR-26b by its mimics reversed the phenotype of EMT in TR cells. Furthermore, we found that miR-26b governed TR-mediate EMT partly due to governing its target Wee1. Notably, overexpression of miR-26b sensitized TR cells to TMZ. These findings suggest that upregulation of miR-26b or targeting Wee1 could serve as novel approaches to reverse chemotherapy resistance in glioma.     

Autophagy inhibition suppresses pulmonary metastasis of HCC in mice via impairing anoikis resistance and colonization of HCC cells

Metastasis is one of the main causes of poor prognosis for hepatocellular carcinoma (HCC), which has been linked to cell-death resistance. Autophagy is an important survival mechanism under conditions of cell stress. We hypothesized that autophagy may play a role in HCC metastasis due to its prosurvival effect. Highly metastatic HCC cell lines with stable autophagy inhibition were established via lentivirus-mediated silencing of BECN1 and ATG5 genes. Mouse models of pulmonary metastasis were then developed using the cells with or without autophagy inhibition. The analysis of lung metastasis by histopathological examination and small animal imaging showed that autophagy inhibition significantly decreased the incidence of pulmonary metastases in vivo. Further invasion, migration, detachment, lung colonization, and epithelial-mesenchymal transition (EMT) assays indicated that autophagy inhibition did not affect cell invasiveness, migration or EMT but attenuated the anoikis-resistance and lung colonization of HCC cells. Investigation of the molecular mechanisms underlying showed that the autophagy-inhibition-mediated anoikis-resistance attenuation was associated with the regulation of apoptotic signaling. As autophagy inhibition was shown to be able to suppress HCC metastasis, an autophagy-based HCC tissue-specific target therapy system (AFP-Cre/LoxP-shRNA) was constructed. In vitro and in vivo analyses showed that the system was able to efficiently inhibit autophagy of HCC cells and tissue in a tissue-specific manner. Further in vivo metastasis assay showed that intratumoral administration of the system could significantly suppress lung metastasis. Together, our findings suggest that autophagy may be involved in HCC metastasis through facilitating anoikis resistance and lung colonization of HCC cells. Autophagy-based HCC tissue-specific target therapy may be a new strategy for the management of HCC metastasis.     

Involvement of anoikis-resistance in the metastasis of hepatoma cells

Acquisition of anoikis-resistance is a pre-requisite for cancer cell metastasis. We have demonstrated that hepatoma cells could resist anoikis by a synoikis-like survival style. In this study, we further suggest that acquisition of anoikis-resistance confer cancer cells more capacity for invasiveness, evading from cancer therapeutic agents and escaping from host immune attacks. We investigated the response of anoikis-resistant hepatoma cells to TNF-related apoptosis-inducing ligand (TRAIL), a typical immune surveillant molecule as well as a potential anticancer agent. Our data indicated that detached hepatoma cells not only resist TRAIL-induced apoptosis, but also domesticate TRAIL to exert a stealth “tumor counterattack” effect. These results reveal that acquisition of anoikis-resistance may act as a selective pressure to superimpose on hepatoma cells more metastatic potential for the development of cancer.     

Curcumin sensitizes non-small cell lung cancer cell anoikis through reactive oxygen species-mediated Bcl-2 downregulation

Anoikis, an apoptosis triggered by loss of cell anchorage, has been shown to be a principal mechanism of inhibition of tumor metastasis. Recently, anti-apoptotic Bcl-2 and Cav-1 proteins have been demonstrated to be highly associated with tumor metastasis and apoptosis resistance. Curcumin, a major active component of turmeric, Curcuma longa, has been shown to inhibit neoplastic evolution and tumor progression; however, the underlying mechanisms are unclear. In this study, we investigated the effect of curcumin on cell anoikis as a possible mechanism of anti-tumorigenic action of curcumin, and evaluated the potential role of Bcl-2 and Cav-1 in this process. Our results showed that ectopic expression of either Bcl-2 or Cav-1 induced anoikis resistance of lung carcinoma H460 cells. Curcumin downregulated Bcl-2 protein during anoikis and sensitized the cells to detachment-induced apoptosis, whereas it had no significant effect on Cav-1 protein expression. Bcl-2 down-regulation as well as anoikis enhancement by curcumin were inhibited by superoxide anion scavenger, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride, but were unaffected by other ROS scavengers including catalase and deferoxamine, suggesting that superoxide anion is a key player in the downregulation of Bcl-2 by curcumin. Furthermore, we provided evidence that curcumin decreased Bcl-2 level through ubiquitin-proteasomal degradation which sensitized cells to detachment-induced apoptosis. These findings indicate a novel pathway for curcumin regulation of Bcl-2 and provide a key mechanism of anoikis regulation that may be exploited for metastatic cancer treatment.     

miR-204 functions as a tumor suppressor by regulating SIX1 in NSCLC

The involvement of miR-204 in lung cancer development is unclear. In our study, we analyzed the expression of miR-204 in tumor- and adjacent-tissue samples from 141 patients with non-small cell lung cancer (NSCLC). MiR-204 expression was decreased in tumor samples compared with non-cancerous tissue-derived controls. Moreover, miR-204 expression negatively correlated with homeobox protein SIX1 expression, tumor size and metastasis. MiR-204 silencing in miR-204-positive NSCLC cell lines promoted cell invasion and proliferation. Concomitantly, MiR-204 overexpression resulted in reduced cell proliferation and invasion, upregulated E-cadherin and downregulated N-cadherin and Vimentin expression. SIX1 was identified as a potential target of miR-204, and SIX1 silencing partially compromised the invasive and proliferative capacity of miR-204-deficient cells. Thus, miR-204 may be involved in the NSCLC development.     

miR-26a Suppresses Tumor Growth and Metastasis by Targeting FGF9 in Gastric Cancer

The role of miR-26a in cancer cells seemed controversial in previous studies. Until now, the role of miR-26a in gastric cancer remains undefined. In this study, we found that miR-26a was strongly downregulated in gastric cancer (GC) tissues and cell lines, and its expression levels were associated with lymph node metastasis and clinical stage, as well as overall survival and replase-free survival of GC. We also found that ectopic expression of miR-26a inhibited GC cell proliferation and GC metastasis in vitro and in vivo. We further identified a novel mechanism of miR-26a to suppress GC growth and metastasis. FGF9 was proved to be a direct target of miR-26a, using luciferase assay and western blot. FGF9 overexpression in miR-26a-expressing cells could rescue invasion and growth defects of miR-26a. In addition, miR-26a expression inversely correlated with FGF9 protein levels in GC. Taken together, our data suggest that miR-26a functions as a tumor suppressor in GC development and progression, and holds promise as a prognostic biomarker and potential therapeutic target for GC.     

LncRNA SNHG12 promotes proliferation and epithelial mesenchymal transition in hepatocellular carcinoma through targeting HEG1 via miR-516a-5p

Hepatocellular carcinoma (HCC) is the most common cancer and its prognosis is poor due to metastasis and recurrence. EMT is associated with metastasis. A deep understanding of regulatory mechanism of EMT is critical. LncRNA is involved in regulation of various biological processes including EMT. This study aimed to investigate the regulatory signal axis among lncRNA SNHG12, miR-516a-5p and the target gene HEG1 during EMT. Cell cycle and apoptosis were analyzed by flow cytometry. Tumorigenesis was analyzed by clone formation assay. Wound healing assay and transwell assay was performed to detect migration and invasion, respectively. Interaction among SNHG12, miR-516a-5p and HEG1 were analyzed by dual luciferase assay and RIP assay. We also detected expression of RNA and protein by QPCR and western blotting. Finally, tumor growth was analyzed by tumorigenesis assay in vivo. Ki-67 and HEG1 level in tumor tissues was analyzed by IHC. SNHG12 and HEG1 were upregulated, miR-516a-5p was downregulated in HCC cell lines. SNHG12 could interact with and inhibit miR-516a-5p. MiR-516a-5p could interact with HEG1 and inhibit HEG1 expression. Knock down SNHG12 inhibited proliferation, migration, invasion, EMT and promoted apoptosis of HCC cells. Such effects were antagonized by inhibiting miR-516a-5p. SNHG12 overexpression lead to opposite results. Similar results were observed in mice. SNHG12 could promote EMT in HCC through targeting and inhibiting miR-516a-5p, which eventually upregulated HEG1 expression, in both cell and mice.     

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5′ base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.     

MicroRNA-340 suppresses osteosarcoma tumor growth and metastasis by directly targeting ROCK1

MicroRNAs (miRNAs) play key roles in cancer development and progression. In the present study, we investigated the role of miR-340 in the progression and metastasis of osteosarcoma (OS). Our results showed that miR-340 was frequently downregulated in OS tumors and cell lines. Overexpression of miR-340 in OS cell lines significantly inhibited cell proliferation, migration, and invasion in vitro, and tumor growth and metastasis in a xenograft mouse model. ROCK1 was identified as a target of miR-340, and ectopic expression of miR-340 downregulated ROCK1 by direct binding to its 3′ untranslated region. siRNA-mediated silencing of ROCK1 phenocopied the effects of miR-340 overexpression, whereas restoration of ROCK1 in miR-340-overexpressing OS cells reversed the suppressive effects of miR-340. Together, these findings indicate that miR-340 acts as a tumor suppressor and its downregulation in tumor tissues may contribute to the progression and metastasis of OS through a mechanism involving ROCK1, suggesting miR-340 as a potential new diagnostic and therapeutic target for the treatment of OS.