MiR-223 promotes the cisplatin resistance of human gastric cancer cells via regulating cell cycle by targeting FBXW7

Background

Increasing evidence showed that miRNAs serve as modulators of human cancer, either as oncogene or tumor suppressors. Cisplatin resistance is the most common cause of chemotherapy failure in gastric cancer (GC). However, the roles of miRNAs in cisplatin resistance of GC remain largely unknown. The aim of the study was to identify a novel miRNA/gene pathway that regulates the sensitivity of GC cells to cisplatin.

Methods

In this study, we chose miR-223 by qRT-PCR analysis, the most significantly up-regulated miRNA in GC, to investigate its formation of DDP-resistant phenotype of GC cells and possible molecular mechanisms.

Results

We found that miR-223 was most significantly up-regulated miRNA in DDP-resistant GC cells compared with parental GC cells. Besides, its expression was also significantly up-regulated in GC tissues. FBXW7 was identified as the direct and functional target gene of miR-223. Overexpression of FBXW7 could mimic the effect of miR-223 down-regulation and silencing of FBXW7 could partially reverse the effect of miR-223 down-regulation on DDP resistance of DDP-resistant GC cells. Besides, miR-223 and FBXW7 could affect the G1/S transition of cell cycle by altering some certain cell cycle regulators. Furthermore, miR-223 was found to be significantly up-regulated in H. pylori infected tissues and cells, suggesting that H. pylori infection may lead to GC development and DDP resistance.

Conclusions

Our findings revealed the roles of miR-223/FBXW7 signaling in the DDP resistance of GC cells and targeting it will be a potential strategic approach for reversing the DDP resistance in human GC.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-015-0145-6) contains supplementary material, which is available to authorized users.     

miR‐106a is frequently upregulated in gastric cancer and inhibits the extrinsic apoptotic pathway by targeting FAS

Emerging evidence has shown the association of aberrantly expressed miR‐106a with cancer development, however, little is known about its potential role in gastric carcinogenesis. In our present study, obviously overexpressed miR‐106a was found in gastric cancer tissues compared with their nontumor counterparts. Suppression of miR‐106a significantly inhibited gastric cancer cell proliferation and triggered apoptosis. Bioinformatic analysis combining with validation experiments identified FAS as a direct target of miR‐106a. Rescue experiments and examination of caspase‐8, PARP and caspase‐3 further approved that miR‐106a could inhibit gastric cancer cell apoptosis through interfering with FAS‐mediated apoptotic pathway. Moreover, a significant inverse correlation was found between miR‐106a and FAS expression not only in gastric cancer cell lines but also in gastric cancer specimens. Taken together, these findings suggest that ectopicly overexpressed miR‐106a may play an oncogenic role in gastric carcinogenesis and impair extrinsic apoptotic pathway through targeting FAS. © 2012 Wiley Periodicals, Inc.     

miR‐193b is an epigenetically regulated putative tumor suppressor in prostate cancer

miRNAs have proven to be key regulators of gene expression and are differentially expressed in various diseases, including cancer. Our aim was to identify epigenetically dysregulated genes in prostate cancer. We performed miRNA expression profiling after relieving epigenetic modifications in 6 prostate cancer cell lines and nonmalignant prostate epithelial cells. Thirty‐eight miRNAs showed increased expression in any prostate cancer cell line after 5‐aza‐2′‐deoxycytidine (5azadC) and trichostatin A (TSA) treatments. Six of these also had decreased expression in clinical prostate cancer samples compared to benign prostatic hyperplasia. Among these, miR‐193b was methylated in 22Rv1 cell line at a CpG island ～1 kb upstream of the miRNA locus. Expressing miR‐193b in 22Rv1 cells using pre‐miR‐193b oligonucleotides caused a significant growth reduction (p < 0.001) resulting from a decrease of cells in S‐phase of the cell cycle (p < 0.01). In addition, the anchorage independent growth was partially inhibited in transiently miR‐193b‐expressing 22Rv1 cells (p < 0.01). Altogether, our data suggest that miR‐193b is an epigenetically silenced putative tumor suppressor in prostate cancer.     

ERGIC3, which is regulated by miR‐203a,is a potential biomarker for non‐small cell lung cancer

In a previous study, we found that ERGIC3 was a novel lung cancer‐related gene by screening libraries of differentially expressed genes. In this study, we developed a new murine monoclonal antibody (mAb) against ERGIC3. This avid antibody (6‐C4) is well suited for immunohistochemistry, immunoblotting and solid‐phase immunoassays. Furthermore, we systematically investigated expressions of ERGIC3 in a broad variety of normal human tissues and various types of tumors by immunohistochemistry. In normal human tissues, 6‐C4 reacted only in some epithelial cells, such as hepatocytes, gastrointestinal epithelium, ducts and acini of the pancreas, proximal and distal tubules of the kidney, and mammary epithelial cells; however, most normal human tissues were not stained. Moreover, almost all carcinomas that originated from the epithelial cells were positive for 6‐C4, whereas all sarcomas were negative. Notably, 6‐C4 strongly stained non‐small cell lung cancer (NSCLC) cells but did not react with normal lung tissues. Hence, ERGIC3 mAb could be used in histopathological diagnosis and cytopathological testing to detect early‐stage NSCLC. We also studied the mechanisms of ERGIC3 regulation in vitro and in vivo by means of bioinformatics analysis, luciferase reporter assay, miRNA expression profiling and miRNA transfection. Results showed that miR‐203a downregulation induced ERGIC3 overexpression in NSCLC cells.     

Gelsolin inhibits malignant phenotype of glioblastoma and is regulated by miR‐654‐5p and miR‐450b‐5p

We have previously shown that gelsolin (GSN) levels are significantly lower in the blood of patients with glioblastoma (GBM) than in healthy controls. Here, we analyzed the function of GSN in GBM and examined its clinical significance. Furthermore, microRNAs involved in GSN expression were also identified. The expression of GSN was determined using western blot analysis and found to be significantly lower in GBM samples than normal ones. Gelsolin was mainly localized in normal astrocytes, shown using immunohistochemistry and immunofluorescence. Higher expression of GSN was correlated with more prolonged progression‐free survival and overall survival. Gelsolin knockdown using siRNA and shRNA markedly accelerated cell proliferation and invasion in GBM in vitro and in vivo. The inactive form of glycogen synthase kinase‐3β was dephosphorylated by GSN knockdown. In GBM tissues, the expression of GSN and microRNA (miR)‐654‐5p and miR‐450b‐5p showed an inverse correlation. The miR‐654‐5p and miR‐450b‐5p inhibitors enhanced GSN expression, resulting in reduced proliferation and invasion. In conclusion, GSN, which inhibits cell proliferation and invasion, is suppressed by miR‐654‐5p and miR‐450b‐5p in GBM, suggesting that these miRNAs can be targets for treating GBM.     

FBXW7/hCDC4 is a general tumor suppressor in human cancer

The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skp1-Cdc53/Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin E1, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of approximately 6%. The highest mutation frequencies were found in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational "hotspots," which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer.     

Genetic polymorphism of miR‐146a is associated with gastric cancer risk: a meta‐analysis

Several studies have investigated the associations between miR‐146a rs2910164 and gastric cancer (GC) risk, but results have been inconclusive. To derive a more precise estimation of the relationship, a meta‐analysis was performed. PubMed and China National Knowledge Infrastructure searches were carried out for relevant studies published before July 2014. Meta‐analysis was performed with the stata , version 11.0. A total of seven case–control studies, including 3283 cases and 4535 controls, were selected. A significant association was found between rs2910164 and GC risk under all genetic models (CC vs. GG, OR = 0.76, 95% CI = 0.66–0.87; CC vs. GC+GG, OR = 0.84, 95% CI = 0.71–0.99; CC+GC vs. GG, OR = 0.82, 95% CI = 0.73–0.91) for the total data. In the subgroup analysis by ethnicity, statistically significant association was found in Asian. This meta‐analysis suggested that the miR‐146a rs2910164 was a risk factor for developing GC.     

miR‐212 is downregulated and suppresses methyl‐CpG‐binding protein MeCP2 in human gastric cancer

To clarify the role of micro (mi) RNAs in gastric carcinogenesis, we studied the expression and function of miRNAs in gastric carcinoma (GC) cells. Initially, we performed microarray analysis using total RNA from 3 human GC cell lines and noncancerous gastric tissue. Among the downregulated miRNAs in GC cells, miR‐212 expression was decreased in all 8 GC cell lines examined and a significant decrease of miR‐212 expression in human primary GC tissues was also observed in 6 of 11 cases. Transfection of the precursor miR‐212 molecule induced decreased growth of 3 GC cell lines. Using 3 different databases, methyl‐CpG‐binding protein MeCP2 was postulated to be a target of miR‐212. As seen on reporter assaying, miR‐212 repressed the construct with the MECP2 3′‐UTR. Ectopic expression of miR‐212 repressed expression of the MeCP2 protein but not the MECP2 mRNA level. These data suggest that downregulation of miR‐212 may be related to gastric carcinogenesis through its target genes, such as MECP2.     

Dickkopf-1 is regulated by the mevalonate pathway in breast cancer

Introduction

Amino-bisphosphonates and statins inhibit the mevalonate pathway, and may exert anti-tumor effects. The Wnt inhibitor dickkopf-1 (DKK-1) promotes osteolytic bone lesions by inhibiting osteoblast functions and has been implicated as an adverse marker in multiple cancers. We assessed the effects of mevalonate pathway inhibition on DKK-1 expression in osteotropic breast cancer.

Methods

Regulation of DKK-1 by bisphosphonates and statins was assessed in human breast cancer cell lines, and the role of the mevalonate pathway and downstream targets was analyzed. Moreover, the potential of breast cancer cells to modulate osteoblastogenesis via DKK-1 was studied in mC2C12 cells. Clinical relevance was validated by analyzing DKK-1 expression in the tissue and serum of women with breast cancer exposed to bisphosphonates.

Results

DKK-1 was highly expressed in receptor-negative breast cancer cell lines. Patients with receptor-negative tumors displayed elevated levels of DKK-1 at the tissue and serum level compared to healthy controls. Zoledronic acid and atorvastatin potently suppressed DKK-1 in vitro by inhibiting geranylgeranylation of CDC42 and Rho. Regulation of DKK-1 was strongest in osteolytic breast cancer cell lines with abundant DKK-1 expression. Suppression of DKK-1 inhibited the ability of breast cancer cells to block WNT3A-induced production of alkaline phosphates and bone-protective osteoprotegerin in preosteoblastic C2C12 cells. In line with the in vitro data, treatment of breast cancer patients with zoledronic acid decreased DKK-1 levels by a mean of 60% after 12 months of treatment.

Conclusion

DKK-1 is a novel target of the mevalonate pathway that is suppressed by zoledronic acid and atorvastatin in breast cancer.     

MAPK/ERK通路影响人胃癌细胞对奥沙利铂敏感性的机制研究

目的:探讨MAPK/ERK通路对人胃癌细胞奥沙利铂敏感性的影响,并进一步探讨其机制.方法:采用MTT法测定奥沙利铂对人胃癌BGC823和SGC7901细胞的增殖抑制影响;MAPK/ERK特异性抑制剂PD98059预处理上述两种细胞后,测定奥沙利铂的药物敏感性.Western blot方法检测胃癌细胞中p-ERK、ERK及谷胱甘肽-S-转移酶π(glutathione S-transferases π,GST-π)蛋白表达.应用SPSS 13.0软件包进行统计学分析.结果:1mg/L-100mg/L奥沙利铂分别作用BGC823和SGC7901细胞,48h的IC50分别为24.26mg/L和18.44 mg/L;20μmol/L的PD98059预处理BGC823和SGC7901细胞,再加入奥沙利铂继续培养48h,IC50分别降至12.42mg/L和7.97mg/L,表明对奥沙利铂的敏感性显著提高.进一步检测发现PD98059处理BGC823和SGC7901细胞24h后,p-ERK蛋白表达明显下调,GST-π蛋白亦显著下调.结论:PD98059通过抑制MAPK/ERK通路,下调GST-π蛋白表达,显著提高人胃癌细胞对奥沙利铂的药物敏感性.     

Overexpression of microRNA-223 regulates the ubiquitin ligase FBXW7 in oesophageal squamous cell carcinoma

Background:

F-box and WD repeat domain-containing 7 (FBXW7) is a cell cycle regulatory gene whose protein product ubiquitinates positive cell cycle regulators such as c-Myc, cyclin E, and c-Jun, thereby acting as a tumour-suppressor gene. This study focused on microRNA-223 (miR-223), which is a candidate regulator of FBXW7 mRNA. The aim of this study was to clarify the clinical significance of miR-223 and FBXW7 in oesophageal squamous cell carcinoma (ESCC) patients, and to elucidate the mechanism by which FBXW7 is regulated by miR-223.

Methods:

The expression levels of miR-223 and the expression of FBXW7 protein was examined using 109 resected specimens to determine the clinicopathological significance. We also investigated the role of miR-223 in the regulation of FBXW7 expression in ESCC cell lines in an in vitro analysis.

Results:

We found that miR-223 expression was significantly higher in cancerous tissues than in the corresponding normal tissues. There was a significant inverse relationship between the expression levels of miR-223 and FBXW7 protein. Moreover, patients with high miR-223 expression demonstrated a significantly poorer prognosis than those with low expression. On the basis of a series of gain-of-function and loss-of-function studies in vitro, we identified FBXW7 as a functional downstream target of miR-223.

Conclusion:

Our present study indicates that high expression of miR-223 had a significant adverse impact on the survival of ESCC patients through repression of the function of FBXW7.