miR‐135b‐ and miR‐146b‐dependent silencing of calcium‐sensing receptor expression in colorectal tumors

Studies have shown that the calcium‐sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco‐2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain‐ and loss‐of‐function studies with the top candidates: miR‐9, miR‐27a, miR‐135b, and miR‐146b. Modulation of miR‐135b or miR‐146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR‐135b and miR‐146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR‐135b and miR‐146b, and this correlated inversely with CaSR expression (miR‐135b: r = ?0.684, p < 0.001 and miR‐146b: r = ?0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR‐135b and miR‐146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.     

微小RNA-148a抑制乳腺癌细胞MDA-MB-231迁移的作用机制

目的:研究微小RNA-148a(miR-148a)在乳腺癌细胞株MDA-MB-231中的表达,探讨上调miR-148a的表达对其迁移的影响及机制.方法:采用实时荧光定量PCR(qRT-PCR)检测正常乳腺上皮细胞MCF-10A和乳腺癌细胞株MDA-MB-231中miR-148a的表达水平.应用脂质体法将miR-148a mimic及阴性对照分别转染MDA-MB-231细胞,qRT-PCR检测转染效率;细胞划痕实验检测转染前后MDA-MB-231细胞迁移能力的变化;Western blot方法检测上皮间质转化(EMT)相关蛋白Slug、Snail和E-cadher-in表达水平的变化.结果:与MCF-10A细胞相比,MDA-MB-231细胞中miR-148a的表达水平明显降低(P<0.05);与阴性对照组相比,miR-148a转染组中miR-148a的表达水平显著升高(P<0.05);过表达miR-148a后,MDA-MB-231细胞迁移能力明显下降,Slug和Snail蛋白表达明显降低,E-cadherin蛋白表达明显增加.结论:miR-148a可通过调控Slug、Snail及E-cadherin蛋白的表达抑制EMT,进而抑制乳腺癌细胞的迁移能力.     

Extracellular calcium and calcium sensing receptor function in human colon carcinomas: promotion of E-cadherin expression and suppression of beta-catenin/TCF activation

Ca(2+) has chemopreventive activity against colon cancer, albeit its mechanisms of action are not understood. In this study, we showed that four different human colon carcinoma cell lines (FET, SW480, MOSER, and CBS) expressed the human parathyroid calcium sensing receptor (CaSR) and that a function of extracellular Ca(2+) and the CaSR in these cells was the promotion of E-cadherin expression and suppression of beta-catenin/T cell factor activation. We also found that human colonic crypt epithelial cells expressed the CaSR, and histologically differentiated carcinomas (i.e., where three-dimensional, crypt-like structures were present) expressed less receptor by comparison, whereas an almost complete loss of CaSR expression was observed in undifferentiated tumors. These results suggest that extracellular Ca(2+) and the CaSR may function to regulate the differentiation of colonic epithelial cells and that disruption of this ligand receptor system may contribute to abnormal differentiation and malignant progression. In addition, the promotion of E-cadherin and suppression of beta-catenin/T cell factor may be an important mechanism underlying the chemopreventive action of Ca(2+) in colon cancer.     

Alterations of microRNAs and their targets are associated with acquired resistance of MCF‐7 breast cancer cells to cisplatin

Cancer cells that develop resistance to chemotherapeutic agents are a major clinical obstacle in the successful treatment of breast cancer. Acquired cancer chemoresistance is a multifactorial phenomenon, involving various mechanisms and processes. Recent studies suggest that chemoresistance may be linked to drug‐induced dysregulation of microRNA function. Furthermore, mounting evidence indicates the existence of similarities between drug‐resistant and metastatic cancer cells in terms of resistance to apoptosis and enhanced invasiveness. We studied the role of miRNA alterations in the acquisition of cisplatin‐resistant phenotype in MCF‐7 human breast adenocarcinoma cells. We identified a total of 103 miRNAs that were overexpressed or underexpressed (46 upregulated and 57 downregulated) in MCF‐7 cells resistant to cisplatin. These differentially expressed miRNAs are involved in the control of cell signaling, cell survival, DNA methylation and invasiveness. The most significantly dysregulated miRNAs were miR‐146a, miR‐10a, miR‐221/222, miR‐345, miR‐200b and miR‐200c. Furthermore, we demonstrated that miR‐345 and miR‐7 target the human multidrug resistance‐associated protein 1. These results suggest that dysregulated miRNA expression may underlie the abnormal functioning of critical cellular processes associated with the cisplatin‐resistant phenotype.     

Tumor‐suppressive microRNA‐135a inhibits cancer cell proliferation by targeting the c‐MYC oncogene in renal cell carcinoma

Recently, many studies have suggested that microRNAs (miRNAs) are involved in cancer cell development, invasion, and metastasis of various types of human cancers. In a previous study, miRNA expression signatures from renal cell carcinoma (RCC) revealed that expression of microRNA‐135a (miR‐135a) was significantly reduced in cancerous tissues. The aim of this study was to investigate the functional significance of miR‐135a and to identify miR‐135a‐mediated molecular pathways in RCC cells. Restoration of mature miR‐135a significantly inhibited cancer cell proliferation and induced G₀/G₁ arrest in the RCC cell lines caki2 and A498, suggesting that miR‐135a functioned as a potential tumor suppressor. We then examined miR‐135a‐mediated molecular pathways using genome‐wide gene expression analysis and in silico analysis. A total of 570 downregulated genes were identified in miR‐135a transfected RCC cell lines. To investigate the biological significance of potential miR‐135a‐mediated pathways, we classified putative miR‐135a‐regulated genes according to the Kyoto Encyclopedia of Genes and Genomics pathway database. From our in silico analysis, 25 pathways, including the cell cycle, pathways in cancer, DNA replication, and focal adhesion, were significantly regulated by miR‐135a in RCC cells. Moreover, based on the results of this analysis, we investigated whether miR‐135a targeted the c‐MYC gene in RCC. Gain‐of‐function and luciferase reporter assays showed that c‐MYC was directly regulated by miR‐135a in RCC cells. Furthermore, c‐MYC expression was significantly upregulated in RCC clinical specimens. Our data suggest that elucidation of tumor‐suppressive miR‐135a‐mediated molecular pathways could reveal potential therapeutic targets in RCC.     

Hypoxia‐induced downregulation of miR‐30c promotes epithelial‐mesenchymal transition in human renal cell carcinoma

MicroRNAs (miRNAs), which negatively regulate protein expression by binding protein‐coding mRNAs, have been integrated into cancer development and progression as either oncogenes or tumor suppressor genes. miR‐30c was reported to be downregulated in several types of cancer. However, its role in human renal cell carcinoma (RCC) remains largely unknown. Here, we show that miR‐30c is significantly downregulated in human RCC tissues and cell lines. We found that miR‐30c downregulation could be induced by hypoxia in RCC cells in a hypoxia‐inducible factors (HIFs) dependent manner. Repression of miR‐30c through its inhibitor resulted in reduction of E‐cadherin production and promotion of epithelial‐mesenchymal transition (EMT), while overexpression of miR‐30c inhibited EMT in RCC cells. We identified Slug as a direct target of miR‐30c in RCC cells. Slug was upregulated in RCC tissues and its expression could be induced by hypoxia, which is consistent with downregulation of miR‐30c by hypoxia. Forced overexpression of Slug in 786‐O cells reduced E‐cadherin production, and promoted EMT as well as cell migration. Moreover, Slug overexpression abrogated the inhibitory role of miR‐30c in regulating EMT and cell migration, indicating miR‐30c regulates EMT through Slug in RCC cells. Our findings propose a model that hypoxia induces EMT in RCC cells through downregulation of miR‐30c, which leads to subsequent increase of Slug expression and repression of E‐cadherin production, and suggest a potential application of miR‐30c in RCC treatment.     

miR‐25‐3p reverses epithelial‐mesenchymal transition via targeting Sema4C in cisplatin‐resistance cervical cancer cells

Acquisition of epithelial‐mesenchymal transition (EMT) has recently been proposed as an important contributor of drug resistance in cervical cancer cells. However, the underlying mechanisms are still unclear. MicroRNAs play a crucial role in regulating EMT. The aim of this study was to explore the potential role of miR‐25‐3p in regulating EMT in cisplatin‐resistant (CR) cervical cancer cells. To this end, we established stable CR cervical cancer cells, HeLa‐CR and CaSki‐CR, and investigated the function of miR‐25‐3p in regulating EMT. It is found that CR cervical cancer cells possessed more EMT characteristics and demonstrated higher migratory abilities and invasiveness. miR‐25‐3p downregulation was also seen in HeLa‐CR and CaSki‐CR cells. Of note, ectopic expression of miR‐25‐3p reversed the EMT phenotype and sensitized CR cells to cisplatin via targeting Sema4C. Furthermore, stable overexpression of miR‐25‐3p in HeLa‐CR cells suppressed tumor growth in mice, downregulated Sema4C and Snail, and upregulated E‐cadherin compared with the control group. These results suggest that miR‐25‐3p is an important regulator of cervical cancer EMT and chemoresistance. Thus, upregulation of miR‐25‐3p could be a novel approach to treat cervical cancers that are resistant to chemotherapy.     

Calcium and calcium sensing receptor modulates the expression of thymidylate synthase, NAD(P)H:quinone oxidoreductase 1 and survivin in human colon carcinoma cells: promotion of cytotoxic response to mitomycin C and fluorouracil

Ca(2+) and the cell-surface calcium sensing receptor (CaSR) constitute a novel and robust ligand/receptor system in regulating the proliferation and differentiation of colonic epithelial cells. Here we show that activation of CaSR by extracellular Ca(2+) (or CaSR agonists) enhanced the sensitivity of human colon carcinoma cells to mitomycin C (MMC) and fluorouracil (5-FU). Activation of CaSR up-regulated the expression of MMC activating enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and down-regulated the expression of 5-FU target, thymidylate synthase (TS) and the anti-apoptotic protein survivin. Cells that were resistant to drugs expressed little or no CaSR but abundant amount of survivin. Disruption of CaSR expression by shRNA targeting the CaSR abrogated these modulating effects of CaSR activation on the expression of NQO1, TS, survivin and cytotoxic response to drugs. It is concluded that activation of CaSR can enhance colon cancer cell sensitivity to MMC and 5-FU and can modulate the expression of molecules involved in the cellular responses to these cytotoxic drugs.     

Significance of P‐cadherin overexpression and possible mechanism of its regulation in intrahepatic cholangiocarcinoma and pancreatic cancer

It has become evident that P‐cadherin, one of the classical cadherins, contributes to the malignant behavior of several types of cancer. In this study, we analyzed the expression of P‐cadherin and its clinicopathological and prognostic values in intrahepatic cholangiocarcinoma (ICC) and pancreatic cancer. Furthermore, we investigated the functional role of P‐cadherin in these cancer cells by knockdown and overexpression in vitro and by analyzing the correlation between the P‐cadherin expression and its promoter methylation status. Thirty of 59 ICC cases (51%) and 36 of 73 pancreatic cancer cases (49%) stained positive for P‐cadherin with mainly membranous distribution in tumor cells by immunohistochemistry. P‐cadherin expression was significantly correlated with several clinicopathological factors, which reflect tumor behavior, and was identified as an independent adverse prognostic factor for disease‐free survival in patients with ICC (relative risk [RR] 2.93, P = 0.04) and pancreatic cancer (RR 2.68, P = 0.005) via multivariate analyses. P‐cadherin downregulation by siRNA suppressed migration and invasion, and P‐cadherin overexpression induced the opposite effects in both ICC and pancreatic cancer cells, without any effects on cell proliferation. P‐cadherin expression was related to its promoter methylation status in both cell lines and cancer tissues. In summary, P‐cadherin overexpression may serve as a useful biomarker of invasive phenotype and poor prognosis; P‐cadherin expression was found to be regulated by its promoter methylation. These results suggest that P‐cadherin represents a novel therapeutic target for the treatment of ICC and pancreatic cancer.     

Induced miR‐31 by 5‐fluorouracil exposure contributes to the resistance in colorectal tumors

Drug resistance makes treatment difficult in cancers. The present study identifies and analyzes drug resistance‐related miRNA in colorectal cancer. We established 4 types of 5‐fluorouracil (5‐FU)‐resistant colon cancer cell lines in vitro and in vivo. We then analyzed the miRNA expression profile by miRNA array in these 4 cell lines, and identified the drug resistance‐related miRNAs. We examined the expression levels of the identified miRNA in 112 colorectal tumor samples from the patients. We identified 12 possible miRNAs involved in 5‐FU resistance by miRNA arrays. We then examined the relationship between miR‐31, which was the most promising among them, and drug resistance. The ectopic expression of mimic miR‐31 showed significant 5‐FU resistance in the parental DLD‐1 cells, while anti–miR‐31 caused significant growth inhibition in DLD/F cells; that is, 5‐FU‐resistant colon cancer cell line DLD‐1 under exposure to 5‐FU. When we exposed high doses of 5‐FU to parent or 5‐FU‐resistant cells, the expression levels of miR‐31 were raised higher than those of controls. Notably, the expression levels of miR‐31 were positively correlated with the grade of clinical stages of colorectal tumors. The protein expression levels of factors inhibiting hypoxia‐inducible factor 1 were downregulated by transfection of mimic miR‐31 into DLD‐1 cells. This study provides evidence supporting the association of miR‐31 with 5‐FU drug resistance and clinical stages of colorectal tumors.