Downregulation of HOXA1 gene affects small cell lung cancer cell survival and chemoresistance under the regulation of miR-100.

Chemoresistance is often developed in small cell lung cancer (SCLC) patients and leads to poor prognosis. Hox genes, a highly conserved family, play a crucial role in apoptosis, receptor signalling and differentiation. MicroRNAs (miRNAs) have also been shown to play a crucial role in these biological processes by regulating the target genes. Several studies reported that both Hox genes and miRNAs are involved in chemoresistance. The aim of our study is to characterise the clinical significance and functional roles of HOXA1 in SCLC. Expression of HOXA1 was examined in 63 cases of SCLC tissues and 29 cases of blood by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methods. Multivariate analysis confirmed the prognostic significance of HOXA1 in SCLC patients. Restoration of HOXA1 expression was carried out in SCLC multidrug resistant cell line H69AR and its parental cell line H69 to assess its influence on chemoresistance. Luciferase reporter assay was used to assess HOXA1 as a target of miR-100. The results showed that HOXA1 was expressed in 46% (29/63) of SCLC. Low HOXA1 expression was associated with the poor prognosis of SCLC (P<0.05 by the Fisher's Exact Test) and the shorter survival rate (P<0.001 by the Kaplan-Meier method). HOXA1 expression on both mRNA and protein levels significantly correlated with chemotherapy response. Enforced expression of HOXA1 in resistant H69AR cells led to increased chemosensitivity through increasing cell apoptosis and cell-cycle arrest. Inhibition of HOXA1 expression using HOXA1 siRNA in H69 cells resulted in cell resistance to therapeutic drugs through reducing drug-induced cell apoptosis accompanied with cell cycle arrest. Expression of endogenous miR-100 was significantly elevated in resistant H69AR cells and negatively related with HOXA1 expression. The expression of HOXA1 in SCLC tissues correlated inversely with the expression levels of miR-100. Reporter assays confirmed that miR-100 targeted predicted sites in 3'-untranslated region (3'-UTR) of HOXA1 gene. Our data suggested that HOXA1-mediated SCLC chemoresistance is under the regulation of miR-100. HOXA1 may be a prognostic predictor and potential therapeutic target in human SCLC.

Xiao F ，Bai Y ，Chen Z ，Li Y ，Luo L ，Huang J ，Yang J ，Liao H ，Guo L ... -  《-》

Zinc finger E-box-binding homeobox 2 (ZEB2) regulated by miR-200b contributes to multi-drug resistance of small cell lung cancer.

Zinc finger E-box-binding homeobox 2 (ZEB2) was closely related to the oncogenesis, development and response to chemotherapy of cancer. However, its biological functions in small cell lung cancer (SCLC) remain unknown. The aim of this study is to investigate the roles of ZEB2 in chemoresistance of SCLC and its possible molecular mechanism. Expression of ZEB2 was examined in sixty-eight cases of SCLC tissues by immunohistochemistry. Knockdown of ZEB2 was carried out in SCLC multidrug resistant cells (H69AR) to assess its influence on chemoresistance. The results showed that ZEB2 was expressed in 23.5% (16/68) of SCLC. Overexpression of ZEB2 was associated with the poor pathologic stage of SCLC (P < 0.001 by the Fisher's Exact Test) and the shorter survival time (by the Kaplan-Meier method). Inhibition of ZEB2 expression using small interfering RNA in H69AR cells sensitized cancer cells to chemotherapeutic drugs through increasing drug-induced cell apoptosis accompanied with S phase arrest. In silico analysis demonstrated that there are complementary binding sites between miR-200b and ZEB2 3'-UTR, and identified miR-200b as a potential regulator of ZEB2. We found that miR-200b was down-regulated in the resistant cells and enforced expression of miR-200b by miRNA mimics increased cell sensitivity. Overexpression of miR-200b led to the downregulation of ZEB2 at protein level. Luciferase reporter gene assay showed that 3'UTR ZEB2 activity was regulated by miR-200b. Our results suggest that ZEB2 modulates drug resistance and is regulated by miR-200b. All findings provide insight into the ZEB2 signaling mechanism and ZEB2 may be a potentially novel target for multi-drug resistance in SCLC.

Fang S ，Zeng X ，Zhu W ，Tang R ，Chao Y ，Guo L ... -  《-》

Gene expression profiling of drug-resistant small cell lung cancer cells by combining microRNA and cDNA expression analysis.

MicroRNAs (miRNAs) are now known to play important roles in the regulation of gene expression for developmental timing, cell proliferation and apoptosis. Therefore, it is likely that they also modulate sensitivity and resistance to anti-cancer drugs. To better understand the molecular mechanisms of multidrug resistance in SCLC and identify novel molecular markers, we evaluated the expression of 856 miRNAs and approximately 22,000 genes using miRNA microarray and cDNA microarray in cellular models of SCLC which were widely used as sensitive (NCI-H69) and resistant cell lines (NCI-H69AR) to chemotherapy. We also analysed the correlations between miRNA and mRNA expression patterns. Further studies were tested to determine whether the differentially expressed miRNAs were involved in multidrug resistance in SCLC. Our results showed that 61 miRNAs are presented significantly (>3-fold) including up-regulation of 24 miRNAs and down-regulation of 37 miRNAs. Among these miRNAs, 48 of 61 differentially expressed miRNAs were firstly reported to be closely associated with drug resistance and 37.7% (24/61) of miRNA genes were organised as 10 clusters in total 61 significantly expressed miRNAs. We also found that only 27 of 69 miRNAs were significantly correlated with 604 of 21,522 70 mRNA transcripts by MAS database. The sensitivity to anti-cancer drugs Cisplatin, Etoposide and Doxorubicin greatly increased or reduced following transfection of the drug-resistant H69AR cells with the mimics or antagomirs of miR-134, miR-379 and miR-495, respectively. miR-134 increases the cell survival by inducing G1 arrest in H69AR cells. MRP1/ABCC1 is negatively regulated by miR-134 and down-regulation of MRP1/ABCC1 at the protein level largely correlates with elevated levels of miR-134 in H69AR cells. Our results support for the first time a substantial role for miRNAs in multidrug resistance in SCLC. miR-134 could be a causal factor of the down-regulation of MRP1/ABCC1 in H69AR cells. These findings provide valuable information for potential utility of these miRNAs as specific diagnostic biomarkers and novel therapeutic approaches for drug resistance of SCLC.

Guo L ，Liu Y ，Bai Y ，Sun Y ，Xiao F ，Guo Y ... -  《-》

miR-7 modulates chemoresistance of small cell lung cancer by repressing MRP1/ABCC1.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs and have been shown to play important roles in various biological processes including cell growth, differentiation and apoptosis by regulating the target genes. miR-7 has been described not only as a tumour suppressor gene but also as an oncogene in human cancers. The aim of this study was to investigate the functional roles of miR-7 in chemoresistance of SCLC and its underlying mechanism. By using a bioinformatic assay, we found that MRP1/ABCC1 was a potential target gene of miR-7. Expression of miR-7 and MRP1/ABCC1 was examined in 44 SCLC samples by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry methods. Low-level expression of miR-7 was associated significantly with drug responsiveness and overall survival rate of patients with SCLC, but not with gender, age and stage. There was an inverse relationship between miR-7 and MRP1/ABCC1 expression. Downregulation of MRP1/ABCC1 level was revealed after transfection with a miR-7 mimic in H69 AR cells. Transfection of a miR-7 inhibitor into H69 cells restored MRP1/ABCC1 expression. A dual-luciferase reporter assay confirmed that miR-7 targeted predicted sites in the 3'-untranslated region (3'-UTR) of the MRP1/ABCC1 gene. Our data suggested that miR-7 mediated SCLC chemoresistance by repressing MRP1/ABCC1 and may be a prognostic predictor and potential therapeutic target in human SCLC.

Liu H ，Wu X ，Huang J ，Peng J ，Guo L ... -  《-》

Upregulation of the inwardly rectifying potassium channel Kir2.1 (KCNJ2) modulates multidrug resistance of small-cell lung cancer under the regulation of miR-7 and the Ras/MAPK pathway.

Liu H ，Huang J ，Peng J ，Wu X ，Zhang Y ，Zhu W ，Guo L ... -  《Molecular Cancer》