Yiguo Jiang

MicroRNA-622 functions as a tumor suppressor by targeting K-Ras and enhancing the anticarcinogenic effect of resveratrol

Aberrant expression of microRNA (miRNA) has been previously demonstrated to play an important role in a wide range of cancer types and further elucidation of its role in the mechanisms underlying tumorigenesis, anticarcinogenesis and potential chemotherapeutics is warranted. We chose the anti-benzo[a]pyrene-7,8-diol-9,10-epoxide-transformed human bronchial epithelial cell line 16HBE-T to study miRNAs involved in anticarcinogenesis. In resveratrol-treated cells, we found that miR-622 was upregulated, whereas it was downregulated in 16HBE-T cells, suggesting that miR-622 potentially acts as a tumor suppressor. Increasing the level of miR-622 by transient transfection-induced inhibition of proliferation and G(0) arrest in 16HBE-T cells and the lung cancer cell line H460 as demonstrated by cell viability and cell cycle analysis. MiR-622 dramatically suppressed the ability of 16HBE-T cells to form colonies in vitro and to develop tumors in nude mice. According to bioinformatics analysis, K-Ras messenger RNA was predicted as a putative miR-622 target; this was confirmed by western blot and luciferase reporter assays. Cell growth retardation was inhibited upon knockdown of K-Ras and an increase in the level of miR-622 in 16HBE-T cells. Furthermore, miR-622 inhibitor partially impaired the growth of 16HBE-T cells as demonstrated by luciferase reporter activity and K-Ras protein expression in 16HBE-T cells. In summary, miR-622 functions as a tumor suppressor by targeting K-Ras and impacting the anticancer effect of resveratrol. Therefore, miR-622 is potentially useful as a clinical therapy. MiR-622 impacts the K-Ras signal pathway and the potentially anticarcinogenic or chemotherapeutic properties warrant further investigation.

MicroRNA-200b targets protein kinase Cα and suppresses triple-negative breast cancer metastasis

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis and lacks effective targeted therapies. The microRNA-200 (miR-200) family is found to inhibit or promote breast cancer metastasis; however, the underlying mechanism is not well understood. This study was performed to investigate the effect and mechanism of miR-200b on TNBC metastasis and identify targets for developing more efficient treatment for TNBC. We found that miR-200 family expression levels are significantly lower in highly migratory TNBC cells and metastatic TNBC tumors than other types of breast cancer cells and tumors. Ectopically expressing a single member (miR-200b) of the miR-200 family drastically reduces TNBC cell migration and inhibits tumor metastasis in an orthotopic mouse mammary xenograft tumor model. We identified protein kinase Cα (PKCα) as a new direct target of miR-200b and found that PKCα protein levels are inversely correlated with miR-200b levels in 12 kinds of breast cancer cells. Inhibiting PKCα activity or knocking down PKCα levels significantly reduces TNBC cell migration. In contrast, forced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration and tumor metastasis. Further mechanistic studies revealed that PKCα downregulation by miR-200b results in a significant decrease of Rac1 activation in TNBC cells. These results show that loss of miR-200b expression plays a crucial role in TNBC aggressiveness and that miR-200b suppresses TNBC cell migration and tumor metastasis by targeting PKCα. Our findings suggest that miR-200b and PKCα may serve as promising therapeutic targets for metastatic TNBC.

Akt Activation Is Responsible for Enhanced Migratory and Invasive Behavior of Arsenic-Transformed Human Bronchial Epithelial Cells

Background: Arsenic is one of the most common environmental contaminants. Long-term exposure to arsenic causes human bronchial epithelial cell (HBEC) malignant transformation and lung cancer. However, the mechanism of arsenic lung carcinogenesis is not clear, and the migratory and invasive properties of arsenic-transformed cells (As-transformed cells) have rarely been studied. Objectives: This study was designed to investigate the migratory and invasive behavior of As-transformed HBECs and the underlying mechanism. Methods: As-transformed p53lowHBECs were generated by exposing p53-knockdown HBECs to sodium arsenite (2.5 μM) for 16 weeks. Cell migration was assessed by transwell migration and wound-healing assay. Cell invasion was evaluated using Matrigel-coated transwell chambers. Gene overexpression, small interfering RNA (siRNA) knockdowns, and pharmacological inhibitors were used to determine the potential mechanism responsible for enhanced cell migration and invasion. Results: Transwell migration and invasion assays revealed that As-transformed p53lowHBECs were highly migratory and invasive. Akt (also known as protein kinase B) and extracellular signal–regulated protein kinase 1/2 (Erk1/2) were strongly activated in As-transformed p53lowHBECs. Stable expression of microRNA 200b in As-transformed p53lowHBECs abolished Akt and Erk1/2 activation and completely suppressed cell migration and invasion. Pharmacological inactivation of Akt but not Erk1/2 significantly decreased cell migration and invasion. Inhibition of Akt reduced the expression of epithelial-to-mesenchymal transition–inducing transcription factors zinc-finger E-box–binding homeobox factor 1 (ZEB1) and ZEB2. siRNA knockdown of ZEB1 and ZEB2 impaired As-transformed p53lowHBEC migration and invasion. Conclusions: Akt activation plays a critical role in enabling As-transformed HBEC migration and invasion by promoting ZEB1 and ZEB2 expression.

MicroRNA Expression Profiles and MiR-10a Target in Anti-benzo[a] pyrene-7,8-diol-9,10-epoxide-transformed Human 16HBE Cells

OBJECTIVE To screen miRNA profiles of malignantly transformed human bronchial epithelial cells, 16HBE-T, induced by anti-benzo[a]pyrene-trans-7,8-diol-9,10-epoxide (anti-BPDE), and to analyze putative miR-10a targets in 16HBE-T. METHODS A novel microarray platform was employed to screen miRNA profiles of 16HBE-T cells transformed by anti-BPDE. Microarray data for miR-10a and miR-320 were validated using quantitative real time polymerase chain reaction (QRT-PCR). The expression of a putative target for miR-10a, HOXA1, was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and QRT-PCR. RESULTS In comparison with the vehicle-treated cells (16HBE-N), 16HBE-T exhibited differential expression of 54 miRNAs, in which, 45 were over-expressed and 9 were down-regulated. The five most highly expressed miRNAs were miR-494, miR-320, miR-498, miR-129, and miR-106a. The lowest expressed miRNAs were miR-10a, miR-493-5p, and miR-363*. Three members of miR-17-92 cluster, miR-17-5p, miR-20a, and miR-92, showed significantly higher abundance in 16BHE-T as miR-21, miR-141, miR-27a, miR-27b, miR-16 and miRNAs of the let-7 family. The putative target for miR-10a, HOXA1 mRNA was up-regulated 3-9-fold in 16HBE-T, as compared with 16HBE-N. CONCLUSION The findings of the study provide information on differentially expressed miRNA in malignant 16HBE-T, and also suggest a potential role of these miRNAs in cell transformation induced by anti-BPDE. HOXA1 is similarly up-regulated, suggesting that miR-10a is associated with the process of HOXA 1-mediated transformation.

miR-106a–mediated Malignant Transformation of Cells Induced by Anti-benzo[a]pyrene-trans-7,8-diol-9,10-epoxide

microRNAs (miRNAs) are an abundant class of small noncoding RNAs that function primarily as oncogenes and tumor suppressors by mediating translational repression or mRNA degradation via binding target genes. In this study, malignant human bronchial epithelial cells transformed by anti-benzo[a]pyrene-trans-7,8-diol-9,10-epoxide were used to help characterize the possible mechanisms of miRNA function in chemical carcinogenesis. The expression level of miR-106a was measured by the real-time, reverse transcriptase polymerase chain reaction. We used the miR-106a inhibitor and the miR-106a mimic to downregulate or upregulate miR-106a activity in malignantly transformed cells to determine the effects of miR-106a on the biological properties of the cell. We observed overrepresentation of miR-106a in transformed cells compared with control cells. Silencing miR-106a by transfection with the miR-106a inhibitor suppressed cell proliferation, induced cell cycle arrest and apoptosis, and inhibited anchorage-independent growth and tumor growth in nude mice. Increasing miR-106a in malignantly transformed cells by transfection with the miR-106a mimic gave the opposite results. Moreover, untransformed cells showed a reduction of cell cycle arrest and apoptosis rate followed by transfection with the miR-106a mimic. Bioinformatic analysis showed that tumor suppressor RB1 is one of predictive targets of miR-106a. We confirmed this target by Western blot and dual luciferase assay. Our findings suggest that miR-106a might function as an oncogene in transformation induced by a chemical carcinogen. Thus, knock down of miR-106a in malignant cells is a potential therapeutic strategy.

Alteration of serum miR-206 and miR-133b is associated with lung carcinogenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The alteration of microRNA (miRNA) expression plays an important role in chemical carcinogenesis. Presently, few reports have been published that concern the significance of circulating miRNAs in lung carcinogenesis induced by environmental carcinogens. The purpose of this study was to identify serum miRNAs that could be associated with lung carcinogenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Male F344 rats were systemically administered with NNK. The rat serum differential expression profiles of miRNAs were analyzed by small RNA solexa sequencing. Using quantitative real-time PCR, the differentially expressed serum miRNAs were identified in each individual rat. Serum miR-206 and miR-133b were selected for further identification in rat serum at different stages of lung carcinogenesis; we detected the levels of serum miR-206 and miR-133b in lung cancer tissues induced by NNK. NNK causes significant alteration of serum miRNA expression. Compared to the control group, serum miR-206 and miR-133b were significantly up-regulated in the early stage of NNK-induced lung carcinogenesis. miR-206 and miR-133b exhibited low-expression in lung cancer tissues. Our results demonstrate that lung carcinogen NNK exposure changes the expression of serum miRNAs. Serum miR-206 and miR-133b could be associated with lung carcinogenesis induced by NNK.

The role of miR-506 in transformed 16HBE cells induced by anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide

Growing evidence indicates that the alteration of microRNA (miRNA) expression in tumors that is induced by chemical carcinogens plays an important role in tumor development and progression. However, the mechanism underlying miRNA involvement in lung carcinogenesis induced by anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-BPDE) remains unclear. In our study, we used the malignant transformation of human bronchial epithelial cells (16HBE-T) induced by anti-BPDE to explore the mechanisms of human lung carcinogenesis. We found that expression of miR-506 was reduced in 16HBE-T transformed malignant human bronchial epithelial cells compared with 16HBE normal human bronchial epithelial cells. Restoration of miR-506 in 16HBE-T cells led to a decrease in cell proliferation, G0/G1 phase cell cycle arrest, as well as significantly suppressed anchorage-dependent growth in vitro and tumor growth inhibition in a nude mouse xenograft model. In addition, we provided novel evidence regarding the role miR-506 potentially plays in negatively regulating the protein and mRNA expression level of N-Ras in cancer cells. Together, these findings revealed that miR-506 acts as an anti-oncogenic miRNA (anti-oncomir) in malignantly transformed cells. The identification of tumor suppressive miRNAs could provide new insight into the molecular mechanisms of chemical carcinogenesis.

Inactivation of Rac1 reduces Trastuzumab resistance in PTEN deficient and insulin-like growth factor I receptor overexpressing human breast cancer SKBR3 cells.

Drug resistance remains to be a big challenge in applying anti-HER2 monoclonal antibody Trastuzumab for treating breast cancer with HER2 overexpression. Amplification of insulin-like growth factor I receptor (IGF-IR) and deletion of tumor suppressor phosphatase and tensin homolog (PTEN) are implicated in Trastuzumab resistance, however, the underlying mechanisms have not been clearly defined. Activation of Rac1, a member of Rho GTPase family, is capable of causing cytoskeleton reorganization, regulating gene expression and promoting cell proliferation. To investigate the mechanism of Trastuzumab resistance, PTEN knockdown and IGF-IR overexpressing stable cell lines were generated in HER2 overexpression human breast cancer SKBR3 cells. Rac1 was highly activated in PTEN deficient and IGF-IR overexpressing Trastuzumab-resistant cells in a HER2-independent manner. Inactivation of Rac1 by using a Rac1 inhibitor NSC23766 or siRNA knocking down the expression of Tiam1, a guanine nucleotide exchange factor for Rac, significantly reduced Trastuzumab resistance in SKBR3 cells. Inhibition of Rac1 had no effect on the levels of phosphor-HER2 and phosphor-Akt, but significantly decreased the levels of cyclin D1 in Trastuzumab-resistant cells. Inhibition of Akt with an Akt inhibitor also significantly reduced Trastuzumab resistance. However, simultaneous inhibition of both Rac1 and Akt resulted in a significantly more decrease of Trastuzumab resistance than inactivation of Rac1 or Akt alone. These results suggest that Rac1 activation is critically involved in Trastuzumab resistance caused by PTEN deletion or IGF-IR overexpression. Simultaneous inhibition of Rac1 and Akt may represent a promising strategy in reducing Trastuzumab resistance in HER2 overexpression breast cancer.

A novel long noncoding RNA AK001796 acts as an oncogene and is involved in cell growth inhibition by resveratrol in lung cancer.

Lung cancer is the most common form of cancer throughout the world. The specific targeting of long noncoding RNAs (lncRNAs) by resveratrol opened a new avenue for cancer chemoprevention. In this study, we found that 21 lncRNAs were upregulated and 19 lncRNAs were downregulated in lung cancer A549 cells with 25 μmol/L resveratrol treatment determined by microarray analysis. AK001796, the lncRNA with the most clearly altered expression, was overexpressed in lung cancer tissues and cell lines, but its expression was downregulated in resveratrol-treated lung cancer cells. By monitoring cell proliferation and growth in vitro and tumor growth in vivo, we observed a significant reduction in cell viability in lung cancer cells and a slow growth in the tumorigenesis following AK001796 knockdown. We also found that AK001796 knockdown caused a cell-cycle arrest, with significant increases in the percentage of cells in G0/G1 in lung cancer cells. By using cell cycle pathway-specific PCR arrays, we detected changes in a number of cell cycle-related genes related to lncRNA AK001796 knockdown. We further investigated whether AK001796 participated in the anticancer effect of resveratrol and the results showed that reduced lncRNA AK001796 level potentially impaired the inhibitory effect of resveratrol on cell proliferation. To our knowledge, this is the first study to report the changes in an lncRNA expression profile induced by resveratrol in lung cancer.

LncRNA-DQ786227-mediated cell malignant transformation induced by benzo(a)pyrene.

It has recently been found that the new class of transcripts, long non-coding RNAs (lncRNAs), are pervasively transcribed in the genome. LncRNAs are a large family of non-coding RNAs and regulate many protein-coding genes. Growing evidence indicates that lncRNAs may play an important functional role in cancer biology. Emerging data have shown that lncRNAs are closely related to the occurrence and development of lung cancer. However, the function and mechanism of lncRNAs in lung cancer remain elusive. Here, we investigated the role of a novel lncRNA in transformed human bronchial epithelial cells induced by benzo(a)pyrene. After establishing the transformed cell model using the BEAS-2B cell line in vitro, we found that expression of lncRNA-DQ786227 was high and changed during the transformation of BEAS-2B cells. Silencing of lncRNA-DQ786227 expression in malignant transformed BEAS-2B cells led to inhibition of cell proliferation and colony formation, and increased apoptosis. LncRNA-DQ786227 dramatically promoted the ability of BEAS-2B-T cells to form colonies in vitro and develop tumors in nude mice. These findings revealed that lncRNA-DQ786227 acts as an oncogene in malignantly transformed BEAS-2B cells induced by benzo(a)pyrene. The identification of lncRNA could provide new insight into the molecular mechanisms of chemical carcinogenesis.