MicroRNA-1291 targets the FOXA2-AGR2 pathway to suppress pancreatic cancer cell proliferation and tumorigenesis.

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Better understanding of pancreatic cancer biology may help identify new oncotargets towards more effective therapies. This study investigated the mechanistic actions of microRNA-1291 (miR-1291) in the suppression of pancreatic tumorigenesis. Our data showed that miR-1291 was downregulated in a set of clinical pancreatic carcinoma specimens and human pancreatic cancer cell lines. Restoration of miR-1291 expression inhibited pancreatic cancer cell proliferation, which was associated with cell cycle arrest and enhanced apoptosis. Furthermore, miR-1291 sharply suppressed the tumorigenicity of PANC-1 cells in mouse models. A proteomic profiling study revealed 32 proteins altered over 2-fold in miR-1291-expressing PANC-1 cells that could be assembled into multiple critical pathways for cancer. Among them anterior gradient 2 (AGR2) was reduced to the greatest degree. Through computational and experimental studies we further identified that forkhead box protein A2 (FOXA2), a transcription factor governing AGR2 expression, was a direct target of miR-1291. These results connect miR-1291 to the FOXA2-AGR2 regulatory pathway in the suppression of pancreatic cancer cell proliferation and tumorigenesis, providing new insight into the development of miRNA-based therapy to combat pancreatic cancer.

N-methylnicotinamide and nicotinamide N-methyltransferase are associated with microRNA-1291-altered pancreatic carcinoma cell metabolome and suppressed tumorigenesis.

The cell metabolome comprises abundant information that may be predictive of cell functions in response to epigenetic or genetic changes at different stages of cell proliferation and metastasis. An unbiased ultra-performance liquid chromatography-mass spectrometry-based metabolomics study revealed a significantly altered metabolome for human pancreatic carcinoma PANC-1 cells with gain-of-function non-coding microRNA-1291 (miR-1291), which led to a lower migration and invasion capacity as well as suppressed tumorigenesis in a xenograft tumor mouse model. A number of metabolites, including N-methylnicotinamide, involved in nicotinamide metabolism, and l-carnitine, isobutyryl-carnitine and isovaleryl-carnitine, involved in fatty acid metabolism, were elevated in miR-1291-expressing PANC-1. Notably, N-methylnicotinamide was elevated to the greatest extent, and this was associated with a sharp increase in nicotinamide N-methyltransferase (NNMT) mRNA level in miR-1291-expressing PANC-1 cells. In addition, expression of NNMT mRNA was inversely correlated with pancreatic tumor size in the xenograft mouse model. These results indicate that miR-1291-altered PANC-1 cell function is associated with the increase in N-methylnicotinamide level and NNMT expression, and in turn NNMT may be indicative of the extent of pancreatic carcinogenesis.

Small nucleolar RNA-derived microRNA hsa-miR-1291 modulates cellular drug disposition through direct targeting of ABC transporter ABCC1.

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is an important membrane transporter that contributes to cellular disposition of many endobiotic and xenobiotic agents, and it can also confer multidrug resistance. This study aimed to investigate the role of human noncoding microRNA-1291 (hsa-miR-1291) in regulation of ABCC1 and drug disposition. Bioinformatics analyses indicated that hsa-miR-1291, localized within the small nucleolar RNA H/ACA box 34 (SNORA34), might target ABCC1 3'-untranslated region (3'UTR). Using splinted ligation small RNA detection method, we found that SNORA34 was processed into hsa-miR-1291 in human pancreatic carcinoma PANC-1 cells. Luciferase reporter assays showed that ABCC1 3'-UTR-luciferase activity was decreased by 20% in cells transfected with hsa-miR-1291 expression plasmid, and increased by 40% in cells transfected with hsa-miR-1291 antagomir. Furthermore, immunoblot study revealed that ABCC1 protein expression was sharply reduced in hsa-miR-1291-stably transfected PANC-1 cells, which was attenuated by hsa-miR-1291 antagomir. The change of ABCC1 protein expression was associated with an alternation in mRNA expression. In addition, hsa-miR-1291-directed downregulation of ABCC1 led to a greater intracellular drug accumulation and sensitized the cells to doxorubicin. Together, our results indicate that hsa-miR-1291 is derived from SNORA34 and modulates cellular drug disposition and chemosensitivity through regulation of ABCC1 expression. These findings shall improve the understanding of microRNA-controlled epigenetic regulatory mechanisms underlying multidrug resistance and interindividual variability in pharmacokinetics.

Noncoding microRNAs: small RNAs play a big role in regulation of ADME?

There are considerable interindividual variations in drug absorption, distribution, metabolism and excretion (ADME) in humans, which may lead to undesired drug effects in pharmacotherapy. Some of the mechanistic causes are known, e.g., genetic polymorphism, inhibition and induction of ADME enzymes and transporters, while others such as posttranscriptional regulation of ADME genes are under active study. MicroRNAs (miRNAs) are a large group of small, noncoding RNAs that control posttranscriptional expression of target genes. More than 1000 miRNAs have been identified in the human genome, which may regulate thousands of protein-coding genes. Some miRNAs directly or indirectly control the expression of xenobiotic-metabolizing cytochrome P450 enzymes, ATP-binding cassette or solute carrier transporters and/or nuclear receptors. Consequently, intervention of miRNA epigenetic signaling may alter ADME gene expression, change the capacity of drug metabolism and transport, and influence the sensitivity of cells to xenobiotics. In addition, the expression of some ADME regulatory miRNAs is significantly changed in cells following the exposure to a given drug, and the consequent changes in ADME gene expression might result in distinct ADME properties and drug response. In this review, we summarized recent findings on the role of noncoding miRNAs in epigenetic regulation of ADME genes and discussed the potential impact on pharmacokinetics and pharmacodynamics.

MicroRNA expression is differentially altered by xenobiotic drugs in different human cell lines.

Several noncoding microRNAs (miR or miRNA) have been shown to regulate the expression of drug-metabolizing enzymes and transporters. Xenobiotic drug-induced changes in enzyme and transporter expression may be associated with the alteration of miRNA expression. Therefore, this study investigated the impact of 19 xenobiotic drugs (e.g. dexamethasone, vinblastine, bilobalide and cocaine) on the expression of ten miRNAs (miR-18a, -27a, -27b, -124a, -148a, -324-3p, -328, -451, -519c and -1291) in MCF-7, Caco-2, SH-SY5Y and BE(2)-M17 cell systems. The data revealed that miRNAs were differentially expressed in human cell lines and the change in miRNA expression was dependent on the drug, as well as the type of cells investigated. Notably, treatment with bilobalide led to a 10-fold increase of miR-27a and a 2-fold decrease of miR-148a in Caco-2 cells, but no change of miR-27a and a 2-fold increase of miR-148a in MCF-7 cells. Neuronal miR-124a was generally down-regulated by psychoactive drugs (e.g. cocaine, methadone and fluoxetine) in BE(2)-M17 and SH-SY5Y cells. Dexamethasone and vinblastine, inducers of drug-metabolizing enzymes and transporters, suppressed the expression of miR-27b, -148a and -451 that down-regulate the enzymes and transporters. These findings should provide increased understanding of the altered gene expression underlying drug disposition, multidrug resistance, drug-drug interactions and neuroplasticity.

Breast cancer resistance protein BCRP/ABCG2 regulatory microRNAs (hsa-miR-328, -519c and -520h) and their differential expression in stem-like ABCG2+ cancer cells.

Recent studies have shown that a number of microRNAs (miRNA or miR) may regulate human breast cancer resistance protein (BCRP/ABCG2), an important efflux transporter responsible for cellular drug disposition, whereas their effects on ABCG2 protein expression are not compared. In this study, we first identified a new proximal miRNA response element (MRE) for hsa-miR-519c within ABCG2 3'-untranslated region (3'UTR) through computational analyses. This miR-519c MRE site was confirmed using dual luciferase reporter assay and site-directed mutagenesis. Immunoblot analyses indicated that ABCG2 protein expression was significantly down-regulated in MCF-7/MX100 cells after transfection with hsa-miR-328- or -519c expression plasmids, and was markedly up-regulated in MCF-7 cells after transfection with miR-328 or -519c antagomir. However, ABCG2 protein expression was unchanged in MCF-7/MX100 cells after transfection with hsa-miR-520h expression plasmids, which was associated with undetectable miR-520h expression. Furthermore, ABCG2 mRNA degradation was accelerated dramatically in cells transfected with miR-519c expression plasmid, suggesting the involvement of mRNA degradation mechanism. Intervention of miR-328 or -519c signaling led to significant change in intracellular mitoxantrone accumulation, as determined by flow cytometry analyses. In addition, we separated RB143 human retinoblastoma cells into stem-like (ABCG2+) and non-stem-like (ABCG2-) populations through immunomagnetic selection, and found that miR-328, -519c and -520h levels were 9-, 15- and 3-fold lower in the ABCG2+ cells, respectively. Our data suggest that miR-519c and -328 have greater impact on ABCG2 expression than miR-520h in MCF-7 human breast cancer cells, and the presence of proximal miR-519c MRE explains the action of miR-519c on shortened ABCG2 3'UTR.

MicroRNAs regulate CYP3A4 expression via direct and indirect targeting.

CYP3A4 metabolizes many drugs on the market. Although transcriptional regulation of CYP3A4 is known to be tightly controlled by some nuclear receptors (NR) including vitamin D receptor (VDR/NR1I1), posttranscriptional regulation of CYP3A4 remains elusive. In this study, we show that noncoding microRNAs (miRNAs) may control posttranscriptional and transcriptional regulation of CYP3A4 by directly targeting the 3'-untranslated region (3'UTR) of CYP3A4 and indirectly targeting the 3'UTR of VDR, respectively. Luciferase reporter assays showed that CYP3A4 3'UTR-luciferase activity was significantly decreased in human embryonic kidney 293 cells transfected with plasmid that expressed microRNA-27b (miR-27b) or mouse microRNA-298 (mmu-miR-298), whereas the activity was unchanged in cells transfected with plasmid that expressed microRNA-122a or microRNA-328. Disruption of the corresponding miRNA response element (MRE) within CYP3A4 3'UTR led to a 2- to 3-fold increase in luciferase activity. Immunoblot analyses indicated that CYP3A4 protein was down-regulated over 30% by miR-27b and mmu-miR-298 in LS-180 and PANC1 cells. The decrease in CYP3A4 protein expression was associated with significantly decreased CYP3A4 mRNA levels, as determined by quantitative real-time PCR (qPCR) analyses. Likewise, interactions of miR-27b or mmu-miR-298 with VDR 3'UTR were supported by luciferase reporter assays. The mmu-miR-298 MRE site is well conserved within the 3'UTR of mouse, rat, and human VDR. Down-regulation of VDR by the two miRNAs was supported by immunoblot and qPCR analyses. Furthermore, overexpression of miR-27b or mmu-miR-298 in PANC1 cells led to a lower sensitivity to cyclophosphamide. Together, these findings suggest that CYP3A4 gene expression may be regulated by miRNAs at both the transcriptional and posttranscriptional level.

MicroRNA-328 negatively regulates the expression of breast cancer resistance protein (BCRP/ABCG2) in human cancer cells.

Breast cancer resistance protein (BCRP/ABCG2) is a molecular determinant of pharmacokinetic properties of many drugs in humans. To understand post-transcriptional regulation of ABCG2 and the role of microRNAs (miRNAs) in drug disposition, we found that microRNA-328 (miR-328) might readily target the 3'-untranslated region (3'-UTR) of ABCG2 when considering target-site accessibility. We then noted 1) an inverse relation between the levels of miR-328 and ABCG2 in MCF-7 and MCF-7/MX100 breast cancer cells and 2) that miR-328 levels could be rescued in MCF-7/MX100 cells by transfection with miR-328 plasmid. Luciferase reporter assays showed that ABCG2 3'-UTR-luciferase activity was decreased more than 50% in MCF-7/MX100 cells after transfection with miR-328 plasmid, the activity was increased over 100% in MCF-7 cells transfected with a miR-328 antagomir, and disruption of miR-328 response element within ABCG2 3'-UTR led to a 3-fold increase in luciferase activity. Furthermore, the level of ABCG2 protein was down-regulated when miR-328 was over-expressed, and the level was up-regulated when miR-328 was inhibited by selective antagomir. Altered ABCG2 protein expression was associated with significantly declined or elevated levels of ABCG2 3'-UTR and coding sequence mRNAs, suggesting possible involvement of the mechanism of mRNA cleavage. Finally, miR-328-directed down-regulation of ABCG2 expression in MCF-7/MX100 cells resulted in an increased mitoxantrone sensitivity, as manifested by a significantly lower IC(50) value (2.46 +/- 1.64 microM) compared with the control (151 +/- 32 microM). Together, these findings suggest that miR-328 targets ABCG2 3'-UTR and, consequently, controls ABCG2 protein expression and influences drug disposition in human breast cancer cells.

[Influence of expression of six transmembrane epithelial antigen of the prostate-1 on intracellular reactive oxygen species level and cell growth: an in vitro experiment].

OBJECTIVE: To investigate the STEAP1 gene function of the newly discovered gene six transmembrAne epithelial antigen of the prostate-1 (STEAP1). METHODS: Total RNA was obtained from human prostate cancer tissue and underwent PCR amplification. The full length of STEAP1 gene thus obtained was cloned. Mammalian expression vector pcDNA3. 1-STEAP1 was constructed and stably transfected into the human thyroid epithelial cells of the line FRTAP2320. A growth curve of the transfected cells was drawn. The intracellular reactive oxygen species (ROS) level was determined by flow cytometry (FC) with dichlorodihydrofluorescein diacetate (DCFHDA). RESULTS: The growth curve showed that the STEAP1 transfected cells grew faster than the control cells. FC showed that the fluorescence intensity of he intracellular ROS of the STEAP1 transfected cells was 42.13 +/- 1.13, significantly higher than those of the cell transfected with blank plasmid (10.02 +/- 1.42) and un-transfected cells (13.02 +/- 2.42, both P < 0.01). CONCLUSION: STEAP1 promotes the cell growth through inducing the intracellular ROS level.

[3,3-diindolylmethane enhances the inhibitory effect of idarubicin on the growth of human prostate cancer cells].

OBJECTIVE: To study the effects of idarubicin (IDA) combined with 3, 3-diindolylmethane (DIM) on the growth inhibition of human prostate cancer cells. METHODS: Human prostate cancer cells of the line PC-3M were cultured and then divided into the following groups: control group with solvent added into the culture fluid; IDA groups, with IDA of the terminal concentrations of 0.5, 1 or 5 mg/L added into the culture fluid; DIM groups, with DIM of the terminal concentrations of 30, 60 or 100 micromol/L added into the culture fluid; and DIM + IDA groups, with 0. 5 mg/L IDA and DIM 30, 60 or 100 micromol/L added into the culture fluid. 48 h later the cell growth inhibition rate was detected by MTT assay. Flow cytometry and acridine orange staining were used to detect the cell cycle and apoptosis. RT-PCR and Western blotting were used to detect the mRNA and protein expression of caspase 9, an apoptosis gene. RESULTS: Both IDA and DIM dose-dependently inhibited the growth of the PC-3M cells. The growth inhibition rate of the 60 micromol/L DIM + 0.5 mg/L IDA group was 69.9%, almost 10 times as that of the 0.5 mg/L IDA group. The apoptosis rate of the 60 micromol/L DIM + 0. 5 mg/L IDA group was 47.0%, significantly higher than that of the 0.5 mg/L IDA group (3.2%, P < 0.05). RT-PCR and Western blotting showed that the combination of DIM and IDA significantly enhanced the mRNA and protein expression of caspase 9. CONCLUSION: DIM enhances the growth inhibition effect of IDA on human prostate cancer cells by the mechanism of induction of apoptosis.

Application of surface-enhanced laser desorption/ionization time-of-flight-based serum proteomic array technique for the early diagnosis of prostate cancer.

AIM: To identify the serum biomarkers of prostate cancer (PCa) by protein chip and bioinformatics. METHODS: Serum samples from 83 PCa patients and 95 healthy men were taken from a mass screening in Changchun, China. Protein profiling was carried out using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The data of spectra were analyzed using two bioinformatics tools. RESULTS: Eighteen serum differential proteins were identified in the PCa group compared with the control group (P < 0.01). There were four proteins at the higher serum level and 14 proteins at the lower serum level in the PCa group. A decision tree classification algorithm that used an eight-protein mass pattern was developed to correctly classify the samples. A sensitivity of 92.0% and a specificity of 96.7% for the study group were obtained by comparing the PCa and control groups. CONCLUSION: We identified new serum biomarkers of PCa. SELDI-TOF MS coupled with a decision tree classification algorithm will provide a highly accurate and innovative approach for the early diagnosis of PCa.

Mass screening of prostate cancer in a Chinese population: the relationship between pathological features of prostate cancer and serum prostate specific antigen.

AIM: To investigate the pathological features of the prostate biopsy through mass screening for prostate cancer in a Chinese cohort and their association with serum prostate specific antigen (PSA). METHODS: A total of 12027 Chinese men in Changchun were screened for prostate cancer by means of the serum total prostate specific antigen tPSA test (by Elisa assay). Transrectal ultrasound-guided systematic six-sextant biopsies were performed on those whose serum tPSA value was > 4.0 ng/mL and those who had obstructive symptoms (despite their tPSA value) and were subject to subsequent pathological analysis with the aid of the statistic software SPSS 10.0 (SPSS. Inc., Chicago. USA). RESULTS: Of the 12027 cases, 158 (including 137 patients whose serum tPSA values were 4.0 ng/mL and 21 patients [serum tPSA < 4.0 ng/mL] who had obstructive symptoms) undertook prostate biopsy. Of the 158 biopsies, 41 cases of prostatic carcinoma were found (25.9 %, 41/158). The moderately differentiated carcinoma and poorly differentiated carcinoma accounted for 61% and 34%, respectively. A significant linear positive correlation between the serum tPSA and the Gleason scores in the 41 cases of prostatic carcinoma (r = 0.312, P < 0.01) was established. A significant linear positive correlation between the serum tPSA value of the 41 prostatic carcinoma and the positive counts of carcinoma in sextant biopsies was established (r = 0.406, P < 0.01), indicating a significant linear relationship between serum tPSA and the size of tumor. CONCLUSION: This study was the first to conduct mass screening for prostate cancer by testing for serum tPSA values and the first to investigate the pathological features of prostate cancer in a cohort of Chinese men. Our results reveal that the moderately differentiated carcinoma is the most common type of prostate cancer. This study also has shown that the serum tPSA value in prostate cancer is associated with the Gleason score and the size of tumor.

[Proteomic analysis of prostate cancer using surface enhanced laser desorption/ionization mass spectrometry].

OBJECTIVE: To identify the serum biomarkers of prostate cancer by using protein chip and bioinformatics. METHODS: Eighty three prostate cancer (PCA) patients and ninety five healthy people from mass screen in Changchun were detected by surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS). The data of spectra were analyzed by bioinformatics tools-Biomarker Wizard and Biomarker Pattern. RESULTS: Compared with the spectra of healthy people, there were 18 potential markers detected in the spectra of the PCA patients, the protein expression was high in 4 of which and low in the 10 of which. The softwares Biomarkerwizard and Biomarker Pattern automatically, under given conditions, selected 8 biomarker proteins to be used to establish a five layer decision tree differentiate to diagnose PCA and differentiate PCA from healthy people with a specificity of 92.632% and a sensitivity of 96.386%. CONCLUSION: New serum biomarkers of PCA have been identified, and this SELDI mass spectrometry coupled with decision tree classification algorithm will provide a highly accurate and innovative approach for the early diagnosis of PCA.

Mass screening of 12,027 elderly men for prostate carcinoma by measuring serum prostate specific antigen.

BACKGROUND: The incidence of prostate carcinoma (Pca) has been increasing in China. We detected Pca in elderly men in Changchun, north China and the significance of prostate specific antigen (PSA) in mass screening and clinical staging of Pca. METHODS: Serum PSA from 12,027 men over 50 years old from Changchun was analyzed. In case of serum PSA greater than 4.0 ng/ml, the patient was suspected of potentially suffering from Pca, and transrectal six-point puncture prostate biopsies were performed under ultrasound guidance. Pathological examinations were performed on the biopsy tissue, and ABCD and TNM clinical stagings were used in accordance with international standards. Correlations between serum PSA level and clinical stage were analyzed. RESULTS: PSA was greater than 4.0 ng/ml in 813 patients (6.8% of the 12,027 men). Transrectal six-point prostate puncture biopsies guided by ultrasound were performed in 273 patients (33.6% of the 813 patients who were tested positive in the initial mass screening). Of these 273 patients, 69 cases of Pca (25.3% of 273) were confirmed by biopsy in the second screening, with an overall detection rate for Pca of 0.57% (69/12,027). The total number of patients in stages A, B, T1, or T2 was 57.9%, and over 20% of them suffered from late stage Pca with lymph node and bone metastasis. An obvious positive correlation was observed between ABCD staging, TNM staging, and serum PSA level. CONCLUSIONS: Serum PSA level is not only the golden standard for mass screening of Pca, but also the predictor for clinical stage of Pca. PSA testing revealed asymptomatic Pca cases in early, middle, and later stages in the elderly, suggesting that mass screening is of paramount importance.

[Pathological features of prostate cancer in Chinese cohort detected through mass screening and their relationships with serum prostate specific antigen].

OBJECTIVE: To investigate the pathological features of the prostate biopsy through mass screening for prostate cancer in Chinese cohort and their relationships with serum prostate specific antigen (PSA). METHODS: ELISA was used to measured the serum PSA in 12 027 Chinese men aged 50 and over in Changchun, Jilin Province. Transrectal ultrasound guided six-sextant biopsy was performed on 137 cases with the serum PSA value > 4.0 micro g/L and 21 cases with the serum PSA < 4.0 micro g/L but with urethral obstructive symptoms. Pathological analysis was done with the aid of statistic software SPSS 10.0. RESULTS: Of the 158 biopsies, 41 cases of prostatic carcinoma were found (25.9%, 41/158). The moderately differentiated carcinoma and poorly differentiated carcinoma accounted for 61% and 34% respectively. In these 41 cases of prostate cancer, a significant linear positive correlation between the serum PSA and the Gleason score (r = 0.329, P < 0.05) and a significant linear positive correlation between the serum PSA and the positive counts of carcinoma in six-sextant biopsy (r = 0.425, P = 0.006) were found. CONCLUSION: The moderately differentiated carcinoma is the most common type of prostate cancer. The approach of mass screening opens a new avenue for the early detection of prostate cancer. Serum PSA value is not only associated with the pathological grading but also with the extension of tumor.