Feng Zhi

Identification of Circulating MicroRNAs as Potential Biomarkers for Detecting Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The disease is characterized by various cytogenetic and molecular abnormalities with distinct prognoses and gene expression profiles. Emerging evidence has suggested that circulating microRNAs (miRNAs) could serve as noninvasive biomarkers for cancer detection; however, little is known about circulating miRNA profiles in AML patients. In this study, a genome-wide serum miRNA expression analysis was performed using Solexa sequencing for initial screen, followed by validation with real-time PCR assays. The analysis was conducted on training and verification sets of serum samples from 140 newly diagnosed AML patients and 135 normal adult donors. After a two-phase selection and validation process, 6 miRNAs, miR-10a-5p, miR-93-5p, miR-129-5p, miR-155-5p, miR-181b-5p and miR-320d, were found to have significantly different expression levels in AML compared with control serum samples. Furthermore, unsupervised clustering analysis revealed the remarkable ability of the 6-miRNA profile to differentiate between AML patients and normal controls. The areas under the ROC curve for the selected miRNAs ranged from 0.8129 to 0.9531. More importantly, miR-181b-5p levels in serum were significantly associated with overall survival. These data demonstrated that the expression patterns of circulating miRNAs were systematically altered in AML and miR-181b-5p may serve as a predictor for overall survival in AML patients.

Identification of 9 serum microRNAs as potential noninvasive biomarkers of human astrocytoma

BACKGROUND Circulating microRNAs (miRNAs) are emerging as promising biomarkers for human cancer. In the current study, we investigated the potential use of serum miRNAs as biomarkers for diagnosis and prognosis in a cohort of Chinese astrocytoma patients. METHODS An initial screening of the circulating miRNA expression profile was performed on pooled serum samples from 10 preoperative patients and 10 healthy controls using a TaqMan low-density array. The selected serum miRNAs were then validated in 90 preoperative patients and 110 healthy controls who were randomly divided into a training set and a validation set. An additional double-blind test was performed in 50 astrocytomas and 50 controls to assess the serum miRNA-based biomarker accuracy in predicting astrocytoma. The differentially expressed miRNAs were evaluated in paired preoperative and postoperative serum samples from 73 astrocytoma patients. The correlation of the miRNA levels with survival in astrocytoma samples was estimated. RESULTS Nine serum miRNAs were significantly increased in the astrocytoma patients. The biomarker composed of these 9 miRNAs had high sensitivity, specificity, and accuracy. These 9 miRNAs were markedly decreased in the serum after operation. The upregulation of miR-20a-5p, miR-106a-5p, and miR-181b-5p was associated with advanced clinical stages of astrocytoma. Kaplan-Meier survival analysis showed that the high expression of miR-19a-3p, miR-106a-5p, and miR-181b-5p was significantly associated with poor patient survival. Finally, the combined 3-miRNAs panel was an important prognostic predictor, independent of other clinicopathological factors. CONCLUSIONS The results indicated the potential of serum miRNAs as novel diagnostic and prognostic biomarkers for human astrocytoma.

miR-106a-5p Inhibits the Proliferation and Migration of Astrocytoma Cells and Promotes Apoptosis by Targeting FASTK

Astrocytomas are common malignant intracranial tumors that comprise the majority of adult primary central nervous system tumors. MicroRNAs (miRNAs) are small, non-coding RNAs (20–24 nucleotides) that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs. In our previous studies, we found that the downregulation of miR-106a-5p in astrocytomas is associated with poor prognosis. However, its specific gene target(s) and underlying functional mechanism(s) in astrocytomas remain unclear. In this study, we used mRNA microarray experiments to measure global mRNA expression in the presence of increased or decreased miR-106a-5p levels. We then performed bioinformatics analysis based on multiple target prediction algorithms to obtain candidate target genes that were further validated by computational predictions, western blot analysis, quantitative real-time PCR, and the luciferase reporter assay. Fas-activated serine/threonine kinase (FASTK) was identified as a direct target of miR-106a-5p. In human astrocytomas, miR-106a-5p is downregulated and negatively associated with clinical staging, whereas FASTK is upregulated and positively associated with advanced clinical stages, at both the protein and mRNA levels. Furthermore, Kaplan-Meier analysis revealed that the reduced expression of miR-106a-5p or the increased expression of FASTK is significantly associated with poor survival outcome. These results further supported the finding that FASTK is a direct target gene of miR-106a-5p. Next, we explored the function of miR-106a-5p and FASTK during astrocytoma progression. Through gain-of-function and loss-of-function studies, we demonstrated that miR-106a-5p can significantly inhibit cell proliferation and migration and can promote cell apoptosis in vitro. The knockdown of FASTK induced similar effects on astrocytoma cells as those induced by the overexpression of miR-106a-5p. These observations suggest that miR-106a-5p functions as a tumor suppressor during the development of astrocytomas by targeting FASTK.

A microRNA expression signature predicts meningioma recurrence

The aberrant expression of microRNAs (miRNAs) is associated with a variety of diseases, including cancer. In our study, we examined the miRNA expression profile of meningiomas, which is a common type of benign intracranial tumor derived from the protective meninges membranes that surround the brain and spinal cord. To define a typical human meningioma miRNA profile, the expression of 200 miRNAs in a training sample set were screened using quantitative reverse transcription polymerase chain reaction analysis, and then significantly altered miRNAs were validated in a secondary independent sample set. Kaplan–Meier and univariate/multivariate Cox proportional hazard regression analyses were performed to assess whether miRNA expression could predict the recurrence of meningioma after tumor resection. After a two‐phase selection and validation process, 14 miRNAs were found to exhibit significantly different expression profiles in meningioma samples compared to normal adjacent tissue (NAT) samples. Unsupervised clustering analysis indicated that the 14‐miRNA profile differed between tumor and NAT samples. Downregulation of miR‐29c‐3p and miR‐219‐5p were found to be associated with advanced clinical stages of meningioma. Kaplan–Meier analysis showed that high expression of miR‐190a and low expression of miR‐29c‐3p and miR‐219‐5p correlated significantly with higher recurrence rates in meningioma patients. Cox proportional hazard regression analysis revealed that miR‐190a expression level is an important prognostic predictor that is independent of other clinicopathological factors. Our results suggest that the use of miRNA profiling has significant potential as an effective diagnostic and prognostic marker in defining the expression signature of meningiomas and in predicting postsurgical outcomes.

MicroRNA-124-3p regulates cell proliferation, invasion, apoptosis, and bioenergetics by targeting PIM1 in astrocytoma.

The PIM1 protein is an important regulator of cell proliferation, the cell cycle, apoptosis, and metabolism in various human cancers. MicroRNAs (miRNAs) are powerful post‐transcriptional gene regulators that function through translational repression or transcript destabilization. Therefore, we aimed to identify whether a close relationship exists between PIM1 and miRNAs. PIM1 protein levels and mRNA levels were significantly upregulated in astrocytoma tissues, indicating the oncogenic role of PIM1 in astrocytoma. Further bioinformatics analysis indicated that miR‐124‐3p targeted the 3′‐UTR of PIM1. We also observed an inverse correlation between the miR‐124‐3p levels and PIM1 protein or mRNA levels in astrocytoma samples. Next, we experimentally confirmed that miR‐124‐3p directly recognizes the 3′‐UTR of the PIM1 transcript and regulates PIM1 expression at both the protein and mRNA levels. Furthermore, we examined the biological consequences of miR‐124‐3p targeting PIM1 in vitro. We showed that the repression of PIM1 in astrocytoma cancer cells by miR‐124‐3p suppressed proliferation, invasion, and aerobic glycolysis and promoted apoptosis. We observed that the restoration or inhibition of PIM1 activity resulted in effects that were similar to those induced by miR‐124‐3p inhibitors or mimics in cancer cells. Finally, overexpression of PIM1 rescued the inhibitory effects of miR‐124‐3p. In summary, these findings aid in understanding the tumor‐suppressive role of miR‐124‐3p in astrocytoma pathogenesis through the inhibition of PIM1 translation.

Tubeimoside-1 induces glioma apoptosis through regulation of Bax/Bcl-2 and the ROS/Cytochrome C/Caspase-3 pathway.

Background Tubeimoside-1 (TBMS1) is a natural compound isolated from tubeimoside, which has been widely used as a traditional Chinese herbal medicine. The purpose of the present study is to investigate the anti-tumor effect and the underling mechanism of TBMS1 on glioma cancer cells. Methods The MTT assay was performed to evaluate the effect of TBMS1 on glioma cell proliferation. The fluorescent microscopy and flow cytometry analysis were performed to evaluate the effect of TBMS1 on glioma cell apoptosis. The Western blot analysis was used to evaluate the protein change. Results TBMS1 inhibited glioma cancer cell proliferation in a dose- and time-dependent manner. Fluorescent microscopy and flow cytometry analysis demonstrated that TBMS1 induced glioma cell apoptosis in a concentration-dependent manner. Western blotting showed that TBMS1 induced apoptosis by increasing the expression of Bax and downregulating the level of Bcl-2. Furthermore, we found that TBMS1 induced apoptosis by increasing the concentration of reactive oxygen species through the release of Cytochrome C and activation of Caspase-3. Conclusion These findings indicate that TBMS1 may be developed as a possible therapeutic agent for the management of glioma.

Attenuating Oxidative Stress by Paeonol Protected against Acetaminophen-Induced Hepatotoxicity in Mice

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. The purpose of this study was to investigate whether paeonol protected against APAP-induced hepatotoxicity. Mice treated with paeonol (25, 50, 100 mg/kg) received 400 mg/kg acetaminophen intraperitoneally (i.p.) and hepatotoxicity was assessed. Pre-treatment with paeonol for 6 and 24 h ameliorated APAP-induced hepatic necrosis and significantly reduced the serum alanine aminotransferase (ALT) and aspartate transaminase (AST) levels in a dose-dependent manner. Post-treatment with 100 mg/kg paeonol ameliorated APAP-induced hepatic necrosis and reduced AST and ALT levels in the serum after APAP administration for 24 h. Western blot revealed that paeonol inhibited APAP-induced phosphorylated JNK protein expression but not p38 and Erk1/2. Moreover, paeonol showed anti-oxidant activities with reducing hepatic MDA contents and increasing hepatic SOD, GSH-PX and GSH levels. Paeonol dose-dependently prevented against H2O2 or APAP-induced LDH releasing and ROS production in primary mouse hepatocytes. In addition, the mRNA levels of pro-inflammatory genes such as TNF-α, MCP-1, IL-1β and IL-6 in the liver were dose-dependently reduced by paeonol pre-treatment. Pre-treatment with paeonol significantly inhibited IKKα/β, IκBα and p65 phosphorylation which contributed to ameliorating APAP-induced hepatic inflammation. Collectively, the present study demonstrates paeonol has a protective ability against APAP-induced hepatotoxicity and might be an effective candidate compound against drug-induced acute liver failure.

Serum level of miR-10-5p as a prognostic biomarker for acute myeloid leukemia

MicroRNAs (miRNAs) are a type of small non-coding RNA molecule that play important roles in tumor initiation, chemotherapy response, promotion, and progression by negatively interfering with gene expression. The aim of the present study was to investigate the serum expression status and explore the prognostic significance of miR-10a-5p in acute myeloid leukemia (AML). The serum expression level of miR-10a-5p in de novo AML was significantly higher, compared with that in controls. The area under the receiver operator characteristic (ROC) curve was of 0.831 in the diagnostic value of miR-10a-5p. In the complete remission (CR) group, the serum expression level of miR-10a-5p was similar to that of healthy subjects and demonstrated a significant downtrend when compared to that on the day of diagnosis. Nevertheless, miR-10a-5p expression was found to significantly increase in cases of relapsed AML when compared individually to the CR population. On analysis of the association of miR-10a-5p expression with the clinical characteristics at diagnosis in AML patients, lower CR rates occurred more frequently in the high-expression group. In addition, high miR-10a-5p expression was associated with poorer overall survival (OS). These data suggest that miR-10a-5p may serve as a biomarker useful to improving the management of AML patients.

δ-Opioid Receptor Activation and MicroRNA Expression of the Rat Cortex in Hypoxia

Prolonged hypoxic/ischemic stress may cause cortical injury and clinically manifest as a neurological disability. Activation of the δ-opioid receptor (DOR) may induce cortical protection against hypoxic/ischemic insults. However, the mechanisms underlying DOR protection are not clearly understood. We have recently found that DOR activation modulates the expression of microRNAs (miRNAs) in the kidney exposed to hypoxia, suggesting that DOR protection may involve a miRNA mechanism. To determine if the miRNAs expressed in the cortex mediated DOR neuroprotection, we examined 19 miRNAs that were previously identified as hypoxia- and DOR-regulated miRNAs in the kidney, in the rat cortex treated with UFP-512, a potent and specific DOR agonist under hypoxic condition. Of the 19 miRNAs tested, 17 were significantly altered by hypoxia and/or DOR activation with the direction and amplitude varying depending on hypoxic duration and times of DOR treatment. Expression of several miRNAs such as miR-29b, -101b, -298, 324-3p, -347 and 466b was significantly depressed after 24 hours of hypoxia. Similar changes were seen in normoxic condition 24 hours after DOR activation with one-time treatment of UFP-512. In contrast, some miRNAs were more tolerant to hypoxic stress and showed significant reduction only with 5-day (e.g., miR-31 and -186) or 10-day (e.g., miR-29a, let-7f and -511) exposures. In addition, these miRNAs had differential responses to DOR activation. Other miRNAs like miRs-363* and -370 responded only to the combined exposure to hypoxia and DOR treatment, with a notable reduction of >70% in the 5-day group. These data suggest that cortical miRNAs are highly yet differentially sensitive to hypoxia. DOR activation can modify, enhance or resolve the changes in miRNAs that target HIF, ion transport, axonal guidance, free radical signaling, apoptosis and many other functions.

Upregulation of the long non-coding RNA SNHG1 predicts poor prognosis, promotes cell proliferation and invasion, and reduces apoptosis in glioma

Long non-coding RNAs (lncRNAs), which are non-coding RNAs with a length above 200 nucleotides, have emerged as novel and important gene expression modulators in carcinogenesis. Recent evidence indicates that the lncRNA small nucleolar RNA host gene 1 (SNHG1) functions as an oncogene in several types of human cancers. However, its function in the development of glioma remains unknown. The aim of this research was to investigate the clinical aspects and biological mechanisms of SNHG1 in glioma. SNHG1 expression was measured in glioma tissues and cell lines by quantitative real-time PCR (qRT-PCR). The association between SNHG1 expression in tissues and clinicopathological characteristics and prognosis in glioma patients was also explored. Gain-of-function and loss-of-function studies using SNHG1 cDNA and siRNA, respectively, were used to investigate the role of SNHG1 in cell proliferation, invasion and apoptosis in glioma. SNHG1 was highly expressed in glioma tissues, and its upregulation was closely related to old age. Kaplan-Meier analysis showed that high expression of SNHG1 was significantly associated with poor overall survival (OS). Functionally, ectopic expression of SNHG1 enhanced cell proliferation and cell invasion and reduced cell apoptosis in vitro, while SNHG1 knockdown reversed these effects. Taken together, our findings indicate that SNHG1 functions as an oncogene in glioma and may serve as a novel therapeutic target in future treatments.