Prognostic risk signature based on the expression of three m6A RNA methylation regulatory genes in kidney renal papillary cell carcinoma.

In this study, we investigated the prognostic significance of the expression of N6-methyladenosine (m6A) RNA methylation regulatory genes in kidney renal papillary cell carcinoma (KIRP). RNA-sequencing data analysis showed that 14 of 20 major m6A RNA methylation regulatory genes were differentially expressed in the KIRP tissues from The Cancer Genome Atlas (TCGA) database. We constructed a prognostic risk signature with three m6A RNA methylation regulatory genes, IGF2BP3, KIAA1429 and HNRNPC, based on the results from univariate and LASSO Cox regression analyses. Multivariate Cox regression analysis confirmed that the risk score based on the three-gene prognostic risk signature was an independent predictive factor in KIRP. The overall survival of high-risk KIRP patients was significantly shorter than the low-risk KIRP patients. Expression of the three prognostic risk-related genes correlated with the AJCC and TNM stages of KIRP patients from TCGA and GEPIA datasets. ROC curve analysis showed that the three-gene prognostic risk signature precisely predicted the 1-year, 3-year and 5-year survival of KIRP patients. These findings demonstrate that expression of three prognostic risk-related m6A RNA methylation regulatory genes accurately predicts survival outcomes in KIRP patients.

Reciprocal regulation of hnRNP C and CELF2 through translation and transcription tunes splicing activity in T cells.

RNA binding proteins (RBPs) frequently regulate the expression of other RBPs in mammalian cells. Such cross-regulation has been proposed to be important to control networks of coordinated gene expression; however, much remains to be understood about how such networks of cross-regulation are established and what the functional consequence is of coordinated or reciprocal expression of RBPs. Here we demonstrate that the RBPs CELF2 and hnRNP C regulate the expression of each other, such that depletion of one results in reduced expression of the other. Specifically, we show that loss of hnRNP C reduces the transcription of CELF2 mRNA, while loss of CELF2 results in decreased efficiency of hnRNP C translation. We further demonstrate that this reciprocal regulation serves to fine tune the splicing patterns of many downstream target genes. Together, this work reveals new activities of hnRNP C and CELF2, provides insight into a previously unrecognized gene regulatory network, and demonstrates how cross-regulation of RBPs functions to shape the cellular transcriptome.

Function of HNRNPC in breast cancer cells by controlling the dsRNA-induced interferon response.

Elevated expression of RNA binding protein HNRNPC has been reported in cancer cells, while the essentialness and functions of HNRNPC in tumors were not clear. We showed that repression of HNRNPC in the breast cancer cells MCF7 and T47D inhibited cell proliferation and tumor growth. Our computational inference of the key pathways and extensive experimental investigations revealed that the cascade of interferon responses mediated by RIG-I was responsible for such tumor-inhibitory effect. Interestingly, repression of HNRNPC resulted in accumulation of endogenous double-stranded RNA (dsRNA), the binding ligand of RIG-I. These up-regulated dsRNA species were highly enriched by Alu sequences and mostly originated from pre-mRNA introns that harbor the known HNRNPC binding sites. Such source of dsRNA is different than the recently well-characterized endogenous retroviruses that encode dsRNA In summary, essentialness of HNRNPC in the breast cancer cells was attributed to its function in controlling the endogenous dsRNA and the down-stream interferon response. This is a novel extension from the previous understandings about HNRNPC in binding with introns and regulating RNA splicing.

Japanese encephalitis virus induces human neural stem/progenitor cell death by elevating GRP78, PHB and hnRNPC through ER stress.

Japanese encephalitis virus (JEV), which is a causative agent of sporadic encephalitis, harbours itself inside the neural stem/progenitor cells. It is a well-known fact that JEV infects neural stem/progenitor cells and decreases their proliferation capacity. With mass spectrometry-based quantitative proteomic study, it is possible to reveal the impact of virus on the stem cells at protein level. Our aim was to perceive the stem cell proteomic response upon viral challenge. We performed a two-dimensional gel electrophoresis-based proteomic study of the human neural stem cells (hNS1 cell line) post JEV infection and found that 13 proteins were differentially expressed. The altered proteome profile of hNS1 cell line revealed sustained endoplasmic reticulum stress, which deteriorated normal cellular activities leading to cell apoptosis. The proteomic changes found in hNS1 cell line were validated in vivo in the subventricular zone of JE infected BALB/c mice. Congruent alterations were also witnessed in multipotent neural precursor cells isolated from human foetus and in autopsy samples of human brain clinically diagnosed as cases of JE patients. Endoplasmic reticulum resident chaperone GRP78, mitochondrial protein Prohibitin and heterogeneous nuclear ribonucleoprotein hnRNPC (C1/C2) have been shown to interact with viral RNA. Hence it is proposed that these are the principle candidates governing endoplasmic reticulum stress-induced apoptosis in JEV infection.

RALY RNA binding protein-like reduced expression is associated with poor prognosis in clear cell renal cell carcinoma.

The molecular mechanisms involved in the progression of clear cell renal cell carcinomas (ccRCCs) are still unclear. The aim of this study was to analyse the relationships between expression of RALYL and clinical characteristics. In 41 paired samples of ccRCCs and adjacent normal tissues, we used real-time qPCR to evaluate the expression of RALYL mRNA. RALYL protein levels were determined in 146 samples of ccRCC and 37 adjacent normal tissues by immunohistochemistry. Statistical analysis was used to explore the relationships between expression of RALYL and the clinical characteristics (gender, age, tumor size, T stage, N stage, M stage, survival times and survival outcome) in ccRCC. In addition, these patients were follow-up period 64 months (range: 4~116 months) to investigate the influence on prognosis. We found significantly differences between ccRCC tissues and normal tissues (p<0.001, paired-sample t test) in mRNA levels of RALYL. Immunohistochemistry analyses in 146 ccRCC samples and 37 adjacent normal tissues showed significantly lower RALYL protein levels in ccRCC samples (χ2-test, p<0.001), inversely correlating with tumour size (p=0.024), T stage (0.005), N stage (p<0.001) as well as M stage (p=0.019), but not age (p=0.357) and gender (p=0.348). Kaplan-Meier survival analysis demonstrated that people with lower level of RALYL expression had a poorer survival rate than those with a higher level of RALYL expression, significantly different by the log-rank test (p=0.011). Cox regression analysis indicated that RALYL expression (p=0.039), N stage (p=0.008) and distant metastasis (p<0.001) were independent prognosis factors for the overall survival of ccRCC patients. We demonstrated that the expression of RALYL was significantly low in ccRCC and correlated with a poor prognosis in a large number of clinical samples. Our findings showed that RALYL may be a potential therapeutic target as well as a poor prognostic factor.

The most negatively charged low-density lipoprotein L5 induces stress pathways in vascular endothelial cells.

BACKGROUND/AIMS: L5, the most negatively charged species of low-density lipoprotein (LDL), has been implicated in atherogenesis by inducing apoptosis of endothelial cells (ECs) and inhibiting the differentiation of endothelial progenitor cells. In this study, we compared the effects of LDL charge on cellular stress pathways leading to atherogenesis. METHODS: We isolated L5 and L1 (the least negatively charged LDL) from the plasma of patients with familial hypercholesterolemia and used JC-1 staining to examine the effects of L5 and L1 on the mitochondrial membrane potential (DCm) in human umbilical vein ECs (HUVECs). Additionally, we characterized the gene expression profiles of 7 proteins involved in various types of cellular stress. RESULTS: The DCm was severely compromised in HUVECs treated with L5. Furthermore, compared with L1, L5 induced a decrease in mRNA and protein expression of the endoplasmic reticulum (ER) chaperone proteins ORP150, Grp94, and Grp58, mitochondrial proteins Prdx3 and ATP synthase, and an increase in the expression of the pro-inflammatory protein hnRNP C1/C2. CONCLUSIONS: Our work suggests that L5, but not L1, may promote the destruction of ECs that occurs during atherogenesis by causing mitochondrial dysfunction and modulating the expression of key proteins to promote inflammation, ER dysfunction, oxidative stress, and apoptosis.

Comparative proteomic analysis of human placenta derived from assisted reproductive technology.

The aim of this study was to use proteomics-based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2-DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, alpha-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.

Posttranscriptional regulation of urokinase receptor expression by heterogeneous nuclear ribonuclear protein C.

Interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, is a key regulatory step in uPA-mediated cell proliferation and migration. Our previous studies demonstrated that posttranscriptional stabilization of uPAR mRNA by uPA contributes to the induction of cell surface uPAR expression, and heterogeneous nuclear ribonuclear protein C1 (hnRNPC) binds to a 110 nt sequence of uPAR mRNA 3'-UTR, thereby preventing its degradation. These observations indicate that hnRNPC could be involved in the induction of uPAR expression by uPA. In the present study, we investigated this possibility and confirmed that uPA increased the binding of hnRNPC to the 3'-UTR of uPAR mRNA. Furthermore, uPA induced tyrosine phosphorylation of hnRNPC and uPAR expression through mRNA stabilization. Inhibition of hnRNPC tyrosine phosphorylation abolished its interaction with uPAR mRNA and suppressed mRNA stabilization and cell surface uPAR expression. Deletion experiments revealed that hnRNPC binds to uPAR mRNA through its RNA binding domain (RBD). Site-directed mutagenesis studies further indicated that phosphorylation of tyrosine residue 57 (Y57) present in RBD of hnRNPC by uPA is essential for uPAR 3'-UTR mRNA binding and uPAR expression. Increased hnRNPC interaction with the uPAR mRNA 3'-UTR through phosphorylation of Y57 represents a novel mechanism by which uPA regulates posttranscriptional uPAR mRNA turnover and cell surface uPAR expression.