Hypoxia-induced lncRNA MRVI1-AS1 accelerates hepatocellular carcinoma progression by recruiting RNA-binding protein CELF2 to stabilize SKA1 mRNA.

BACKGROUND: Long non-coding RNAs (lncRNAs) perform a vital role during the progression of hepatocellular carcinoma (HCC). Here, we aimed to identify a novel lncRNA involved in HCC development and elucidate the underlying molecular mechanism. METHODS: The RT-qPCR and TCGA dataset analysis were applied to explore the expressions of MRVI1-AS1 in HCC tissues and cell lines. Statistical analysis was applied to analyze the clinical significance of MRVI1-AS1 in HCC. The functions of MRVI1-AS1 in HCC cells metastasis and growth were explored by transwell assays, wound healing assay, MTT assay, EdU assay, the intravenous transplantation tumor model, and the subcutaneous xenograft tumor model. Microarray mRNA expression analysis, dual luciferase assays, and actinomycin D treatment were used to explore the downstream target of MRVI1-AS1 in HCC cells. RIP assay was applied to assess the direct interactions between CELF2 and MRVI1-AS1 or SKA1 mRNA. Rescue experiments were employed to validate the functional effects of MRVI1-AS1, CELF2, and SKA1 on HCC cells. RESULTS: MRVI1-AS1 was found to be dramatically upregulated in HCC and the expression was strongly linked to tumor size, venous infiltration, TNM stage, as well as HCC patients' outcome. Cytological experiments and animal experiments showed that MRVI1-AS1 promoted HCC cells metastasis and growth. Furthermore, SKA1 was identified as the downstream targeted mRNA of MRVI1-AS1 in HCC cells, and MRVI1-AS1 increased SKA1 expression by recruiting CELF2 protein to stabilize SKA1 mRNA. In addition, we found that MRVI1-AS1 expression was stimulated by hypoxia through a HIF-1-dependent manner, which meant that MRVI1-AS was a direct downstream target gene of HIF-1 in HCC. CONCLUSION: In a word, our findings elucidated that hypoxia-induced MRVI1-AS1 promotes metastasis and growth of HCC cells via recruiting CELF2 protein to stabilize SKA1 mRNA, pointing to MRVI1-AS1 as a promising clinical application target for HCC therapy.

Novel Functional Features of cGMP Substrate Proteins IRAG1 and IRAG2.

The inositol triphosphate-associated proteins IRAG1 and IRAG2 are cGMP kinase substrate proteins that regulate intracellular Ca2+. Previously, IRAG1 was discovered as a 125 kDa membrane protein at the endoplasmic reticulum, which is associated with the intracellular Ca2+ channel IP3R-I and the PKGIß and inhibits IP3R-I upon PKGIß-mediated phosphorylation. IRAG2 is a 75 kDa membrane protein homolog of IRAG1 and was recently also determined as a PKGI substrate. Several (patho-)physiological functions of IRAG1 and IRAG2 were meanwhile elucidated in a variety of human and murine tissues, e.g., of IRAG1 in various smooth muscles, heart, platelets, and other blood cells, of IRAG2 in the pancreas, heart, platelets, and taste cells. Hence, lack of IRAG1 or IRAG2 leads to diverse phenotypes in these organs, e.g., smooth muscle and platelet disorders or secretory deficiency, respectively. This review aims to highlight the recent research regarding these two regulatory proteins to envision their molecular and (patho-)physiological tasks and to unravel their functional interplay as possible (patho-)physiological counterparts.

Loss of PKGIß/IRAG1 Signaling Causes Anemia-Associated Splenomegaly.

Inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1) is a substrate protein of the NO/cGMP-signaling pathway and forms a ternary complex with the cGMP-dependent protein kinase Iß (PKGIß) and the inositol triphosphate receptor I (IP3R-I). Functional studies about IRAG1 exhibited that IRAG1 is specifically phosphorylated by the PKGIß, regulating cGMP-mediated IP3-dependent Ca2+-release. IRAG1 is widely distributed in murine tissues, e.g., in large amounts in smooth muscle-containing tissues and platelets, but also in lower amounts, e.g., in the spleen. The NO/cGMP/PKGI signaling pathway is important in several organ systems. A loss of PKGI causes gastrointestinal disorders, anemia and splenomegaly. Due to the similar tissue distribution of the PKGIß to IRAG1, we investigated the pathophysiological functions of IRAG1 in this context. Global IRAG1-KO mice developed gastrointestinal bleeding, anemia-associated splenomegaly and iron deficiency. Additionally, Irag1-deficiency altered the protein levels of some cGMP/PKGI signaling proteins-particularly a strong decrease in the PKGIß-in the colon, spleen and stomach but did not change mRNA-expression of the corresponding genes. The present work showed that a loss of IRAG1 and the PKGIß/IRAG1 signaling has a crucial function in the development of gastrointestinal disorders and anemia-associated splenomegaly. Furthermore, global Irag1-deficient mice are possible in vivo model to investigate PKGIß protein functions.

P53-induced MRVI1 mediates carcinogenesis of colorectal cancer.

Background: Colorectal cancer (CRC) is one of the most prevalent cancer types worldwide. Despite the advancements in current diagnosis and treatment strategies of CRC, the incidence and mortality rates of CRC have been rising. To explore novel mechanism of CRC, this study focused on the expression pattern and functional mechanism of murine retrovirus integration site 1 (MRVI1) in CRC.Methods: Tumor tissues and adjacent normal tissues were collected from CRC patients, and the expression levels of MRVI1 were determined by RT-PCR and Western blot. MRVI1 knockdown was achieved by shRNA in HCT116 and HT29 cells, followed by CCK-8 assay to detect the cell proliferation, and caspase-3 activity assay combined with nucleosome ELISA assay to detect cell apoptosis. Transwell assay was used to detect cell invasion and luciferase reporter assay was used to validate the activation of the MRVI1 promoter by p53.Results: MRVI1 was downregulated in CRC tissues and several CRC cell lines. Knockdown of MRVI1 enhanced the proliferation and apoptosis, while promoted invasion and stemness of CRC cells. Mechanism study revealed that MRVI1 was transcriptionally activated by p53 at its upstream. In addition, p53-induced inhibition of CRC prognosis depended on MRVI1.Conclusion: MRVI1 inhibited the prognosis of CRC via a mechanism involving p53 activation. MRVI1 could serve as a potential target for clinical diagnosis and treatment of CRC.

MRVI1 and NTRK3 Are Potential Tumor Suppressor Genes Commonly Inactivated by DNA Methylation in Cervical Cancer.

The abnormally methylated tumor suppressor genes (TSGs) associated with cervical cancer are unclear. DNA methylation data, RNA-seq expression profiles, and overall survival data were downloaded from TCGA CESC database. DMGs and DEGs were obtained through CHAMP and DESeq packages, respectively. TSGs were downloaded from TSGene 2.0. Candidate hypermethylated/down-regulated TSGs were further evaluated and pyrosequencing was used to confirm their difference in methylation levels of selected TSGs in cervical cancer patients. A total of 25946 differentially methylated CpGs corresponding to 2686 hypermethylated genes and 4898 hypomethylated genes between cervical cancer and adjacent normal cervical tissues were found in this study. Besides, 693 DEGs (109 up-regulated and 584 down-regulated) were discovered in cervical cancer tissues. Then, 192 hypermethylated/down-regulated genes were obtained in cervical cancer compared to adjacent tissues. Interestingly, 26 TSGs were found in hypermethylated/down-regulated genes. Among these genes, low expression of MRVI1 and NTRK3 was associated with poor overall survival in cervical cancer. Moreover, GEO data showed that MRVI1 and NTRK3 were significantly decreased in cervical cancer tissues. The expression levels of MRVI1 and NTRK3 were negatively correlated with the methylation levels of their promoter CpG sites. Additionally, elevated methylation levels of MRVI1 and NTRK3 promoter were further verified in cervical cancer tissues by pyrosequencing experiments. Finally, the ROC results showed that the promoter methylation levels of MRVI1 and NTRK3 had the ability to discriminate cervical cancer from healthy samples. The study contributes to our understanding of the roles of MRVI1 and NTRK3 in cervical cancer.

miR-940 potentially promotes proliferation and metastasis of endometrial carcinoma through regulation of MRVI1.

The specific functions and clinical significance of miR-940 in endometrial carcinoma (EC) have not been studied. First, we assessed the expression of miR-940 and MRVI1 in EC tissues collected from The Cancer Genome Atlas (TCGA) database and EC cell lines. miR-940 was significantly overexpressed in EC tissues and cell lines, particularly in RL95-2 cells. Correlation analysis showed that miR-940 expression level was remarkably associated with age, grade, and death. Moreover, the overall survival (OS) rate in the miR-940 low expression group was higher, compared with miR-940 high expression group. Univariate and multivariate models demonstrated that miR-940 expression, stage, and age were predictive indicators of OS. Moreover, there was no significance of the proliferation ability among the three EC cell lines (RL95-2, ISK, and KLE). To reveal the biological roles of miR-940, we respectively transfected RL95-2 cells with miR-940 mimics, miR-940 inhibitors, and control to further investigate the cell proliferation ability, and migration as well as invasion potential of RL95-2 cells. The transfection of miR-940 mimics significantly increased the proliferation and migration/invasion ability of RL95-2 cells. MRVI1 was predicted to be a potential target of miR-940 by means of in silico analysis followed by validation using luciferase reporter assays. MRVI1 was correlated with good prognosis. Moreover, forced expression of MRVI1 in miR-940 mimic transfected cells abolished the facilitation of miR-940 on cell proliferation, migration, and invasion of RL95-2 and KLE cells. In conclusion, our study demonstrates that miR-940 might function as a reliable diagnostic and prognostic signature in EC.