Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

[Overexpression of MKRN2 Inhibits the Growth of Ovarian Cancer Cells].

Ovarian cancer has a high mortality with low five-year survival rates. The role of the E3 ligase Makorin ring finger protein 2 (MKRN2) in ovarian cancer is unknown. This study investigated the impact of MKRN2 on the growth of ovarian cancer. MKRN2 expression in ovarian cancer tissue was analyzed by immunohistochemistry. Overexpression of MKRN2 was induced in two ovarian cancer cell lines (SKOV3 and CAOV3) by lentivirus transfection, and expression levels were verified by western blotting. Proliferation and growth were determined by CCK-8 and colony formation assays, while migration was examined using transwell assays and apoptosis by flow cytometry. Xenograft tumors of transfected SKOV3 cells were established in mice, and immunohistochemistry and TUNEL assays measured MKRN2 levels and apoptosis in tumor cells. Reduced levels of MKRN2 in cancerous tissue relative to non-cancerous ovarian tissues. Lentiviral-based MKRN2 overexpression in SKOV3 and CAOV3 cells reduced tumor-associated behavior while inducing apoptosis in vitro. In xenograft tumors, MKRN2 overexpression inhibited ovarian cancer growth and increased apoptosis in vivo. These findings imply the MKRN2 involvement in ovarian carcinogenesis and suggest its potential for treating the disease.

Ubiquitination of IGF2BP3 by E3 ligase MKRN2 regulates the proliferation and migration of human neuroblastoma SHSY5Y cells.

Neuroblastoma (NB) is a paediatric tumour that shows great biomolecule and clinical heterogeneity, and patients with NB often develop various neurological complications. Currently, the disease is mainly treated by surgery and still lacks specific therapeutic drugs; therefore, targets are urgently needed. Makorin ring finger protein 2 (MKRN2) is an E3 ligase whose effects on neuroblastoma have not been illustrated. shRNAs for MKRN2 have been designed, and MKRN2-knockdown human neuroblastoma SHSY5Y cells were established. MKRN2 knockdown promotes the proliferation and migration of SHSY5Y cells. Because MKRN2 is an E3 ligase, we performed a series of experiments, and Insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) was identified as a new substrate for MKRN2. IGF2BP3 is an RNA-binding protein that regulates the stability of many mRNAs, including CD44 and PDPN, and our study demonstrated that MKRN2 regulates the expression of CD44 and PDPN in an IGF2BP3-dependent manner. These results suggest that MKRN2 might be a potential therapeutic target for neuroblastoma.

MKRN2 knockout causes male infertility through decreasing STAT1, SIX4, and TNC expression.

Makorin-2 (Mkrn2) is an evolutionarily conserved gene whose biological functions are not fully known. Although recent studies have shed insights on the potential causes of male infertility, its underlining mechanisms still remain to be elucidated. We developed a Mrkn2 knockout mice model to study this gene and found that deletion of Mkrn2 in mice led to male infertility. Interestingly, the expression level of signal transducer and activator of the transcription (STAT)1 was significantly decreased in MKRN2 knockout testis and MEF cells. Co-IP assay showed an interaction between MKRN2 and STAT1. Moreover, our results further indicated that MKRN2 regulated the expression level of SIX4 and tenascin C (TNC) via the EBF transcription factor 2 (EBF2) in mice. The results of our study will provide insights into a new mechanism of male infertility.

MKRN1/2 serve as tumor suppressors in renal clear cell carcinoma by regulating the expression of p53.

BACKGROUND: Kidney renal clear cell carcinoma (KIRC) belongs to renal cell carcinoma which is a very aggressive malignant tumor with poor prognosis and high mortality. The MKRN family includes three members MKRN1, MKRN2 and MKRN3, which are closely related to cancers, and have been involved in many studies. OBJECTIVE: This study aimed to explore the roles of MKRN family in KIRC. METHODS: The expression of MKRNs was analyzed using the UALCAN database, prognostic analysis was performed with the GEPIA2 and Kaplan-Meier Plotter database, and correlation analysis was assessed by GEPIA2. The CCK-8 and colony formation assay were performed to detect cell proliferation, wound healing assays were performed to detect cell migration, cell cycles were detected by flow cytometry analysis, GST pull-down and co-immunoprecipitation assays were performed to detect the interaction of proteins, and the expression of MKRNs, p53 and other proteins were detect by immunoblotting analysis or quantitative PCR (qPCR). RESULTS: MKRN1 and MKRN2 were lowly expressed in KIRC samples compared to the corresponding normal tissues, and KIRC patients with high levels of MKRN1 and MKRN2 showed higher overall survival (OS) and disease free survival (DFS) rates. The overexpression of MKRN1 and MKRN2 inhibited the proliferation of human KIRC cells by arresting the cell cycles, but shows little effect on cells migration. The expression of MKRN1 and MKRN2 are correlated, and MKRN1 directly interacts with MKRN2. Moreover, both MKRN1 and MKRN2 were closely correlated with the expression of TP53 in KIRC tumor, and promoted the expression of p53 both at protein and mRNA levels. CONCLUSIONS: Our study suggests that MKRN1 and MKRN2 serve as tumor suppressors in KIRC, and act as promising therapeutic targets for KIRC treatment.

MKRN2 inhibits the proliferation of gastric cancer by downregulating PKM2.

Cumulative evidence suggests that dysfunction of ubiquitinating enzymes is responsible for multiple types of diseases including cancer. However, what role the ubiquitinating enzyme plays in gastric cancer remains unknown. In this study, using bioinformatics analysis and a series of experimental analyses, we found that an E3 ubiquitin-protein, MKRN2 was down-regulated in gastric cancer tissues. Kaplan-Meier survival analysis showed the low MKRN2 expression significantly indicated poor prognosis. Overexpression of MKRN2 notably inhibited cell proliferation in vitro and in vivo. Conversely, knockdown of MKRN2 had the opposite effects in vitro. Additionally, the mechanical analysis indicated that MKRN2 promoted ubiquitination-mediated degradation of PKM2 and attenuated its effect on ERK. Overall, the present study suggests that MKRN2 may be a potential therapeutic target for gastric cancer.

MKRN2 Physically Interacts with GLE1 to Regulate mRNA Export and Zebrafish Retinal Development.

The mammalian mRNA nuclear export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through ribonucleoprotein remodeling. We conduct a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network reveals interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assess MKRN2 epistasis with GLE1 in a zebrafish model. Morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescue retinal developmental defects seen upon GLE1 depletion, consistent with a functional association between GLE1 and MKRN2. Using ribonomic approaches, we show that MKRN2 binds selectively to the 3' UTR of a diverse subset of mRNAs and that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, we suggest that MKRN2 interacts with GLE1 to selectively regulate mRNA nuclear export and retinal development.

Integrated analysis of exosomal lncRNA and mRNA expression profiles reveals the involvement of lnc-MKRN2-42:1 in the pathogenesis of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a common movement disorder for which diagnosis mainly depends on the medical history and clinical symptoms. Exosomes are now considered an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids, and genetic material. Long noncoding (lnc) RNA in exosomes plays a critical role in many diseases, including neurodegenerative disease. AIM: To study expression differences for lncRNAs in peripheral blood exosomes of PD patients compared with healthy individuals and to look for lncRNAs that might be related to the pathogenesis of PD. MATERIALS AND METHODS: We recruited PD patients along with age- and sex-matched healthy individuals as healthy controls and evaluated levels of lncRNAs extracted from exosomes in plasma samples via next-generation sequencing and real-time quantitative PCR. Correlation analysis was conducted for the clinical characteristics of PD patients and the expression of selected lncRNAs. RESULTS: We found 15 upregulated and 24 downregulated exosomal lncRNAs in the PD group. According to bioinformatics analyses, we chose lnc-MKRN2-42:1 for further study. Interestingly, lnc-MKRN2-42:1 was positively correlated with the MDS-UPDRS III score for PD patients. CONCLUSION: Our study suggested that lnc-MKRN2-42:1 may be involved in the occurrence and development of PD.

MKRN2 inhibits migration and invasion of non-small-cell lung cancer by negatively regulating the PI3K/Akt pathway.

BACKGROUND: Makorin RING zinc finger-2 (MKRN2) belongs to the makorin RING zinc finger family and is a novel ubiquitin E3 ligase targeting the p65 subunit of NF-κB to negatively regulate inflammatory responses; however, the relationship between MKRN2 and tumorigenesis remains unclear. In this study, we clarified the role of MKRN2 in non-small cell lung cancer (NSCLC). METHODS: Tumor specimens collected from 261 NSCLC patients from 2013 to 2017 were retrieved from the Pathology Archive of the First Affiliated Hospital of China Medical University, and we performed assays to evaluate MKRN2 expression and to determine the impact of MKRN2 silencing and overexpression on NSCLC-cell migration and invasion. RESULTS: We demonstrated that MKRN2 expression was associated with lymph node metastasis, p-TNM stage, cancer-cell differentiation, and poor prognosis. By altering the expression of MKRN2 in selected cell lines, we found that MKRN2 inhibited cell migration and invasion through downregulation of the PI3K/Akt pathway. CONCLUSIONS: These results suggested that MKRN2 inhibited NSCLC progression by reducing the metastatic potential of cancer cells. Our findings provide critical insight into the association of MKRN2 expression with favorable clinicopathological characteristics in NSCLC patients and suggested that MKRN2 plays a role in inhibiting NSCLC development.