Promoting proliferation and tumorigenesis of breast cancer: KCND2's significance as a prognostic factor.

In recent years, the potassium voltage-gated channel subfamily D (KCND) channels, particularly KCND2 (also known as Kv4.2), have been suggested to play a role in a variety of cancers, but their role in breast cancer has not yet been revealed. We analyzed RNA sequencing data from The Cancer Genome Atlas database and the Genotype-Tissue Expression database to investigate the differential expression of KCND2 in breast cancer and normal breast tissue. In addition, we leveraged GO and KEGG analysis techniques to gain a better understanding of the potential functional enrichment of 500 genes related to KCND2. Our findings were validated using collected tissue samples and clinical data from hospitals showed that KCND2 is a crucial independent factor in the prognosis of breast cancer patients. The higher the expression of KCND2, the shorter the survival time of breast cancer patients. Colony formation assay confirmed that KCND2 promotes the proliferation of breast cancer cells, whereas transwell assay and wound healing assay verified that KCND2 promoted breast cancer invasion and migration. In addition, 5-Ethynyl-2'-deoxyuridine (EdU) and flow cytometry revealed that KCND2 affected the cycle changes of breast cancer cells and contributed to the G1/S phase transition of breast cancer cells. Overall, our study demonstrates that KCND2 holds a promising potential as a significant target for breast cancer diagnosis and therapy.

Long noncoding RNA FAM66C promotes tumor progression and glycolysis in intrahepatic cholangiocarcinoma by regulating hsa-miR-23b-3p/KCND2 axis.

Long noncoding RNAs (lncRNAs) are known to be the important regulators in cancer progression. However, the role of lncRNA FAM66C (FAM66C) is yet to be investigated in intrahepatic cholangiocarcinoma (ICC). This study aimed to investigate the effects and related mechanisms of FAM66C in ICC. Human ICC tissues and cell lines were collected. The expression levels of FAM66C, hsa-miR-23b-3p (miR-23b-3p), and KCND2 were detected by qRT-RCR. The transfection experiments were employed to measure the effect of FAM66C on cell viabilities, migration, and invasion in ICC cells by CCK-8, transwell assays. Glycolysis was investigated by glucose consumption, lactate production and ATP levels. The dual-luciferase reporter and RNA pull down assays were conducted as a means of confirming the interactions between FAM66C, miR-23b-3p, and KCND2. Furthermore, the levels of the EMT-associated proteins (KCND2, GLUT1, PKM2, and LDHA) in ICC cells were detected by western blot. FAM66C was increased in ICC tissues and cells, increased cell viability, glycolysis, migration and invasion, and decreased apoptosis were shown in FAM66C overexpressing cells. Mechanistic analyses revealed that FAM66C regulated the downstream target gene KCND2 by sponging miR-23b-3p. FAM66C effect on ICC was further validated in murine xenograft assays. FAM66C knockdown cells gave rise to tumors that were smaller in size, consistent with the role of FAM66C as a promoter of in vivo tumor growth. These data revealed that FAM66C was able to drive ICC tumor progression and glycolytic activity via the miR-23b-3p/KCND2 axis, indicating FAM66C may be a viable target for treating ICC.

KCND2 upregulation might be an independent indicator of poor survival in gastric cancer.

AIM: We aimed to investigate the expression of voltage-gated potassium channels KCND1/KCND2/KCND3 in gastric cancer (GC) and normal stomach tissues and to investigate the prognostic value of the upregulated gene KCND2. PATIENTS & METHODS: A retrospective analysis was performed using data from large available databases. RESULTS: KCND2 was significantly upregulated at the mRNA and protein levels in GC compared with that in normal stomach tissues. High KCND2 RNA expression was independently associated with shorter overall survival (HR: 1.634, 95% CI: 1.135-2.352; p = 0.008) and recurrence-free survival (HR: 2.644, 95% CI: 1.438-4.863; p = 0.002). Data mining in the Kaplan-Meier plotter confirmed the prognostic value of KCND2. CONCLUSION: KCND2 upregulation is a valuable prognostic biomarker in GC patients, in terms of overall survival and recurrence-free survival.

KCND2: A prognostic biomarker and regulator of immune function in gastric cancer.

BACKGROUND: Gastric cancer is a highly heterogeneous disease, which makes it challenging to develop effective targeted therapies. Although the potassium voltage-gated channel subfamily D (KCND) channels, particularly KCND2 (also known as Kv4.2), have found evidence of involvement in the occurrence and development of various cancers, there are still some limitations in our understanding of KCND2's roles in gastric cancer. METHODS: We analyzed the correlation between KCND2 expression and clinical features as well as immune infiltration using the Cancer Genome Atlas (TCGA) database. Functional assays of KCND2 were conducted using Cell counting Kit-8 (CCK8), clone formation assay and cell cycle analysis. Additionally, immunofluorescence, flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR) techniques were used to investigate tumor proliferation and immune cell infiltration at different levels of KCND2 expression in vivo. RESULTS: KCND2 was markedly elevated in gastric cancer and its expression appeared to link to different grades, T stages, and N stages. In addition, KCND2 was an independent predictor of prognosis, and its elevated levels in TCGA database revealed a more unfavorable prognosis for patients with gastric cancer. KCND2 strengthened the viability at the cellular level by boosting the proliferation of gastric cancer cells and reducing their death rate. Additionally, it also highlights that KCND2 the abilities of proliferating of gastric cancer cells by stimulating NF-κB both in cell and animal levels. In addition, the findings provided proof that in animal levels, KCND2 might regulate the immune system by associating with promoting M2 macrophages, which are known to play critical roles in cancer progression. Mechanistically, KCND2 was found to lead to the infiltration of M2 macrophages through activation of NF-κB, ultimately promoting the advancement of gastric cancer. CONCLUSION: Overall, these findings suggest that KCND2 is likely to be available as an underlying therapeutic target for gastric cancer.

Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons.

Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated K+ channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Transcriptome Analysis of Cardiac Hypertrophic Growth in MYBPC3-Null Mice Suggests Early Responders in Hypertrophic Remodeling.

Rationale: With a prevalence of 1 in 200 individuals, hypertrophic cardiomyopathy (HCM) is thought to be the most common genetic cardiac disease, with potential outcomes that include severe hypertrophy, heart failure, and sudden cardiac death (SCD). Though much research has furthered our understanding of how HCM-causing mutations in genes such as cardiac myosin-binding protein C (MYBPC3) impair contractile function, it remains unclear how such dysfunction leads to hypertrophy and/or arrhythmias, which comprise the HCM phenotype. Identification of early response mediators could provide rational therapeutic targets to reduce disease severity. Our goal was to differentiate physiologic and pathophysiologic hypertrophic growth responses and identify early genetic mediators in the development of cardiomegaly in the cardiac myosin-binding protein C-null (cMyBP-C-/-) mouse model of HCM. Methods and Results: We performed microarray analysis on left ventricles of wild-type (WT) and cMyBPC-/- mice (n = 7 each) at postnatal day (PND) 1 and PND 9, before and after the appearance of an overt HCM phenotype. Applying the criteria of ≥2-fold change, we identified genes whose change was exclusive to pathophysiologic growth (n = 61), physiologic growth (n = 30), and genes whose expression changed ≥2-fold in both WT and cMyBP-C-/- hearts (n = 130). Furthermore, we identified genes that were dysregulated in PND1 cMyBP-C-/- hearts prior to hypertrophy, including genes in mechanosensing pathways and potassium channels linked to arrhythmias. One gene of interest, Xirp2, and its protein product, are regulated during growth but also show early, robust prehypertrophic upregulation in cMyBP-C-/- hearts. Additionally, the transcription factor Zbtb16 also shows prehypertrophic upregulation at both gene and protein levels. Conclusion: Our transcriptome analysis generated a comprehensive data set comparing physiologic vs. hypertrophic growth in mice lacking cMyBP-C. It highlights the importance of extracellular matrix pathways in hypertrophic growth and early dysregulation of potassium channels. Prehypertrophic upregulation of Xirp2 in cMyBP-C-/- hearts supports a growing body of evidence suggesting Xirp2 has the capacity to elicit both hypertrophy and arrhythmias in HCM. Dysregulation of Xirp2, as well as Zbtb16, along with other genes associated with mechanosensing regions of the cardiomyocyte implicate stress-sensing in these regions as a potentially important early response in HCM.

Update on the implication of potassium channels in autism: K(+) channelautism spectrum disorder.

Autism spectrum disorders (ASDs) are characterized by impaired ability to properly implement environmental stimuli that are essential to achieve a state of social and cultural exchange. Indeed, the main features of ASD are impairments of interpersonal relationships, verbal and non-verbal communication and restricted and repetitive behaviors. These aspects are often accompanied by several comorbidities such as motor delay, praxis impairment, gait abnormalities, insomnia, and above all epilepsy. Genetic analyses of autistic individuals uncovered deleterious mutations in several K(+) channel types strengthening the notion that their intrinsic dysfunction may play a central etiologic role in ASD. However, indirect implication of K(+) channels in ASD has been also reported. For instance, loss of fragile X mental retardation protein (FMRP) results in K(+) channels deregulation, network dysfunction and ASD-like cognitive and behavioral symptoms. This review provides an update on direct and indirect implications of K(+) channels in ASDs. Owing to a mounting body of evidence associating a channelopathy pathogenesis to autism and showing that nearly 500 ion channel proteins are encoded by the human genome, we propose to classify ASDs - whose susceptibility is significantly enhanced by ion channels defects, either in a monogenic or multigenic condition - in a new category named " c hannel A utism S pectrum D isorder" (channelASD; cASD) and introduce a new taxonomy (e.g., Kv x.y-channelASD and likewise Nav x.y-channelASD, Cav x.y-channelASD; etc.). This review also highlights some degree of clinical and genetic overlap between K(+) channelASDs and K(+) channelepsies, whereby such correlation suggests that a subcategory characterized by a channelASD-channelepsy phenotype may be distinguished. Ultimately, this overview aims to further understand the different clinical subgroups and help parse out the distinct biological basis of autism that are essential to establish patient-tailored treatments.

MRNA and miRNA expression patterns associated to pathways linked to metal mixture health effects.

Metals are a threat to human health by increasing disease risk. Experimental data have linked altered miRNA expression with exposure to some metals. MiRNAs comprise a large family of non-coding single-stranded molecules that primarily function to negatively regulate gene expression post-transcriptionally. Although several human populations are exposed to low concentrations of As, Cd and Pb as a mixture, most toxicology research focuses on the individual effects that these metals exert. Thus, this study aims to evaluate global miRNA and mRNA expression changes induced by a metal mixture containing NaAsO2, CdCl2, Pb(C2H3O2)2·3H2O and to predict possible metal-associated disease development under these conditions. Our results show that this metal mixture results in a miRNA expression profile that may be responsible for the mRNA expression changes observed under experimental conditions in which coding proteins are involved in cellular processes, including cell death, growth and proliferation related to the metal-associated inflammatory response and cancer.