Adenovirus expressing nc886, an anti-interferon and anti-apoptotic non-coding RNA, is an improved gene delivery vector.

Recombinant adenovirus (rAdV) vector is the most promising vehicle to deliver an exogenous gene into target cells and is preferred for gene therapy. Exogenous gene expression from rAdV is often too inefficient to induce phenotypic changes and the amount of administered rAdV must be very high to achieve a therapeutic dose. However, it is often hampered because a high dose of rAdV is likely to induce cytotoxicity by activating immune responses. nc886, a 102-nucleotide non-coding RNA that is transcribed by RNA polymerase III, acts as an immune suppressor and a facilitator of AdV entry into the nucleus. Therefore, in this study, we have constructed an rAdV expressing nc886 (AdV:nc886) to explore whether AdV:nc886 overcomes the aforementioned drawbacks of conventional rAdV vectors. When infected into mouse cell lines and mice, AdV:nc886 expresses a sufficient amount of nc886, which suppresses the induction of interferon-stimulated genes and apoptotic pathways triggered by AdV infection. As a result, AdV:nc886 is less cytotoxic and produces more rAdV-delivered gene products, compared with the parental rAdV vector lacking nc886. In conclusion, this study demonstrates that the nc886-expressing rAdV could become a superior gene delivery vehicle with greater safety and higher efficiency for in vivo gene therapy.

Epigenetics of the non-coding RNA nc886 across blood, adipose tissue and skeletal muscle in offspring exposed to diabetes in pregnancy.

BACKGROUND: Diabetes in pregnancy is associated with increased risk of long-term metabolic disease in the offspring, potentially mediated by in utero epigenetic variation. Previously, we identified multiple differentially methylated single CpG sites in offspring of women with gestational diabetes mellitus (GDM), but whether stretches of differentially methylated regions (DMRs) can also be identified in adolescent GDM offspring is unknown. Here, we investigate which DNA regions in adolescent offspring are differentially methylated in blood by exposure to diabetes in pregnancy. The secondary aim was to characterize the RNA expression of the identified DMR, which contained the nc886 non-coding RNA. METHODS: To identify DMRs, we employed the bump hunter method in samples from young (9-16 yr, n = 92) offspring of women with GDM (O-GDM) and control offspring (n = 94). Validation by pyrosequencing was performed in an adult offspring cohort (age 28-33 years) consisting of O-GDM (n = 82), offspring exposed to maternal type 1 diabetes (O-T1D, n = 67) and control offspring (O-BP, n = 57). RNA-expression was measured using RT-qPCR in subcutaneous adipose tissue and skeletal muscle. RESULTS: One significant DMR represented by 10 CpGs with a bimodal methylation pattern was identified, located in the nc886/VTRNA2-1 non-coding RNA gene. Low methylation status across all CpGs of the nc886 in the young offspring was associated with maternal GDM. While low methylation degree in adult offspring in blood, adipose tissue, and skeletal muscle was not associated with maternal GDM, adipose tissue nc886 expression was increased in O-GDM compared to O-BP, but not in O-T1D. In addition, adipose tissue nc886 expression levels were positively associated with maternal pre-pregnancy BMI (p = 0.006), but not with the offspring's own adiposity. CONCLUSIONS: Our results highlight that nc886 is a metastable epiallele, whose methylation in young offspring is negatively correlated with maternal obesity and GDM status. The physiological effect of nc886 may be more important in adipose tissue than in skeletal muscle. Further research should aim to investigate how nc886 regulation in adipose tissue by exposure to GDM may contribute to development of metabolic disease.

Non-coding 886 (nc886/vtRNA2-1), the epigenetic odd duck - implications for future studies.

Non-coding 886 (nc886, vtRNA2-1) is the only human polymorphically imprinted gene, in which the methylation status is not determined by genetics. Existing literature regarding the establishment, stability and consequences of the methylation pattern, as well as the nature and function of the nc886 RNAs transcribed from the locus, are contradictory. For example, the methylation status of the locus has been reported to be stable through life and across somatic tissues, but also susceptible to environmental effects. The nature of the produced nc886 RNA(s) has been redefined multiple times, and in carcinogenesis, these RNAs have been reported to have conflicting roles. In addition, due to the bimodal methylation pattern of the nc886 locus, traditional genome-wide methylation analyses can lead to false-positive results, especially in smaller datasets. Herein, we aim to summarize the existing literature regarding nc886, discuss how the characteristics of nc886 give rise to contradictory results, as well as to reinterpret, reanalyse and, where possible, replicate the results presented in the current literature. We also introduce novel findings on how the distribution of the nc886 methylation pattern is associated with the geographical origins of the population and describe the methylation changes in a large variety of human tumours. Through the example of this one peculiar genetic locus and RNA, we aim to highlight issues in the analysis of DNA methylation and non-coding RNAs in general and offer our suggestions for what should be taken into consideration in future analyses.

Fermented black ginseng extract prevents UVB-induced inflammation by regulating the nc886-PKR pathway in human keratinocytes.

BACKGROUND: Continuous exposure of the skin to ultraviolet B (UVB) rays can cause inflammation and photodamage. In previous studies, we observed that the upregulation of nc886, a noncoding RNA (ncRNA), can alleviate UVB-induced inflammation through suppression of the protein kinase RNA (PKR) pathway. We aim to investigate the effect of fermented black ginseng extract (FBGE), which has been shown to increase the expression of nc886, on UVB-induced inflammation in keratinocytes. METHODS: To confirm the cytotoxicity of FBGE, MTT assay was performed, and no significant cytotoxicity was found on human keratinocytes. The efficacies of FBGE were assessed through qPCR, Western blotting, and ELISA analysis which confirmed regulation of UVB-induced inflammation. RESULTS: The analysis results showed that FBGE inhibited the decrease in nc886 expression and the increase in the methylated nc886 caused by UVB. It also prevented the UVB-induced increase of metalloproteinase-9 (MMP-9), metalloproteinase-1 (MMP-1), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α). Additionally, FBGE suppressed the PKR-MAPK pathways activated by UVB. CONCLUSION: These results implicate that FBGE can alleviate UVB-induced inflammation through regulation of the nc886-PKR pathway.

nc886, an RNA Polymerase III-Transcribed Noncoding RNA Whose Expression Is Dynamic and Regulated by Intriguing Mechanisms.

nc886 is a medium-sized non-coding RNA that is transcribed by RNA polymerase III (Pol III) and plays diverse roles in tumorigenesis, innate immunity, and other cellular processes. Although Pol III-transcribed ncRNAs were previously thought to be expressed constitutively, this concept is evolving, and nc886 is the most notable example. The transcription of nc886 in a cell, as well as in human individuals, is controlled by multiple mechanisms, including its promoter CpG DNA methylation and transcription factor activity. Additionally, the RNA instability of nc886 contributes to its highly variable steady-state expression levels in a given situation. This comprehensive review discusses nc886's variable expression in physiological and pathological conditions and critically examines the regulatory factors that determine its expression levels.

Hypermethylation of nc886 in HPV-positive oropharyngeal cancer and its clinical implications: An epigenome-wide association study.

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has increased rapidly in the United States, driven by rising human papillomavirus (HPV) infections in the U.S. population. HPV-positive OPSCC patients have a better prognosis than HPV-negative patients. To gain insights into the unique biology of HPV(+) OPSCC that may contribute to its clinical behaviors, we performed a multi-stage epigenome-wide methylation profiling of leukocyte and tumor DNA in OPSCC patients and compared the methylation levels of CpG sites between HPV(+) and HPV(-) OPSCC patients. We identified and validated a significantly differentially methylated region (DMR) of 1,355 bp encompassing non-coding RNA 886 (nc886) gene and its promoter region. Nc886 is hypermethylated in both leukocytes and tumor DNA of HPV(+) OPSCC patients. Homozygous knockout of nc886 by CRISPR-Cas9 in head and neck cell lines was lethal, but nc886 could be knocked out on the background of protein kinase R (PKR) knockout. Our data suggest that HPV induces nc886 hypermethylation, and nc886 acts as both a viral sensor and a tumor sensor in OPSCC patients and contribute to the better prognosis of HPV(+) OPSCC patients. Nc886 may become a therapeutic target in OPSCC.

Methylation status of VTRNA2-1/nc886 is stable across populations, monozygotic twin pairs and in majority of tissues.

Aims & methods: The aim of this study was to characterize the methylation level of a polymorphically imprinted gene, VTRNA2-1/nc886, in human populations and somatic tissues.48 datasets, consisting of more than 30 tissues and >30,000 individuals, were used. Results: nc886 methylation status is associated with twin status and ethnic background, but the variation between populations is limited. Monozygotic twin pairs present concordant methylation, whereas ∼30% of dizygotic twin pairs present discordant methylation in the nc886 locus. The methylation levels of nc886 are uniform across somatic tissues, except in cerebellum and skeletal muscle. Conclusion: The nc886 imprint may be established in the oocyte, and, after implantation, the methylation status is stable, excluding a few specific tissues.

Double-stranded RNA induction asa potential dynamic biomarkerfor DNA-demethylating agents.

Hypomethylating agents (HMAs), such as azacitidine and decitabine, induce cancer cell death by demethylating DNAs to promote the expression of tumor-suppressor genes. HMAs also reactivate the transcription of endogenous double-stranded RNAs (dsRNAs) that trigger the innate immune response and subsequent apoptosis via viral mimicry. However, the expression patterns of endogenous dsRNAs and their relevance in the efficacy of HMAs remain largely uninvestigated. Here, we employ amidine-conjugated spiropyran (Am-SP) to examine the dynamic expression pattern of total dsRNAs regulated by HMAs. By analyzing the bone-marrow aspirates of myelodysplastic syndrome or acute myeloid leukemia patients who received the HMAs, we find a dramatic increase in total dsRNA levels upon treatment only in patients who later benefited from the therapy. We further apply our approach in solid tumor cell lines and show that the degree of dsRNA induction correlates with the effectiveness of decitabine in most cases. Notably, when dsRNA induction is accompanied by increased expression of nc886 RNA, decitabine becomes ineffective. Collectively, our study establishes the potential application of monitoring the total dsRNA levels by a small molecule as an analytical method and a dynamic marker to predict the clinical outcome of the HMA therapy.

Are We Studying Non-Coding RNAs Correctly? Lessons from nc886.

Non-coding RNAs (ncRNAs), such as microRNAs or long ncRNAs, have brought about a new paradigm in the regulation of gene expression. Sequencing technologies have detected transcripts with tremendous sensitivity and throughput and revealed that the majority of them lack protein-coding potential. Myriad articles have investigated numerous ncRNAs and many of them claim that ncRNAs play gene-regulatory roles. However, it is questionable whether all these articles draw conclusions through cautious gain- and loss-of function experiments whose design was reasonably based on an ncRNA's correct identity and features. In this review, these issues are discussed with a regulatory ncRNA, nc886, as an example case to represent cautions and guidelines when studying ncRNAs.

A host non-coding RNA, nc886, plays a pro-viral role by promoting virus trafficking to the nucleus.

Elucidation of the interplay between viruses and host cells is crucial for taming viruses to benefit human health. Cancer therapy using adenovirus, called oncolytic virotherapy, is a promising treatment option but is not robust in all patients. In addition, inefficient replication of human adenovirus in mouse hampered the development of an in vivo model for preclinical evaluation of therapeutically engineered adenovirus. nc886 is a human non-coding RNA that suppresses Protein Kinase R (PKR), an antiviral protein. In this study, we have found that nc886 greatly promotes adenoviral gene expression and replication. Remarkably, the stimulatory effect of nc886 is not dependent on its function to inhibit PKR. Rather, nc886 facilitates the nuclear entry of adenovirus via modulating the kinesin pathway. nc886 is not conserved in mouse and, when xenogeneically expressed in mouse cells, promotes adenovirus replication. Our investigation has discovered a novel mechanism of how a host ncRNA plays a pro-adenoviral role. Given that nc886 expression is silenced in a subset of cancer cells, our study highlights that oncolytic virotherapy might be inefficient in those cells. Furthermore, our findings open future possibilities of harnessing nc886 to improve the efficacy of oncolytic adenovirus and to construct nc886-expressing transgenic mice as an animal model.

Nc886 promotes renal cancer cell drug-resistance by enhancing EMT through Rock2 phosphorylation-mediated ß-catenin nuclear translocation.

Drug resistance is a significant challenge in the present treatment regimens of renal cell carcinoma (RCC). Our previous study confirmed that nc886 functions as an oncogene in RCC. Nevertheless, the role and underlying mechanism of nc886 in RCC drug resistance are unclear. In the present study, Sunitinib and Everolimus treatment, respectively, downregulated nc886 expression in a dose-dependent manner in all four renal cancer cell lines. Nc886 overexpression in 786-O cells and ACHN cells significantly reduced the sensitivity of cancer cells to both Sunitinib and Everolimus treatment, respectively, by promoting cell viability and inhibiting cell apoptosis, whereas nc886 silencing increased cancer cell sensitivity. In renal cancer cell line with the highest drug-resistance, 786-O cells, Sunitinib, or Everolimus treatment enhanced the cellular EMT and was further enhanced by nc886 overexpression while attenuated by nc886 silencing. In 786-O cells, nc886 overexpression significantly promoted EMT, ROCK2 phosphorylation, and ß-catenin nucleus translocation under Sunitinib or Everolimus treatment. Moreover, ROCK2 silencing significantly reversed the effects of nc886 overexpression on EMT, ROCK2 phosphorylation, and ß-catenin nucleus translocation, as well as drug-resistant renal cancer cell viability and apoptosis. In conclusion, it was demonstrated that nc886 promotes renal cancer cell proliferation, migration, and invasion, as demonstrated previously. nc886 also promotes renal cancer cell drug-resistance to Sunitinib or Everolimus by promoting EMT through Rock2 phosphorylation-mediated nuclear translocation of ß-catenin.

nc886, a Non-Coding RNA, Is a New Biomarker and Epigenetic Mediator of Cellular Senescence in Fibroblasts.

Functional studies of organisms and human models have revealed that epigenetic changes can significantly impact the process of aging. Non-coding RNA (ncRNA), one of epigenetic regulators, plays an important role in modifying the expression of mRNAs and their proteins. It can mediate the phenotype of cells. It has been reported that nc886 (=vtRNA2-1 or pre-miR-886), a long ncRNA, can suppress tumor formation and photo-damages of keratinocytes caused by UVB. The aim of this study was to determine the role of nc886 in replicative senescence of fibroblasts and determine whether substances capable of controlling nc886 expression could regulate cellular senescence. In replicative senescence fibroblasts, nc886 expression was decreased while methylated nc886 was increased. There were changes of senescence biomarkers including SA-ß-gal activity and expression of p16INK4A and p21Waf1/Cip1 in senescent cells. These findings indicate that the decrease of nc886 associated with aging is related to cellular senescence of fibroblasts and that increasing nc886 expression has potential to suppress cellular senescence. AbsoluTea Concentrate 2.0 (ATC) increased nc886 expression and ameliorated cellular senescence of fibroblasts by inhibiting age-related biomarkers. These results indicate that nc886 has potential as a new target for anti-aging and that ATC can be a potent epigenetic anti-aging ingredient.

Pan-cancer chromatin analysis of the human vtRNA genes uncovers their association with cancer biology.

Background: The vault RNAs (vtRNAs) are a class of 84-141-nt eukaryotic non-coding RNAs transcribed by RNA polymerase III, associated to the ribonucleoprotein complex known as vault particle. Of the four human vtRNA genes, vtRNA1-1, vtRNA1-2 and vtRNA1-3, clustered at locus 1, are integral components of the vault particle, while vtRNA2-1 is a more divergent homologue located in a second locus. Gene expression studies of vtRNAs in large cohorts have been hindered by their unsuccessful sequencing using conventional transcriptomic approaches. Methods: VtRNA expression in The Cancer Genome Atlas (TCGA) Pan-Cancer cohort was estimated using the genome-wide DNA methylation and chromatin accessibility data (ATAC-seq) of their genes as surrogate variables. The association between vtRNA expression and patient clinical outcome, immune subtypes and transcriptionally co-regulated gene programs was analyzed in the dataset. Results: VtRNAs promoters are enriched in transcription factors related to viral infection. VtRNA2-1 is likely the most independently regulated homologue. VtRNA1-1 has the most accessible chromatin, followed by vtRNA1-2, vtRNA2-1 and vtRNA1-3. VtRNA1-1 and vtRNA1-3 chromatin status does not significantly change in cancer tissues. Meanwhile, vtRNA2-1 and vtRNA1-2 expression is widely deregulated in neoplastic tissues and its alteration is compatible with a broad oncogenic role for vtRNA1-2, and both tumor suppressor and oncogenic functions for vtRNA2-1. Yet, vtRNA1-1, vtRNA1-2 and vtRNA2-1 promoter DNA methylation predicts a shorter patient overall survival cancer-wide. In addition, gene ontology analyses of vtRNAs co-regulated genes identify a chromosome regulatory domain, epithelial differentiation, immune and thyroid cancer gene sets for specific vtRNAs. Furthermore, vtRNA expression patterns are associated with cancer immune subtypes and vtRNA1-2 expression is positively associated with cell proliferation and wound healing. Conclusions: Our study presents the landscape of vtRNA chromatin status cancer-wide, identifying co-regulated gene networks and ontological pathways associated with the different vtRNA genes that may account for their diverse roles in cancer.

Methylation status of nc886 epiallele reflects periconceptional conditions and is associated with glucose metabolism through nc886 RNAs.

BACKGROUND: Non-coding RNA 886 (nc886) is coded from a maternally inherited metastable epiallele. We set out to investigate the determinants and dynamics of the methylation pattern at the nc886 epiallele and how this methylation status associates with nc886 RNA expression. Furthermore, we investigated the associations between the nc886 methylation status or the levels of nc886 RNAs and metabolic traits in the YFS and KORA cohorts. The association between nc886 epiallele methylation and RNA expression was also validated in induced pluripotent stem cell (iPSC) lines. RESULTS: We confirm that the methylation status of the nc886 epiallele is mostly binomial, with individuals displaying either a non- or hemi-methylated status, but we also describe intermediately and close to fully methylated individuals. We show that an individual's methylation status is associated with the mother's age and socioeconomic status, but not with the individual's own genetics. Once established, the methylation status of the nc886 epiallele remains stable for at least 25 years. This methylation status is strongly associated with the levels of nc886 non-coding RNAs in serum, blood, and iPSC lines. In addition, nc886 methylation status associates with glucose and insulin levels during adolescence but not with the indicators of glucose metabolism or the incidence of type 2 diabetes in adulthood. However, the nc886-3p RNA levels also associate with glucose metabolism in adulthood. CONCLUSIONS: These results indicate that nc886 metastable epiallele methylation is tuned by the periconceptional conditions and it associates with glucose metabolism through the expression of the ncRNAs coded in the epiallele region.

VTRNA2-1: Genetic Variation, Heritable Methylation and Disease Association.

VTRNA2-1 is a metastable epiallele with accumulating evidence that methylation at this region is heritable, modifiable and associated with disease including risk and progression of cancer. This study investigated the influence of genetic variation and other factors such as age and adult lifestyle on blood DNA methylation in this region. We first sequenced the VTRNA2-1 gene region in multiple-case breast cancer families in which VTRNA2-1 methylation was identified as heritable and associated with breast cancer risk. Methylation quantitative trait loci (mQTL) were investigated using a prospective cohort study (4500 participants with genotyping and methylation data). The cis-mQTL analysis (334 variants ± 50 kb of the most heritable CpG site) identified 43 variants associated with VTRNA2-1 methylation (p < 1.5 × 10-4); however, these explained little of the methylation variation (R2 < 0.5% for each of these variants). No genetic variants elsewhere in the genome were found to strongly influence VTRNA2-1 methylation. SNP-based heritability estimates were consistent with the mQTL findings (h2 = 0, 95%CI: -0.14 to 0.14). We found no evidence that age, sex, country of birth, smoking, body mass index, alcohol consumption or diet influenced blood DNA methylation at VTRNA2-1. Genetic factors and adult lifestyle play a minimal role in explaining methylation variability at the heritable VTRNA2-1 cluster.

Oocyte age and preconceptual alcohol use are highly correlated with epigenetic imprinting of a noncoding RNA (nc886).

Genomic imprinting occurs before fertilization, impacts every cell of the developing child, and may be sensitive to environmental perturbations. The noncoding RNA, nc886 (also called VTRNA2-1) is the only known example of the ∼100 human genes imprinted by DNA methylation, that shows polymorphic imprinting in the population. The nc886 gene is part of an ∼1.6-kb differentially methylated region (DMR) that is methylated in the oocyte and silenced on the maternal allele in about 75% of humans worldwide. Here, we show that the presence or absence of imprinting at the nc886 DMR in an individual is consistent across different tissues, confirming that the imprint is established before cellular differentiation and is maintained into adulthood. We investigated the relationships between the frequency of imprinting in newborns and maternal age, alcohol consumption and cigarette smoking before conception in more than 1,100 mother/child pairs from South Africa. The probability of imprinting in newborns was increased in older mothers and decreased in mothers who drank alcohol before conception. On the other hand, cigarette smoking had no apparent relationship with the frequency of imprinting. These data show an epigenetic change during oocyte maturation which is potentially subject to environmental influence. Much focus has been placed on avoiding alcohol consumption during pregnancy, but our data suggest that drinking before conception may affect the epigenome of the newborn.

Nc886, a Novel Suppressor of the Type I Interferon Response Upon Pathogen Intrusion.

Interferons (IFNs) are a crucial component in the innate immune response. Especially the IFN-ß signaling operates in most cell types and plays a key role in the first line of defense upon pathogen intrusion. The induction of IFN-ß should be tightly controlled, because its hyperactivation can lead to tissue damage or autoimmune diseases. Activation of the IFN-ß promoter needs Interferon Regulatory Factor 3 (IRF3), together with Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Activator Protein 1 (AP-1). Here we report that a human noncoding RNA, nc886, is a novel suppressor for the IFN-ß signaling and inflammation. Upon treatment with several pathogen-associated molecular patterns and viruses, nc886 suppresses the activation of IRF3 and also inhibits NF-κB and AP-1 via inhibiting Protein Kinase R (PKR). These events lead to decreased expression of IFN-ß and resultantly IFN-stimulated genes. nc886's role might be to restrict the IFN-ß signaling from hyperactivation. Since nc886 expression is regulated by epigenetic and environmental factors, nc886 might explain why innate immune responses to pathogens are variable depending on biological settings.

Inhibition of UVB-Induced Inflammation by Laminaria japonica Extract via Regulation of nc886-PKR Pathway.

Continuous exposure to ultraviolet B (UVB) can cause photodamage of the skin. This photodamage can be inhibited by the overexpression of the non-coding RNA, nc886, via the protein kinase RNA-activated (PKR) pathway. The study aims to identify how UVB inhibits nc886 expression, and it also seeks to determine whether substances that can control nc886 expression can influence UV-induced inflammation, and the mechanisms involved. The results suggest that UVB irradiation accelerates the methylation of the nc886 gene, therefore, reducing its expression. This induces the activation of the PKR, which accelerates the expression of metalloproteinase-9 (MMP-9) and cyclooxygenase (COX-2), and the production of MMP-9, prostaglandin-endoperoxide synthase (PGE2), and certain pro-inflammatory cytokines, specifically interleukin-8 (IL-8), and tumor necrosis factor- (TNF-). Conversely, in a model of nc886 overexpression, the expression and production of those inflammatory factors are inhibited. In addition, Laminaria japonica extract (LJE) protect the levels of nc886 against UVB irradiation then subsequently inhibit the production of UV-induced inflammatory factors through the PKR pathway.

RNA polymerase III transcription as a disease factor.

RNA polymerase (Pol) III is responsible for transcription of different noncoding genes in eukaryotic cells, whose RNA products have well-defined functions in translation and other biological processes for some, and functions that remain to be defined for others. For all of them, however, new functions are being described. For example, Pol III products have been reported to regulate certain proteins such as protein kinase R (PKR) by direct association, to constitute the source of very short RNAs with regulatory roles in gene expression, or to control microRNA levels by sequestration. Consistent with these many functions, deregulation of Pol III transcribed genes is associated with a large variety of human disorders. Here we review different human diseases that have been linked to defects in the Pol III transcription apparatus or to Pol III products imbalance and discuss the possible underlying mechanisms.

A Regulatory Noncoding RNA, nc886, Suppresses Esophageal Cancer by Inhibiting the AKT Pathway and Cell Cycle Progression.

nc886 is a regulatory non-coding RNA (ncRNA) whose expression is frequently silenced in malignancies. In the case of esophageal squamous cell carcinoma (ESCC), nc886 silencing is associated with shorter survival of patients, suggesting nc886's tumor suppressor role in ESCC. However, this observation has not been complemented by an in-detail study about nc886's impact on gene expression and cellular phenotypes. Here we have shown that nc886 inhibits AKT, a key protein in a renowned pro-survival pathway in cancer. nc886-silenced cells (nc886- cells) have activated AKT and altered expression of cell cycle genes. nc886- cells tend to have lower expression of CDKN2A and CDKN2C, both of which are inhibitors for cyclin-dependent kinase (CDK), and higher expression of CDK4 than nc886-expressing cells. As a result, nc886- cells are hyperactive in the progression of the G1 to S cell cycle phase, proliferate faster, and are more sensitive to palbociclib, which is a cancer therapeutic drug that targets CDK4/6. Experimentally by nc886 expression and knockdown, we have determined the AKT target genes and cell cycle genes that are controlled by nc886 (nc886-associated gene sets). These gene sets, in combination with pathologic staging and nc886 expression levels, are a vastly superior predictor for the survival of 108 ESCC patients. In summary, our study has elucidated in ESCC how nc886 inhibits cell proliferation to explain its tumor suppressor role and identified gene sets that are of future clinical utility, by predicting patient survival and responsiveness to a therapeutic drug.