Interference of the Circular RNA Sperm Antigen With Calponin Homology and Coiled-Coil Domains 1 Suppresses Growth and Promotes Apoptosis of Breast Cancer Cells Partially Through Targeting miR-1236-3p/Chromobox 8 Pathway.

Noncoding RNAs and RNA modifiers are implicated in cancer radiotherapy. Here, we aimed to investigate the role of sperm antigen with calponin homology and coiled-coil domains 1 (SPECC1)-derived circular RNA (circSPECC1; hsa_circ_0000745) in breast cancer (BC) cells under radiation treatment. Based on quantitative real-time PCR, circSPECC1 was highly upregulated in BC patients' tumors and cells, and circSPECC1 expression was further increased with the dosage of radiation in BC cells. Moreover, circSPECC1 upregulation was found to be concomitant with higher chromobox 8 (CBX8) and lower microRNA (miR)-1236-3p expression. Functionally, 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU) and colony formation assays showed that circSPECC1 interference suppressed cell proliferation and long-term survival in BC cells and irradiated BC cells. Xenograft tumor model experiments showed that circSPECC1 knockdown restrained BC tumor growth in vivo. Meanwhile, flow cytometry assay and western blotting revealed an enhanced apoptosis by silencing circSPECC1. Moreover, miR-1236-3p overexpression, similar to circSPECC1 silencing, displayed anti-growth and proapoptosis roles in irradiated BC cells. Mechanistically, dual-luciferase reporter assay and RNA immunoprecipitation assay identified a target relationship between miR-1236-3p and circSPECC1 or CBX8. Also, CBX8 expression could be modulated by circSPECC1 via miR-1236-3p regulation. Collectively, we indicated that inhibiting circSPECC1 could suppress growth and promote apoptosis of BC cells in both irradiated and nonirradiated conditions at least partially via miR-1236-3p/CBX8 axis, confirming that circSPECC1 might be target to develop anticancer drug in BC.

CBX8 Promotes Epithelial-mesenchymal Transition, Migration, and Invasion of Lung Cancer through Wnt/ß-catenin Signaling Pathway.

BACKGROUND: Lung cancer (LC) is primarily responsible for cancer-related deaths worldwide. Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells acquire mesenchymal features and is associated with the development of tumors. CBX8, a member of the PcG protein family, plays a critical role in various cancers, containing LC. However, specific regulatory mechanisms of CBX8 in LC progression are not fully understood. This study aimed to investigate the regulatory role of CBX8 in LC progression. METHODS: Bioinformatics was used to analyze the relationship between CBX8 level and tumor and the enrichment pathway of CBX8 enrichment. qRT-PCR was used to detect the differential expression of CBX8 in LC cells and normal lung epithelial cells. The effects of knockdown or overexpression of CBX8 on the proliferation, migration and invasion of LC cells were evaluated by CCK- -8 assay and Transwell assay, and the levels of proteins associated with the EMT pathway and Wnt/ ß-catenin signaling pathway were detected by western blot. RESULTS: Bioinformatics analysis revealed that CBX8 was highly expressed in LC and enriched on the Wnt/ß-catenin signaling pathway. The expression level of CBX8 was significantly elevated in LC cells. Knockdown of CBX8 significantly inhibited cell proliferation, migration and invasion, and decreased the expression levels of EMT-related proteins and Wnt/ß-catenin pathway-related proteins. Conversely, overexpression of CBX8 promoted cell proliferation, migration and invasion, and increased the expression levels of EMT-related proteins and Wnt/ß-catenin pathway-related proteins. The Wnt inhibitor IWP-4 alleviated the effects produced by overexpression of CBX8. CONCLUSION: Collectively, these data demonstrated that CBX8 induced EMT through Wnt/ß-- catenin signaling, driving migration and invasion of LC cells.

TRIM28 recruits E2F1 to regulate CBX8-mediated cell proliferation and tumor metastasis of ovarian cancer.

Chromobox protein homolog 8 (CBX8) is a transcriptional suppressor participated in various cancers. However, the function and mechanism of CBX8 in the progression of ovarian cancer (OC) are unclear. In this study, we found that CBX8 was upregulated in OC tissues originating from GEPIA and TNM databases, OC patients' samples from hospital, and OC cell lines. Furthermore, CBX8 knockdown by short hairpin RNA (shRNA) technology markedly inhibited proliferation and invasion, induced migration, cell cycle arrest, and apoptosis in vitro. Mechanistically, CBX8 activated PI3K/AKT/mTOR signaling pathway to take effect. In addition, TRIM28 and E2F1 were enriched in OC tissues from the TNM database and OC patients' samples similar to the results of CBX8. Correlation analysis indicated positive correlations among TRIM28, E2F1, and CBX8. E2F1 was proved to bind to the promoter regions of CBX8 and TRIM28, while TRIM28 recruited E2F1 to increase the expression of CBX8 to further increase cell viability, proliferation, and invasion, and decrease migration, apoptosis, and cell cycle progression. Finally, CBX8 or TRIM28 knockdown repressed tumor growth and metastasis of OC in vivo. Therefore, our study showed that the promoting effect of CBX8 on tumor growth and metastasis of OC was participated in the PI3K/AKT/mTOR signaling, TRIM28 and E2F1. Our findings suggested that CBX8 could serve as a potential marker and therapeutic target for OC patients.

CBX8 promotes lung adenocarcinoma growth and metastasis through transcriptional repression of CDKN2C and SCEL.

Dysregulation of polycomb group (PcG) proteins that mediate epigenetic gene silencing contributes to tumorigenesis. As core components of the polycomb repressive complex 1 (PRC1), chromobox (CBX) proteins recognize H3K27me3 to recruit PRC1 to maintain a repressive transcriptional state. However, the individual biological functions of these CBX proteins in tumorigenesis warrant in-depth investigation. In this study, we analyzed the mRNA expression of CBX family genes across multiple cancers using The Cancer Genome Atlas data and found different expression patterns of the five CBX genes in different types of cancer. This analyses together with the result of immunohistochemistry indicated that CBX8 expression was significantly higher in lung adenocarcinoma (LUAD) tissues compared to adjacent nontumor tissues. Overexpression approaches demonstrated that CBX8 facilitated LUAD cell proliferation and migration in vitro. Consistently, CBX8 knockdown reduced LUAD cell proliferation and migration in both cell culture and mouse models. RNA sequencing combined with real-time RT-PCR assays revealed CDKN2C and SCEL as target genes of CBX8. Furthermore, chromatin immunoprecipitation assays indicated that CBX8 directly bound to the promoters of CDKN2C and SCEL to establish H2AK119ub. CBX8 depletion reduced the enrichment of H2AK119ub on CDKN2C and SCEL promoters. Moreover, depletion of CDKN2C and SCEL restored the repressed growth and invasion ability of LUAD cells caused by CBX8 knockdown. These findings demonstrate that CBX8 promotes LUAD growth and metastasis through the transcriptional repression of CDKN2C and SCEL. Our study uncovers the oncogenic role of CBX8 in LUAD progression and provides a new target for the diagnosis and therapy of LUAD.

High CBX8 Expression Leads to Poor Prognosis in Laryngeal Squamous Cell Carcinoma by Inducing EMT by Activating the Wnt/ß-Catenin Signaling Pathway.

Background: In this study, we detected the expression of chromobox protein homolog 8 (CBX8) in laryngeal squamous cell carcinoma (LSCC) and its influence on the occurrence and progression of LSCC. Methods: Pancancer analysis of CBX8 was analyzed by TCGA database and its expression in LSCC.The expression of CBX8 in 30 pairs of LSCC and adjacent tissues was analyzed by quantitative real-time PCR(qRT-PCR)and immunohistochemical assays, and its association with the prognosis and clinicopathological features of LSCC was further evaluated. A CBX8 knockdown model was constructed in AMC-HN-8 and Hep2 cell lines. The effects of CBX8 on LSCC cell proliferation, migration, invasion and apoptosis were detected by CCK8,EdU,wound healing, Transwell and flow cytometry assays. Levels of apoptosis-related protein, WNT/ß-catenin signaling pathway and epithelial to mesenchymal transition (EMT) proteins, including Bax, Bcl2, ß-catenin, DKK1, GSK3ß, N-cadherin, E-cadherin and Snail1, in LSCC cells were detected by Western blotting. Results: CBX8 was overexpressed in LSCC. High expression of CBX8 in LSCC patients led to shorter overall survival and correlated with tumor stage and lymphatic metastasis. After CBX8 knockdown, the proliferation of AMC-HN-8 and Hep2 cells slowed, and the number of EdU-positive cells decreased. Wound healing slowed down, and the number of Transwell invading cells decreased. The percentage of apoptotic cells increased. The expression levels of Bcl2, ß-catenin, N-cadherin and Snail11 proteins were significantly reduced in the CBX8 knockdown cells, while Bax, DKK1, GSK3ß and E-cadherin significantly increased with their corresponding controls. Conclusion: CBX8 is highly expressed in LSCC and induces the EMT process by activating the Wnt/ß-catenin signaling pathway to promote LSCC cell proliferation and migration and inhibit apoptosis, resulting in poor prognosis.

High Expression of a Cancer Stemness-Related Gene, Chromobox 8 (CBX8), in Normal Tissue Adjacent to the Tumor (NAT) Is Associated with Poor Prognosis of Colorectal Cancer Patients.

BACKGROUND: Several studies have demonstrated that the molecular profile of normal tissue adjacent to the tumor (NAT) is prognostic for recurrence in patients with different cancers. This study investigated the clinical significance of CBX8 gene expression, a cancer stemness-related gene, in tumor and NAT tissue of colorectal cancer (CRC) patients. METHODS: The gene level of CBX8 in paired CRC and NAT specimens from 95 patients was determined by quantitative PCR. CBX8 protein level in CRC and NAT specimens from 66 patients was determined by immunohistochemistry. CBX8 gene and protein levels were correlated with the patients' clinicopathological parameters and circulatory immune cell profiles. The association between CBX8 and pluripotency-associated genes was analyzed using the TCGA database. RESULTS: NAT CBX8 gene level positively correlated with TNM stage, tumor invasion, lymph node metastasis and distant metastasis, indicating its association with tumor progression and metastasis. There was no correlation between NAT CBX8 protein level and clinicopathological parameters. Moreover, a high level of CBX8 gene and protein in NAT both correlated with poor DFS and OS. There was an inverse correlation between CBX8 gene level and post-operative platelet counts and platelet to lymphocyte level, suggesting its association with systematic inflammation. Finally, TCGA analysis showed that CBX8 level was correlated with a couple of pluripotency-associated genes, supporting its association with cancer stemness. CONCLUSIONS: High NAT CBX8 is a poor prognostic factor for tumor progression and survival in CRC patients.

CBX8 interacts with chromatin PTEN and is involved in regulating mitotic progression.

OBJECTIVES: Besides its role in regulating phosphatidylinositol-3 kinase (PI3K) signalling in the cytosol, PTEN also has a nuclear function. In this study, we attempted to understand the mechanism of chromatin PTEN in suppressing chromosomal instability during cell division. MATERIALS AND METHODS: Immunocoprecipitation, ectopic expression, and deletional analyses were used to identify the physical interaction between Chromobox Homolog protein 8 (CBX8) and PTEN, as well as the functional domain(s) of PTEN mediating the interaction. Cell synchronization followed by immunoblotting was employed to study cell cycle regulation of CBX8 and the functional interaction between chromatin PTEN and CBX8. Small interfering RNAs (siRNAs) were used to study the role of PTEN and CBX8 in modulating histone epigenetic markers during the cell cycle. RESULTS: Polycomb group (PcG) proteins including CBXs function to repress gene expression in a wide range of organisms including mammals. We recently showed that PTEN interacted with CBX8, a component of Polycomb Repressing Complex 1 (PRC1), and that CBX8 co-localized with PTEN in the nucleus. CBX8 levels were high, coinciding with its phosphorylation in mitosis. Phosphorylation of CBX8 was associated with monoubiquitinated PTEN and phosphorylated-BubR1 on chromatin. Moreover, CBX8 played an important role in cell proliferation and mitotic progression. Significantly, downregulation of either PTEN or CBX8 induced H3K27Me3 epigenetic marker in mitotic cells. CONCLUSION: CBX8 is a new component that physically interacts with chromatin PTEN, playing an important role in regulating mitotic progression.

Chromobox Homolog 8 (CBX8) in Human Tumor Carcinogenesis and Prognosis: A Pancancer Analysis Using Multiple Databases.

Some emerging studies have suggested that chromobox homolog 8 (CBX8) may play a critical role in carcinogenesis and prognosis in human cancer. Based on The Cancer Genome Atlas (TCGA)'s available data and the Gene Expression Omnibus (GEO) database, we conducted a systematic analysis of the carcinogenic effects of the CBX8 gene. We used TIMER2, GEPIA2, UALCAN, cBioPortal, Kaplan-Meier plotter, OncoLnc, STRING, HPA, and Oncomine data analysis websites and R data analysis software to analyze available data. The results show that the level of expression of CBX8 was significantly different among 27 different types of tumors and adjacent normal tissues. Moreover, we found that CBX8 expression had a close relationship with prognosis in some kinds of cancers. The phosphorylation level of some protein sites (such as S256) was significantly increased in tumors. CD8 + T-cell, B-cell and cancer-associated fibroblast infiltration levels were associated with CBX8 expression. The results of enrichment analysis indicated that the main biological activities of CBX8 are connected to gene transcription and repair of DNA damage. In conclusion, the level of expression of CBX8 was closely related to carcinogenesis and prognosis of some kinds of tumors, which needs further experimental verification.

Construction of a prognostic model with histone modification-related genes and identification of potential drugs in pancreatic cancer.

BACKGROUND: Pancreatic cancer (PC) is a highly fatal and aggressive disease with its incidence and mortality quite discouraging. An effective prediction model is urgently needed for the accurate assessment of patients' prognosis to assist clinical decision-making. METHODS: Gene expression data and clinicopathological data of the samples were acquired from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Gene Expression Omnibus (GEO) databases. Differential expressed genes (DEGs) analysis, univariate Cox regression analysis, least absolute shrinkage and selection operator (LASSO) regression analysis, random forest screening and multivariate Cox regression analysis were applied to construct the risk signature. The effectiveness and independence of the model were validated by time-dependent receiver operating characteristic (ROC) curve, Kaplan-Meier (KM) survival analysis and survival point graph in training set, test set, TCGA entire set and GSE57495 set. The validity of the core gene was verified by immunohistochemistry and our own independent cohort. Meanwhile, functional enrichment analysis of DEGs between the high and low risk groups revealed the potential biological pathways. Finally, CMap database and drug sensitivity assay were utilized to identify potential small molecular drugs as the risk model-related treatments for PC patients. RESULTS: Four histone modification-related genes were identified to establish the risk signature, including CBX8, CENPT, DPY30 and PADI1. The predictive performance of risk signature was validated in training set, test set, TCGA entire set and GSE57495 set, with the areas under ROC curve (AUCs) for 3-year survival were 0.773, 0.729, 0.775 and 0.770 respectively. Furthermore, KM survival analysis, univariate and multivariate Cox regression analysis proved it as an independent prognostic factor. Mechanically, functional enrichment analysis showed that the poor prognosis of high-risk population was related to the metabolic disorders caused by inadequate insulin secretion, which was fueled by neuroendocrine aberration. Lastly, a cluster of small molecule drugs were identified with significant potentiality in treating PC patients. CONCLUSIONS: Based on a histone modification-related gene signature, our model can serve as a reliable prognosis assessment tool and help to optimize the treatment for PC patients. Meanwhile, a cluster of small molecule drugs were also identified with significant potentiality in treating PC patients.

Polycomb Paralog Chromodomain Inhibitors Active against Both CBX6 and CBX8*.

Methyllysine reader proteins bind to methylated lysine residues and alter gene transcription by changing either the compaction state of chromatin or by the recruitment of other multiprotein complexes. The polycomb paralog family of methyllysine readers bind to trimethylated lysine on the tail of histone 3 (H3) via a highly conserved aromatic cage located in their chromodomains. Each of the polycomb paralogs are implicated in several disease states. CBX6 and CBX8 are members of the polycomb paralog family with two structurally similar chromodomains. By exploring the structure-activity relationships of a previously reported CBX6 inhibitor we have discovered more potent and cell permeable analogs. Our current report includes potent, dual-selective inhibitors of CBX6 and CBX8. We have shown that the -2 position in our scaffold is an important residue for selectivity amongst the polycomb paralogs. Preliminary cell-based studies show that the new inhibitors impact cell proliferation in a rhabdoid tumor cell line.

CBX8 acts as an independent RNA-binding protein to regulate the maturation of miR-378a-3p in colon cancer cells.

CBX8 is the core component of the PCG family protein PRC1 complex. It is overexpressed in many solid tumors and plays an important role in the prognosis and biological behaviors of tumors such as occurrence, development, invasion, and metastasis. However, exploration of the role and molecular mechanism of CBX8 in tumors is still in its infancy. Our study found that the down-regulation of CBX8 expression by RNA interference induced differential expression of several microRNAs in human colon cancer cells. The 5 most differentially expressed miRNA precursors (pre-miRNA) (hsa-miR-363-3p, hsa-miR-378a-3p, hsa-miR-371b-3p, hsa-miR-361-3p, and hsa-miR-576-3p) share a common motif sequence: ARAAAKUGCMC. We selected miR-378a-3p and further revealed that the negative regulation of miRNA expression by CBX8 mainly occurs in the processing of pre-miRNA to mature miRNA. CBX8 uses its own RNA-binding domain to interact with pre-miRNA, and is dependent on its own nuclear localization characteristics to limit nucleoplasmic transport of pre-miRNA. Changing the characteristic sequence of pre-miRNA or mutating the RNA-binding domain and nuclear localization signal of CBX8 can effectively weaken the regulation of miR-378a-3p expression by CBX8. However, our experimental results showed that miR-378a-3p inhibited the malignant expression of human colon cancer cells by targeting PDIA4, resulting in increased activity of caspases-3 and -7. In summary, our study suggests that CBX8 acts as an independent RNA-binding protein to regulate miRNA expression. Simultaneously, this study shows the correlation between the CBX8/miR-378a-3p/PDIA4 pathway and the malignant biological properties of colorectal cancer, suggesting this proposed pathway as a possible therapeutic target for human cancers.

Relative mRNA and protein stability of epigenetic regulators in musculoskeletal cell culture models.

Control of gene expression by epigenetic regulators is fundamental to tissue development and homeostasis. Loss-of-function (LOF) studies using siRNAs for epigenetic regulators require that RNA interference rapidly reduces the cellular levels of the corresponding mRNAs and/or proteins. The most abundant chromatin structural proteins (i.e., the core histones H2A, H2B, H3 and H4) have relatively long half-lives and do not turn over rapidly, although their mRNAs are labile. The question arises whether epigenetic regulatory enzymes (e.g., Ezh2) or proteins that interact with histones via selective modifications (e.g., Cbx1 to Cbx8, Brd4) are stable or unstable. Therefore, we performed classical α-amanitin and cycloheximide inhibition assays that block, respectively, mRNA transcription and protein translation in mouse MC3T3 osteoblasts, ATDC5 chondrocytes and C2C12 myoblasts. We find that mRNA levels of Cbx proteins and Ezh2 were significantly depleted after 24 hrs, while their corresponding proteins remained relatively stable. As positive control, the half-life of the labile cyclin D1 protein was found to be less than 1 hr. Our study suggests that histone code readers and writers are relatively stable chromatin-related proteins, which is consistent with their long-term activities in maintaining chromatin organization and phenotype identity. These findings have conceptual ramifications for the interpretation of RNAi experiments that reduce the mRNA but not protein levels of epiregulatory proteins. We propose that siRNAs for at least some epigenetic regulatory proteins may exert their biological effects by blocking translation and new protein synthesis rather than by decreasing pre-existing protein pools.

Biological functions of chromobox (CBX) proteins in stem cell self-renewal, lineage-commitment, cancer and development.

Epigenetic regulatory proteins support mammalian development, cancer, aging and tissue repair by controlling many cellular processes including stem cell self-renewal, lineage-commitment and senescence in both skeletal and non-skeletal tissues. We review here our knowledge of epigenetic regulatory protein complexes that support the formation of inaccessible heterochromatin and suppress expression of cell and tissue-type specific biomarkers during development. Maintenance and formation of heterochromatin critically depends on epigenetic regulators that recognize histone 3 lysine trimethylation at residues K9 and K27 (respectively, H3K9me3 and H3K27me3), which represent transcriptionally suppressive epigenetic marks. Three chromobox proteins (i.e., CBX1, CBX3 or CBX5) associated with the heterochromatin protein 1 (HP1) complex are methyl readers that interpret H3K9me3 marks which are mediated by H3K9 methyltransferases (i.e., SUV39H1 or SUV39H2). Other chromobox proteins (i.e., CBX2, CBX4, CBX6, CBX7 and CBX8) recognize H3K27me3, which is deposited by Polycomb Repressive Complex 2 (PRC2; a complex containing SUZ12, EED, RBAP46/48 and the methyl transferases EZH1 or EZH2). This second set of CBX proteins resides in PRC1, which has many subunits including other polycomb group factors (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, PCGF6), human polyhomeotic homologs (HPH1, HPH2, HPH3) and E3-ubiquitin ligases (RING1 or RING2). The latter enzymes catalyze the subsequent mono-ubiquitination of lysine 119 in H2A (H2AK119ub). We discuss biological, cellular and molecular functions of CBX proteins and their physiological and pathological activities in non-skeletal cells and tissues in anticipation of new discoveries on novel roles for CBX proteins in bone formation and skeletal development.

Upregulated CBX8 Promotes Cancer Metastasis via the WNK2/MMP2 Pathway.

Metastasis is associated with poor prognosis in cancer and is a multistep process that includes invasion and migration. Several epigenetic factors are involved in this process, including chromobox protein homolog 8 (CBX8). Here, we show that CBX8 is overexpressed in many cancers compared with normal tissues. Functional analyses indicated that CBX8 promoted invasion and migration in glioblastoma, breast cancer, and lung cancer in vitro and in vivo. WNK2 was identified as a target gene of CBX8, which interacted with the WNK2 promoter to suppress WNK2 expression and activity. WNK2 acted as an antioncogene, and decreased WNK2 levels resulted in high activity of matrix metalloprotease (MMP)-2 and RAC1, which play a central role in invasion and migration, respectively. There was a positive relationship between MMP2 and RAC1 activity in CBX8-modulated cell lines. In addition, WNK2 negatively regulated MMP2 and RAC1 activity. Collectively, the results indicated that CBX8 promoted invasion and migration by targeting WNK2, which resulted in increased RAC1 and MMP2 expression and activity. Therefore, CBX8 may be a novel therapeutic target to treat metastatic cancers.

m6A modification-mediated CBX8 induction regulates stemness and chemosensitivity of colon cancer via upregulation of LGR5.

BACKGROUND: Colon cancer (CC) cells can exhibit stemness and expansion capabilities, which contribute to resistance to conventional chemotherapies. Aberrant expression of CBX8 has been identified in many types of cancer, but the cause of this aberrant CBX8 expression and whether CBX8 is associated with stemness properties in CC remain unknown. METHODS: qRT-PCR and IHC were applied to examine CBX8 levels in normal and chemoresistant CC tissues. Cancer cell stemness and chemosensitivity were evaluated by spheroid formation, colony formation, Western blot and flow cytometry assays. RNA-seq combined with ChIP-seq was used to identify target genes, and ChIP, IP and dual luciferase reporter assays were applied to explore the underlying mechanisms. RESULTS: CBX8 was significantly overexpressed in chemoresistant CC tissues. In addition, CBX8 could promote stemness and suppress chemosensitivity through LGR5. Mechanistic studies revealed that CBX8 activate the transcription of LGR5 in a noncanonical manner with assistance of Pol II. CBX8 recruited KMT2b to the LGR5 promoter, which maintained H3K4me3 status to promote LGR5 expression. Moreover, m6A methylation participated in the upregulation of CBX8 by maintaining CBX8 mRNA stability. CONCLUSIONS: Upon m6A methylation-induced upregulation, CBX8 interacts with KMT2b and Pol II to promote LGR5 expression in a noncanonical manner, which contributes to increased cancer stemness and decreased chemosensitivity in CC. This study provides potential new therapeutic targets and valuable prognostic markers for CC.

Chromobox homolog 8 (CBX8) Interacts with Y-Box binding protein 1 (YBX1) to promote cellular proliferation in hepatocellular carcinoma cells.

Polycomb group (PcG) proteins have recently been identified as critical regulators in tumor initiation and development. However, the function of CBX8 in human hepatocellular carcinoma (HCC) remains largely unknown. Our study was designed to explore the biological function and clinical implication of CBX8 in HCC. We investigated the interplay between CBX8 and cell cycle through Gene Set Enrichment Analysis and western blotting. Bioinformatics tools and co-immunoprecipitation were used to explore cell cycle regulation. Finally, we studied the expression and clinical significance of CBX8 in HCC through 3 independent datasets. CBX8 was upregulated in HCC and its expression correlated with cell cycle progression. CyclinD1 was downregulated by CBX8 knockdown but upregulated by CBX8 overexpression. YBX1 interacted with CBX8 and regulated the cell cycle. Moreover, targeting YBX1 with specific siRNA impaired CBX8-mediated regulation of CyclinD1. CBX8 overexpression boosted HCC cell growth, while CBX8 knockdown suppressed cell proliferation. Further, YBX1 interacted with CBX8. YBX1 knockdown compromised the proliferation of CBX8 overexpressing cells. CBX8 promotes HCC cell proliferation through YBX1 mediated cell cycle progression and is related to poor HCC prognoses. Therefore, CBX8 may serve as a potential target for the diagnosis and treatment of HCC.

Increased Expression of Circular RNA circ_0005230 Indicates Dismal Prognosis in Breast Cancer and Regulates Cell Proliferation and Invasion via miR-618/ CBX8 Signal Pathway.

BACKGROUND/AIMS: Circular RNAs (circRNAs) are a class of non-coding RNAs. They have been proved to be critically involved in tumorigenesis and progression of malignancies through competing endogenous RNA (ceRNA) mechanism. Nevertheless, the exploration between circRNAs and pathogenesis of breast cancer (BC) is limited. Previously, circ_0005230 was identified upregulated in BC tissues screened by circRNA microarray. In the present study, we aimed to investigate the expression pattern, functional role, and mechanism of circ_0005230 in BC. METHODS: qRT-PCR was conducted to elucidate the expression levels of circ_0005230 in BC tissues and cells. Additionally, the clinical severity and prognostic value were investigated. CCK-8, colony-forming, flow cytometric assays were performed. Animal study was conducted to validate the in vitro data. What's more, Transwell assays were induced to detect the cell metastatic properties of circ_0005230 exerts in BC cells. Luciferase reporter assay was used to measure the mechanism of circ_0005230. RESULTS: circ_0005230 was overexpressed in BC tissue specimens and cell lines. The overexpression of circ_0005230 was related to adverse phenotypes in the patients with BC. In addition, circ_0005230 could be regarded as a prognostic predictor in BC patients. In vitro and in vivo data demonstrated the cell growth promoting role of circ_0005230. Moreover, circ_0005230 could also promote cell migratory and invasive capacities. For the mechanism investigation, circ_0005230 was proved to be a sponge of miR-618, and expression of miR-618 could regulate CBX8 expression via targeting the 3'UTR of CBX8. Rescue assays also illustrated an oncogenic function of circ_0005230 in BC via acting as a miR-618 sponge to promote CBX8 expression. CONCLUSION: circ_0005230/miR-618/CBX8 axis might play a key role in BC tumorigenesis and development.

CircRNA8924 Promotes Cervical Cancer Cell Proliferation, Migration and Invasion by Competitively Binding to MiR-518d-5p /519-5p Family and Modulating the Expression of CBX8.

BACKGROUND/AIMS: Circular RNAs (circRNAs) play a significant role in the development and progression of various human cancers. However, the expression and function of circRNAs in cervical cancer (CC) have rarely been explored. The aim of this study was to investigate the biological function of circRNA8924 in CC and elucidate the possible molecular mechanism involved. METHODS: Quantitative polymerase chain reaction was used to determine mRNA expression of circRNA8924, miR-518d-5p/519-5p and CBX8 in CC tissues and cells. CBX8 protein expression was measured by Western blotting. The CCK-8 assay was used to evaluate cell proliferation, and the transwell assay to determine cell migration and invasion. The luciferase reporter assay was used to determine the direct regulation of miR-518d-5p/519-5p and circRNA8924 or CBX8 Results: The study demonstrated that the expression level of circRNA8924 in CC was significantly higher than that in the adjacent normal tissues (P < 0.001), and that it was also associated with tumor size, FIGO staging and myometrial invasion. The knockdown of circRNA8924 significantly inhibited the proliferation, migration and invasion of CC cells SiHa and HeLa. The expression level of miR-518d-5p/519-5p was negatively correlated with circRNA8924, and circRNA8924 regulated CBX8 by competitively binding to miR-518d-5p/519-5p. CONCLUSIONS: CircRNA8924 is highly expressed in CC tissue and can be considered a competitive endogenous RNA of the miR-518d-5p/519-5p family to promote the malignant biological behavior of CC cells. It is suggested that it may serve as a new biomarker for CC diagnosis and disease progression and provide potential targets for targeted therapy.

PIM1-catalyzed CBX8 phosphorylation promotes the oncogene-induced senescence of human diploid fibroblast.

The proto-oncogene PIM1 encodes Ser/Thr kinase and regulates cell growth, differentiation and apoptosis. However, more and more studies including ours have found that PIM1 can induce senescence in normal human diploid fibroblasts and behave as a tumor suppressor. But the relevant molecular mechanism of this process is not yet clear. It has been reported that Chromobox homolog 8 (CBX8) binds directly to INK4A as a transcriptional repressor, thereby suppressing stress-induced senescence. Here we report that PIM1 can phosphorylate CBX8 to promote its degradation, thereby up-regulating p16, during PIM1-induced cell senescence. Overexpression of CBX8 can inhibit PIM1-induced cell senescence. These data suggest that to promote CBX8 degradation may be an important molecular mechanism of PIM1-induced cell senescence.

CBX8 Suppresses Tumor Metastasis via Repressing Snail in Esophageal Squamous Cell Carcinoma.

The poor clinical outcome and prognosis of esophageal squamous cell carcinoma (ESCC) is mainly attributed to its highly invasive and metastatic nature, making it urgent to further elicit the molecular mechanisms of the metastasis of ESCC. The function of each polycomb chromobox (CBX) protein in cancer is cell-type-dependent. Although CBX8 has been reported to promote the esophageal squamous cell carcinoma (ESCC) tumorigenesis, its role in ESCC metastasis has not been explored yet. In this study, we report that the inhibition of cell migration, invasion, and metastasis in ESCC requires CBX8-mediated repression of Snail, a key transcription factor that induces epithelial-to-mesenchymal transition (EMT), and that CBX8 inversely correlated with Snail in the ESCC tissues. Moreover, this novel function of CBX8 is dependent on its binding with the Snail promoter, which in turn suppresses the transcription of Snail. Collectively, CBX8 may play paradoxical roles in ESCC, inhibiting metastasis while promoting cell proliferation.