Integrated analyzes identify CCT3 as a modulator to shape immunosuppressive tumor microenvironment in lung adenocarcinoma.

BACKGROUND: Chaperonin-containing tailless complex polypeptide 1 (TCP1) subunit 3 (CCT3) has tumor-promoting effects in lung adenocarcinoma (LUAD). This study aims to investigate the molecular mechanisms of CCT3 in LUAD oncogenesis. METHODS: The UALCAN databases, Human Protein Atlas (HPA) and The Cancer Genome Atlas (TCGA) data were used to analyze CCT3 expression in LUAD. Both the Wilcoxon rank-sum test and the regression model were used to investigate the connection between clinicopathologic characteristics of LUAD patients and CCT3 expression. The prognostic value of CCT3 was determined by Cox regression models, the Kaplan-Meier method and Nomogram prediction. Next, we identified the most related genes with CCT3 via GeneMANIA and String databases, and the association between CCT3 and infiltrated immune cells using single-sample Gene Set Enrichment Analysis (ssGSEA). CCT3-related pathway enrichment analysis was investigated by GSEA. Finally, CCT3 roles in cell proliferation and apoptosis of LUAD A549 cells was verified by siRNA (small interfering RNA) mediated CCT3 knockdown. RESULTS: CCT3 was upregulated in LUAD both in mRNA and protein levels. CCT3 overexpression was associated with clinicopathological characteristics including sex, smoking, T- and N-categories, pathological staging, and a poor prognosis of LUAD patients. GeneMANIA and String databases found a set of CCT3-related genes that are connected to the assembly and stability of proteins involved in proteostasis of cytoskeletal filaments, DNA repair and protein methylation. Furthermore, CCT3 was found to be positively correlated with the infiltrating Th2 cells (r = 0.442, p < 0.01) while negatively correlated with mast cells (r = -0.49, p < 0.01) and immature dendritic cells (iDCs, r = -0.401, p < 0.001) according to ssGSEA analyzes. The pathway analysis based on GSEA method showed that the cell cycle pathway, the protein export pathway, the proteasome pathway and the ribosome pathway are enriched in CCT3 high group, whereas the JAK/STAT pathway, B cell receptor pathway, T cell receptor pathway and toll like receptor pathway were enriched in CCT3 low group. Finally, CCT3 knockdown substantially inhibited proliferation while promoted apoptosis of A549 cells. CONCLUSION: Integrated analyzes identify CCT3 as a modulator to shape immunosuppressive tumor microenvironment in LUAD and therefore, a prognostic factor for LUAD.

Circular RNA CCT3 is a unique molecular marker in bladder cancer.

This study surveyed circular RNA CCT3 in bladder cancer (BCa). We recruited 85 BCa patients and 40 normal controls (Normal) and collected clinical specimens for analysis. circRNA CCT3 expression was analyzed by RT-qPCR, diagnostic accuracy was calculated by ROC curves, and survival outcomes were evaluated by survival curves. CircRNA CCT3 was overexpressed or knocked down in cells, thereafter to observe the changes in cell malignant phenotypes. The downstream molecules of circRNA CCT3 were detected. Our data suggest that circRNA CCT3 was upregulated in human BCa and was associated with poor survival outcomes of BCa patients. In cell experiments, overexpressing circRNA CCT3 promoted BCa cell malignancy, whereas silencing circRNA CCT3 did the opposite. In addition, circRNA CCT3 modulated PP2A expression by miR-135a-5p. This study demonstrates that circRNA CCT3 is a diagnostic and prognostic biomarker in BCa patients and is a tumor promoter in BCa.

Loss of PMFBP1 Disturbs Mouse Spermatogenesis by Downregulating HDAC3 Expression.

PURPOSE: Polyamine modulating factor 1 binding protein (PMFBP1) acts as a scaffold protein for the maintenance of sperm structure. The aim of this study was further to identify the new role and molecular mechanism of PMFBP1 during mouse spermatogenesis. METHODS AND RESULTS: We identified a profile of proteins interacting with PMFBP1 by immunoprecipitation combined with mass spectrometry and demonstrated that class I histone deacetylases, particularly HDAC3 and chaperonin-containing TCP1 subunit 3 (CCT3), were potential interaction partners of PMFBP1 based on network analysis of protein-protein interactions and co-immunoprecipitation. Immunoblotting and immunochemistry assays showed that loss of Pmfbp1 would result in a decline in HDACs and change the proteomic profile of mouse testis, in which differently expressed proteins are associated with spermatogenesis and assembly of flagella, which was proved by proteomic analysis of testis tissue obtained from Pmfbp1-/- mice. After integrating with transcriptome data for Hdac3-/- and Sox30-/- round sperm obtained from a public database, RT-qPCR confirmed ring finger protein 151 (Rnf151) and ring finger protein 133 (Rnf133) were key downstream response factors of the Pmfbp1-Hdac axis affecting mouse spermatogenesis. CONCLUSION: Taken together, this study indicates a previously unidentified molecular mechanism of PMFBP1 in spermatogenesis whereby PMFBP1 interacts with CCT3, affecting the expression of HDAC3, followed by the downregulation of RNF151 and RNF133, resulting in an abnormal phenotype of sperm beyond the headless sperm tails. These findings not only advance our understanding of the function of Pmfbp1 in mouse spermatogenesis but also provide a typical case for multi-omics analysis used in the functional annotation of specific genes.

Restraint of chaperonin containing T-complex protein-1 subunit 3 has antitumor roles in non-small cell lung cancer via affection of YAP1.

The implication of chaperonin containing T-complex protein-1 subunit 3 (CCT3) in carcinogenesis has been observed in a diverse malignancies. However, the relevance of CCT3 in non-small cell lung cancer (NSCLC) has not been well addressed. This research is dedicated to investigating the expression pattern and functional role of CCT3 in NSCLC. An elevation in CCT3 levels was observed in NSCLC tissue, which was linked to a reduced overall survival rate. The inhibition of CCT3 by shRNA-mediated gene silencing induced suppressive effects on the transformative phenotypes of NSCLC cells, including the inhibition of cell proliferation and invasion, and the induction of cell cycle arrest and apoptosis. Further investigation revealed that the silencing of CCT3 led to the suppression of Yes-associated protein 1 (YAP1), and decreased the expression of YAP1 target genes in NSCLC cells. The activation of YAP1 via forced expression of constitutively active YAP1 mutant reversed CCT3-restraint-evoked antitumor effects in NSCLC cells. Crucially, NSCLC cells with CCT3 silencing also exhibited weakened oncogenicity in nude mice associated with the down-regulation of YAP1 activation in xenografts. To sum up, these observations of our work show that the inhibition of CCT3 produces antitumor effects in NSCLC via the suppression of YAP1. This study unveils a possible role CCT3/YAP1 axis in NSCLC and suggests CCT3 as a candidate anticancer target.

Suppression of CCT3 Inhibits Tumor Progression by Impairing ATP Production and Cytoplasmic Translation in Lung Adenocarcinoma.

Heat shock proteins are highly expressed in various cancers and exert critical functions in tumor progression. However, their expression patterns and functions in lung adenocarcinoma (LUAD) remain largely unknown. We identified that chaperonin-containing T-complex protein-1 subunit 3 (CCT3) was highly expressed in LUAD cells and was positively correlated with LUAD malignancy in the clinical samples. Animal studies showed that silencing CCT3 dramatically inhibited tumor growth and metastasis of LUAD. Proliferation and migration were markedly suppressed in CCT3-deficient LUAD cells. Moreover, the knockdown of CCT3 promoted apoptosis and cell cycle arrest. Mechanistically, the function of glycolysis was significantly inhibited and the total intracellular ATP levels were reduced by at least 25% in CCT3-deficient cells. In addition, the knockdown of CCT3 decreased the protein translation and led to a significant reduction in eukaryotic translation initiation factor 3 (EIF3G) protein, which was identified as a protein that interacts with CCT3. Impaired protein synthesis and cell growth in EIF3G-deficient cells were consistent with those caused by CCT3 knockdown in LUAD cells. Taken together, our study demonstrated in multiple ways that CCT3 is a critical factor for supporting growth and metastasis of LUAD, and for the first time, its roles in maintaining intracellular ATP levels and cytoplasmic translation are reported. Our novel findings provide a potential therapeutic target for lung adenocarcinoma.

Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila.

Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. We report that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila.

Suppression of CCT3 inhibits the proliferation and migration in breast cancer cells.

BACKGROUND: CCT3 is a subunit of chaperonin-containing TCP-1 (CCT), which folds many proteins involved in cancer development and plays an important role in many cancers. However, the role of CCT3 in breast cancer is still unclear. METHODS: CCT3 expression was knocked down by transfecting breast cancer cells with lentiviral shRNA. The proliferation of breast cancer cells (HCC1937 and MDA-MB-231) was detected by Celigo image cytometry and MTT assay, the migration of the cells was measured by Transwell analysis, cell cycle distribution and apoptosis was detected by flow cytometry, and changes in signal transduction proteins were detected by western blot analysis. RESULTS: The expression of CCT3 was significantly suppressed by transduction with lentiviral shRNA; CCT3 knockdown significantly reduced the proliferation and metastasis ability of breast cancer cells (HCC 1937 and MDA-MB-231), increased the proportion of cells in S phase, and decreased the proportion of cells in G1 phase compared to those in shControl cells. There was no significant change in the number of cells in the G2/M phase. Apoptosis analysis showed that knockdown of CCT3 induced apoptosis in breast cancer cells. Western blot analysis showed that the expression of many signal transduction proteins was changed after suppression of CCT3. A rescue experiment showed that overexpression of NFκB-p65 rescued the cell proliferation and migration affected by CCT3 in breast cancer cells. CONCLUSION: CCT3 is closely related to the proliferation and migration of breast cancer and may be a novel therapeutic target.

Repurposing cyclovirobuxine D as a novel inhibitor of colorectal cancer progression via modulating the CCT3/YAP axis.

BACKGROUND AND PURPOSE: Colorectal cancer (CRC) ranks second in mortality worldwide and requires effective and affordable remedies. Cyclovirobuxine D (CVB-D) is the main effective component of Huangyangning tablet, an approved traditional patent medicine, which is mainly used for cardiovascular treatment. As a multibioactive natural compound, CVB-D possesses underlying anticancer activities. EXPERIMENTAL APPROACH: Cell viability and clone-forming ability were determined in human CRC lines. Western blot, immunofluorescence assay, transmission electron microscopy and senescence-associated ß-galactosidase (SA-ß-Gal) staining were utilized to investigate cell autophagy and senescence. The molecular mechanisms were explored by virtual prediction and experimental validation. Patient-derived xenograft (PDX), dextran sulfate sodium salt (DSS), and azomethane (AOM)/DSS mouse models were employed for in vivo studies. KEY RESULTS: CVB-D inhibited the growth and development of advanced CRC cells / mice by inducing autophagic and senescent activities through the chaperonin containing TCP1 subunit 3 (CCT3)/yes-associated protein (YAP) axis. CVB-D acted as a promising inhibitor of CCT3 by interacting with its ATP site. In PDX tumours, CVB-D showed potential therapeutic effects by targeting CCT3. Treatment with CVB-D alleviated the mouse model of colitis induced by DSS and attenuated AOM/DSS-induced formation of adenomatous polyps by its action on CCT3. CONCLUSIONS AND IMPLICATIONS: Our study has provided a scientific basis for the suggestion that CVB-D may be recognized as a prospective drug candidate for the therapy of CRC in patients.

m6A-modification regulated circ-CCT3 acts as the sponge of miR-378a-3p to promote hepatocellular carcinoma progression.

Background: Circular RNA (circRNA) plays a critical role in tumour progression. Circ-CCT3, a particularly abundant circRNA, was proposed to be involved in tumorigenesis. However, the role of circ-CCT3 in hepatocellular carcinoma remains elusive.Methods: Here, circ-CCT3 (a circRNA derived from exons 3, 4 and 5 of the CCT3 gene, hsa_circ_0004680) was identified by circRNA microarray and validated by qRT-PCR. RNA immunoprecipitation (RIP) was performed to confirm the binding between ALKBH5 along with METTL3 and circ-CCT3. Methylated RNA Immunoprecipitation (MeRIP) was used to detect the N6-methyladenosine (m 2A) levels of circ-CCT3. CircRNAs in vivo precipitation, luciferase reporter assay, biotin-coupled microRNA capture, and fluorescence in situ hybridization were conducted to assess the interaction between circ-CCT3 and miR-378a-3p. The functions of circ-CCT3 in HCC were evaluated both in vitro and in vivo.Results: We demonstrated that circ-CCT3 was highly expressed in HCC which indicated the poor prognosis. Circ-CCT3 expression served as an independent risk factor for overall survival in patients with HCC. Knocking-down of circ-CCT3 inhibited the proliferation, invasion and migration of HCC cells, and angiogenesis of HUVEC. Mechanistically, ALKBH5 and METTL3 could bind and regulate m A-modification of circ-CCT3. Further, circ-CCT3 upregulated the expression of FLT-1 by sponging miR-378a-3p.Conclusions: Circ-CCT3 was significantly up-regulated in HCC and promoted liver cancer development via miR-378a-3p-FLT1 axis. It was also found that circ-CCT3 was under m A-modification mediated by ALKBH5 and METTL3. Our study highlights circ-CCT3 as a potential therapeutic target of HCC treatment, which provides a novel understanding on mechanisms of circRNAs in HCC progression.

Current understanding on the role of CCT3 in cancer research.

Chaperonin containing TCP1 Subunit 3 (CCT3) is an important member of the chaperone protein family, providing a favorable environment for the correct folding of proteins in cell division, proliferation, and apoptosis pathways, which is involved in a variety of biological processes as well as the development and invasion of many malignant tumors. Many malignancies have been extensively examined with CCT3. It is presently used as a possible target for the treatment of many malignancies since it is not only a novel biomarker for the screening and diagnosis of different tumors, but it is also closely associated with tumor progression, prognosis, and survival. Recent studies have shown that the expression of CCT3 is up-regulated in some tumors, such as liver cancer, breast cancer, colon cancer, acute myeloid leukemia, etc. In this paper, we review the role of CCT3 in various tumors.

Insights into the roles and driving forces of CCT3 in human tumors.

CCT3 played a key role in many cancers. This study aimed to further explore the characteristics of CCT3 from a pan-cancer perspective and reveal the driving forces for CCT3. By bioinformatic analysis, we found that the mRNA and protein levels of CCT3 were abnormally elevated in most tumor types and were correlated with poor prognosis. Single-cell sequencing data indicated an abnormal increase of CCT3 expression in both malignant cells and multiple immune cells. In the tumor microenvironment, CCT3 expression was negatively relevant with immune cell infiltration and immune checkpoint genes expression. In colon cancer, knockdown of CCT3 inhibited cell proliferation. Gene set enrichment analysis showed that CCT3 may be oncogenic by regulating amino acid metabolism. Furthermore, we predicted sensitive drugs for CCT3 by virtual screening and sensitivity analysis. Many driver genes such as TP53 and KRAS were essential for CCT3 overexpression. Epigenetic factors, enhancers in particular, were also critical for CCT3 expression. Additionally, we constructed the lncRNA/circRNA-miRNA-CCT3 regulatory network. Collectively, CCT3 had the potential to be a diagnostic and prognostic biomarker for multiple tumor types. CCT3 expression was relevant with an immunosuppressive tumor microenvironment. CCT3 could be a new molecular target for colon cancer. Both genetic and epigenetic factors were responsible for CCT3 expression in tumors.

Upregulation of CCT3 predicts poor prognosis and promotes cell proliferation via inhibition of ferroptosis and activation of AKT signaling in lung adenocarcinoma.

BACKGROUND: Chaperonin containing TCP1 subunit 3 (CCT3) acts as an oncogene in cancers, whereas its role and underlying mechanisms in lung adenocarcinoma (LUAD) are poorly understood. This study investigated the clinical relevance and function of CCT3 in LUAD. METHODS: Clinical relevance of CCT3 in LUAD and lung squamous cell carcinoma (LUSC) was analyzed based on TCGA database. qRT-PCR and Western blot were used to detect mRNA and protein expression, respectively. CCK8 and colony formation were performed to measure cell viability. PI and PI/Annexin V-FITC assay kit was used to determine cell cycle and cell death, respectively. Luciferase activity was performed to check whether CCT3 regulated slc7a11's transcription activity. Ferroptosis was determined by incubating the cells with ferroptosis and apoptosis inducer, their inhibitor and autophagy inhibitor, followed by cell viability examination. RESULTS: We found that CCT3 was overexpressed in LUAD and LUSC tissues. Overexpression of CCT3 predicted the poor prognosis of LUAD patients. Loss-of-function and gain-of-function experiments demonstrated that CCT3 promoted the proliferation and colony formation of LUAD cells. In addition, CCT3 promoted cell cycle progression and suppressed slc7a11-mediated cell ferroptosis, but not apoptosis. We also found that CCT3 activated AKT. MK2206 significantly reduced the viability of CCT3 overexpressed LUAD cells, while had smaller inhibitory effect on the proliferation of control cells, suggesting that CCT3 dictates the sensitivity of LUAD cells to AKT inhibition. CONCLUSION: Our study demonstrates that CCT3 contributes to the proliferation and growth of LUAD cells through inhibition of ferroptosis and activation of AKT.

Corrigendum: Upregulation of CCT-3 Induces Breast Cancer Cell Proliferation Through miR-223 Competition and Wnt/b-Catenin Signaling Pathway Activation.

[This corrects the article DOI: 10.3389/fonc.2020.533176.].

Identification of CCT3 as a prognostic factor and correlates with cell survival and invasion of head and neck squamous cell carcinoma.

BACKGROUND: Recurrent locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC) is associated with dismal prognosis because of its highly invasive behavior and resistance to conventional intensive chemotherapy. The identification of effective markers for early diagnosis and prognosis is important for reducing mortality and ensuring that therapy for HNSCC is effective. Chaperonin-containing TCP-1 3 (CCT3) folds cancer-related proteins to control carcinogenesis. The prognostic value and growth association of CCT3 and HNSCC remain unknown. METHODS: The GEO, Oncomine and UALCAN databases were used to examine CCT3 expression in HNSCC. A few clinical HNSCC samples with normal tissues were used to detect CCT3 expression by using immunohistochemistry method. The TCGA-HNSC dataset was used to evaluate the association between expression of CCT3 and prognosis. The molecular mechanism was investigated with gene set enrichment analysis (GSEA). CCK-8 and wound healing assays were used to detect cell growth and invasion of HNSCC, respectively. RESULTS: CCT3 expression was significantly up-regulated in HNSCC in both mRNA and protein levels. In addition, up-regulated CCT3 expression was associated with various clinicopathological parameters. High expression of CCT3 was significantly correlated with inferior survival of HNSCC patients. Knockdown of CCT3 significantly inhibited cell growth and invasion of HNSCC cell lines. GSEA analysis indicated that CCT3 was closely correlated with tumor-related signaling pathways and HNSCC cell survival. CONCLUSION: Our findings suggest that CCT3 is a biomarker of poor prognosis and related to the process of HNSCC.

Upregulation of CCT3 promotes cervical cancer progression through FN1.

The mechanisms underlying cervical cancer progression have not yet been fully elucidated; thus, further investigations are required. Chaperonin containing TCP1 subunit 3 (CCT3) expression was found to be upregulated in several types of human cancer. However, the roles of CCT3 in cervical cancer remain poorly understood. Thus, the present study aimed to determine the roles of CCT3 in the progression of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). For this purpose, the Tumor Immune Estimation Resource and Gene Expression Profiling Interactive Analysis databases were used to analyze the mRNA and protein expression levels of CCT3 in CESC samples. The effects of CCT3 on the proliferation and migration of CESC in vitro were determined using various experiments, including proliferation, Transwell and flow cytometric assays. The results revealed that CCT3 expression was significantly upregulated in CESC, which was associated with a poor prognosis. The silencing of CCT3 suppressed CESC cell proliferation, migration and invasiveness in vitro. Additionally, CCT3­knockdown promoted CESC cell apoptosis and cell cycle arrest, and suppressed fibronectin 1 (FN1) protein expression. Furthermore, rescue assays demonstrated that CCT3 promoted CESC proliferation and migration via FN1. In conclusion, the findings of the present study demonstrated that CCT3 is closely associated with the progression of CESC. Thus, CCT3 may be considered a novel, promising biomarker, and a possible therapeutic target for CESC.

Chaperonin containing TCP1 subunit 3 (CCT3) promotes cisplatin resistance of lung adenocarcinoma cells through targeting the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway.

Cisplatin resistance remains a major obstacle to effective chemotherapies for non-small cell lung cancer (NSCLC). Chaperonin containing TCP1 subunit 3 (CCT3) has been extensively investigated in various cancers, but not in the context of drug resistance. In the present study, we aimed to investigate the role of CCT3 in cisplatin resistance of lung adenocarcinoma (LUAD) cells. By surveying the Gene Expression Profiling Interactive Analysis (GEPIA) website, we found CCT3 expression to be up-regulated in NSCLCs, which correlated with the poor prognosis of LUAD patients. Furthermore, both mRNA and protein levels of CCT3 were upregulated in the cisplatin-resistant A549/DDP cells compared to the cisplatin-sensitive A549 cells. Importantly, upon cisplatin treatment, short hairpin RNA (shRNA)-mediated CCT3 knockdown significantly inhibited the proliferation, invasion and migration of A549/DDP cells, and induced significant G2/M cell cycle arrest and apoptosis in A549/DDP cells. Moreover, CCT3 knockdown significantly weakened the tumorigenicity of the cisplatin-treated A549/DDP cells in vitro and in vivo. Finally, CCT3 knockdown re-sensitized A549/DDP cells to cisplatin through inhibiting the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway. In conclusion, our results demonstrated that CCT3 could promote cisplatin resistance of LUAD cells via activating the JAK2/STAT3 pathway, indicating that CCT3 may be a novel molecular target for overcoming cisplatin resistance in LUAD patients.

CCT3 suppression prompts apoptotic machinery through oxidative stress and energy deprivation in breast and prostate cancers.

Mediated by chaperon proteins, protein misfolding plays a crucial role in cancer pathogenesis. Chaperonin Containing TCP1 Subunit 3 (CCT3) is one of eight subunits forming eukaryotic chaperons that catalyzes correct folding of the proteins employed in cell division, proliferation, and apoptosis pathway. Moreover, CCT3 expression increases responsively with carcinogenesis. However, how CCT3 drives the cancerous process has not been documented. Here we probed the mechanistic and functional interactions between CCT3 and apoptotic pathways and cell stressors. First, we profiled CCT3 expression levels of different 16 cell lines and found that CCT3 expression levels of CRL-2329 and PC3 were significantly increased. Then, we suppressed CCT3 levels in CRL-2329 and PC3 lines by miR-24-3p, miR-128-3p, and miR-149-5p mimics, and measured apoptotic response of the cell lines to the knockdown of CCT3 by acridine orange/ethidium bromide and Annexin V/PI staining, cell-cycle and mitochondria membrane potential (MMP) analyses, intracellular reactive oxygen species (ROS) measurement and analysis of expression levels of the apoptotic genes. After having suppressed CCT3, the cell cycle was arrested in the G0/G1 phase, MMP was impaired, and the intracellular ROS level was increased. These signs of apoptotic flux were corroborated by morphological images, statistically enhanced expression levels of the apoptotic pathway modulators and intracellular free amino acids profile. The free amino acid profile, which is heavily implicated in energy metabolism and cell division, is fluctuated in the progress of canceration. Strikingly, suppressed CCT3 shifted intracellular levels of glutamine, beta-alanine, glycine, serin, asparagine and sarcosine, which are employed in energy metabolism. Consequently, miRNA-mediated CCT3 suppression spur apoptosis by unbalancing the homeostasis in intracellular ROS and the profile of free amino acids in energy metabolism. Taken together, we anticipate that miRNA-mediated CCT3 suppression might provide a "dual therapeutic strategy" through conventional cellular toxicity as well as energy withdrawal.

Circular RNA circ­CCT3 promotes hepatocellular carcinoma progression by regulating the miR­1287­5p/TEAD1/PTCH1/LOX axis.

Hepatocellular carcinoma (HCC) is characterized by a poor prognosis because of its insensitivity to radiation and chemotherapy. Recently, circular RNAs (circRNAs) have been found to serve important roles in hepatocellular carcinogenesis. circ­CCT3, a novel circRNA, was screened from the differential tissue expression results of a circRNA microarray. Relative expression levels of circ­CCT3 in specimens and cell lines were evaluated by reverse transcription­quantitative PCR and the relationship between circ­CCT3 and prognosis was analyzed by Kaplan­Meier curves. The oncogenic role of circ­CCT3 was confirmed in HCC cells through a cell counting kit­8 (CCK­8) assay, a colony formation assay, acridine orange/ethidium bromide double fluorescence staining, flow cytometry, a wound­healing assay and a Transwell assay. Bioinformatics prediction and luciferase reporter assays validated that circ­CCT3 facilitated HCC progression through the miR­1287­5p/TEA domain transcription factor 1 (TEAD1) axis. TEAD1 could then directly activate patched 1 and lysyl oxidase transcription, as analyzed by chromatin immunoprecipitation and luciferase reporter assays. The present study identified a novel circRNA, circ­CCT3, which may be used as a potential therapeutic target for HCC.

Understanding the Molecular Mechanism of miR-877-3p Could Provide Potential Biomarkers and Therapeutic Targets in Squamous Cell Carcinoma of the Cervix.

No therapeutic targets and molecular biomarkers are available in cervical cancer (CC) management. In other cancer types, micro-RNA-877-3p (miR-877-3p) has been associated with events relevant for CC development. Thus, we aimed to determine miR-877-3p role in CC. miR-877-3p levels were examined by quantitative-PCR in 117 cervical lesions and tumors. Effects on CC cell proliferation, migration, and invasion were evaluated upon anti-miR-877-3p transfection. miR-877-3p dependent molecular mechanism was comprehensively explored by proteomics, dual-luciferase reporter assay, western blot, and immunohistochemistry. Cervical tumors expressed higher miR-877-3p levels than benign lesions. miR-877-3p promoted CC cell migration and invasion, at least partly by modulating cytoskeletal protein folding through the chaperonin-containing T-complex protein 1 complex. Notably, miR-877-3p silencing synergized with paclitaxel. Interestingly, miR-877-3p downregulated the levels of an in silico-predicted target, ZNF177, whose expression and subcellular location significantly distinguished high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix (SCCCs). Cytoplasmic ZNF177 was significantly associated with worse progression-free survival in SCCC. Our results suggest that: (i) miR-877-3p is a potential therapeutic target whose inhibition improves paclitaxel effects; (ii) the expression and location of its target ZNF177 could be diagnostic biomarkers between HSIL and SCCC; and (iii) cytoplasmic ZNF177 is a poor-prognosis biomarker in SCCC.

Upregulation of CCT-3 Induces Breast Cancer Cell Proliferation Through miR-223 Competition and Wnt/ß-Catenin Signaling Pathway Activation.

The clinical significance and the function of chaperonin-containing TCP1 complex 3 (CCT-3) in breast cancer remain unknown. In this study, we found that CCT-3 was markedly overexpressed in breast cancer tissues. Statistical analysis revealed a significant correlation of CCT-3 expression with advanced breast cancer clinical stage and poorer survival. Ablation of CCT-3 knocked down the proliferation and the tumorigenicity of breast cancer cells in vitro and in vivo. CCT-3 may regulate breast cancer cell proliferation through a ceRNA network between miR-223 and ß-catenin, thus affecting Wnt/ß-catenin signaling pathway activation. We also validated that CCT-3 and ß-catenin are novel direct targets of tumor suppressor miR-223. Our results suggest that both mRNA and the protein levels of CCT-3 are potential diagnosis biomarkers and therapeutic targets for breast cancer.