Promoted Colorectal Cancer Cell Proliferation Research Articles

Inorganic Polyphosphate Promotes Colorectal Cancer Growth via TRPM8 Receptor Signaling Pathway

Background: Colorectal cancer (CRC) is characterized by a pro-inflammatory microenvironment and features high-energy-supply molecules that assure tumor growth. A still less studied macromolecule is inorganic polyphosphate (iPolyP), a high-energy linear polymer that is ubiquitous in all forms of life. Made up of hundreds of repeated orthophosphate units, iPolyP is essential for a wide variety of functions in mammalian cells, including the regulation of proliferative signaling pathways. Some evidence has suggested its involvement in carcinogenesis, although more studies need to be pursued. Moreover, iPolyP regulates several homeostatic processes in animals, spanning from energy metabolism to blood coagulation and tissue regeneration. Results: In this study, we tested the role of iPolyP on CRC proliferation, using in vitro and ex vivo approaches, in order to evaluate its effect on tumor growth. We found that iPolyP is significantly increased in tumor tissues, derived from affected individuals enrolled in this study, compared to the corresponding peritumoral counterparts. In addition, iPolyP signaling occurs through the TRPM8 receptor, a well-characterized Na+ and Ca2+ ion channel often overexpressed in CRC and linked with poor prognosis, thus promoting CRC cell proliferation. The pharmacological inhibition of TRPM8 or RNA interference experiments performed in established CRC cell lines, such as Caco-2 and SW620, showed that the involvement of TRPM8 is essential, greater than that of the other two known iPolyP receptors, P2Y1 and RAGE. The presence of iPolyP drives cancer cells towards the mitotic phase of the cell cycle by enhancing the expression of ccnb1, which encodes the Cyclin B protein. In vitro 2D and 3D data reflected the ex vivo results, obtained by the generation of CRC-derived organoids, which increased in size. Conclusions: These results indicate that iPolyP may be considered a novel and unexpected early biomarker supporting colorectal cancer cell proliferation.

CircSEC24B activates autophagy and induces chemoresistance of colorectal cancer via OTUB1-mediated deubiquitination of SRPX2

Circular RNAs (circRNAs) are a type of regulatory RNA that feature covalently closed single-stranded loops. Evidence suggested that circRNAs play important roles in the progression and development of various cancers. However, the impact of circRNA on autophagy-mediated progression of colorectal cancer (CRC) remains unclear. The objective of this project was to investigate the influence of circSEC24B on autophagy and its underlying mechanisms in CRC. To validate the presence and circular structure of circSEC24B in CRC cells and tissues, PCR and Sanger sequencing techniques were employed. Drug resistance and invasive phenotype of CRC cells were evaluated using CCK8, transwell, and Edu assays. Gain- and loss-of-function experiments were conducted to assess the effects of circSEC24B and its protein partner on the growth, invasion, and metastasis of CRC cells in vitro and in vivo. Interactions between circSEC24B, OTUB1, and SRPX2 were analyzed through immunofluorescence, RNA-pulldown, and RIP assays. Mass spectrometry analysis was used to identify potential binding proteins of circRNA in CRC cells. Vectors were constructed to investigate the specific structural domain of the deubiquitinating enzyme OTUB1 that binds to circSEC24B. Results showed that circSEC24B expression was increased in CRC tissues and cell lines, and it enhanced CRC cell proliferation and autophagy levels. Mechanistically, circSEC24B promoted CRC cell proliferation by regulating the protein stability of SRPX2. Specifically, circSEC24B acted as a scaffold, facilitating the binding of OTUB1 to SRPX2 and thereby enhancing its protein stability. Additionally, evidence suggested that OTUB1 regulated SRPX2 expression through an acetylation-dependent mechanism. In conclusion, this study demonstrated that circSEC24B activated autophagy and induced chemoresistance in CRC by promoting the deubiquitination of SRPX2, mediated by the deubiquitinating enzyme OTUB1.

Exploring the Effect of Fidgetin-Like 1 on Colorectal Cancer Through Tissue Chip and In Vitro Experiments

Fidgetin-like 1 (FIGNL1) is extensively overexpressed in a variety of cancers. It facilitates non‑small cell lung cancer tumor cell proliferation and hepatocellular carcinoma formation due to abnormal DNA repair. Clinically relevant data indicates that its high expression is linked with the poor prognosis of patients with renal clear-cell carcinoma, low-grade gliomas, and hepatocellular carcinoma. Nevertheless, the scope of FIGNL1’s involvement in cancer, particularly colorectal cancer (CRC), remains unclear. To investigate the function of FIGNL1 in CRC. Cell culture study. The TCGA database and immunohistochemistry analysis were employed to investigate FIGNL1 expression in CRC tissue. A cell viability assay was performed using the Cell Counting Kit-8. The cell migration and invasion were evaluated using the transwell assay. Small interfering RNA (siRNA) transfection was conducted to knockdown FIGNL1 expression. Infection with FIGNL1 overexpression lentivirus was performed to promote FIGNL1 overexpression. The STRING database was employed for predicting protein interaction. FIGNL1 was substantially upregulated in human CRC tissues and was associated with TNM stages and lymph node metastasis in patients. The inhibition of CRC cell proliferation, migration, and invasion in Caco-2 cells was achieved by silencing FIGNL1 using siRNA. Additional investigations suggested that FIGNL1 overexpression could promote CRC cell proliferation, migration, and invasion via P38 signaling pathway activation in Colo-205 cells. Subsequent experiments demonstrated that FIGNL1-mediated P38 phosphorylation was contingent upon SPIDR interaction. These results implied that FIGNL1 was a potential anticancer drug target, which also offered a novel strategy for future CRC treatment.

CLK3 promotes tumor proliferation by activating MYC signaling

Colorectal cancer (CRC) ranks among the leading causes of cancer-related mortality worldwide, posing a significant public health challenge. Despite advancements in treatment strategies, prognosis for advanced CRC remains poor. Here, we investigate the role of CLK3 and its interaction with the c-Myc signaling pathway in CRC progression. Our study reveals significant overexpression of CLK3 in CRC tumor tissues, correlating with disease advancement, and demonstrates that CLK3 promotes CRC cell proliferation, mediated by its activation of MYC signaling through upregulation of c-MYC expression. In vivo experiments confirm the oncogenic role of CLK3, with its loss resulting in decreased tumor growth and c-MYC expression. These findings highlight CLK3 as a potential therapeutic target in CRC, offering insights into novel treatment strategies.

METTL3 in cancer-associated fibroblasts-derived exosomes promotes the proliferation and metastasis and suppresses ferroptosis in colorectal cancer by eliciting ACSL3 m6A modification

BackgroundCancer-associated fibroblasts (CAFs) have been reported that can affect cancer cell proliferation, metastasis, ferroptosis, and immune escape. METTL3-mediated N6-methyladenine (m6A) modification is involved in the tumorigenesis of colorectal cancer (CRC). Herein, we investigated whether METTL3-dependent m6A in CAFs-derived exosomes (exo) affected CRC progression.MethodsqRT-PCR and western blotting analyses detected levels of mRNAs and proteins. Cell proliferation and metastasis were evaluated using MTT, colony formation, transwell, and wound healing assays, respectively. Cell ferroptosis was assessed by detecting cell viability and the levels of Fe+, reactive oxygen species, and glutathione after erastin treatment. Exosomes were isolated from CAFs by ultracentrifugation. The m6A modification profile was determined by methylated RNA immunoprecipitation assay and the interaction between METTL3 and ACSL3 (acyl-CoA synthetase 3) was verified using dual-luciferase reporter assay. Animal models were established for in vivo analysis.ResultsCAFs promoted CRC cell proliferation and metastasis, and suppressed cell ferroptosis. METTL3 was enriched in CAFs and was packaged into exosomes. The m6A modification and METTL3 expression were increased in CRC samples. Knockdown of METTL3 in CAFs-exo suppressed CRC cell proliferation and metastasis, and induced cell ferroptosis. Mechanistically, METTL3 induced ACSL3 m6A modification and stabilized its expression. The anticancer effects mediated by METTL3-silenced CAFs-exo could be rescued by ACSL3 overexpression. Moreover, in vivo assay also showed that CAFs-exo with decreased METTL3 could hinder CRC growth and metastasis in mice models.ConclusionCAFs promoted the proliferation and metastasis, and restrained the ferroptosis in CRC by exosomal METTL3-elicited ACSL3 m6A modification.

The ubiquitin-like protein UBTD1 promotes colorectal cancer progression by stabilizing c-Myc to upregulate glycolysis

Dysfunction of the ubiquitin-proteasome system (UPS) is involved in the pathogenesis of various malignancies including colorectal cancer (CRC). Ubiquitin domain containing 1 (UBTD1), a ubiquitin-like protein, regulates UPS-mediated protein degradation and tumor progression in some cancer types. However, the biological function and mechanism of UBTD1 are far from being well elucidated, and its role in CRC has not been explored yet. In our study, we analyzed CRC patients’ clinical information and UBTD1 expression data, and found that the expression of UBTD1 in cancer tissue was significantly higher than that in adjacent normal tissue. Higher UBTD1 expression was significantly associated with poorer survival and more lymph node metastasis. Overexpression of UBTD1 could facilitate, while knockdown could inhibit CRC cell proliferation and migration, respectively. RNA-seq and proteomics indicated that c-Myc is an important downstream target of UBTD1. Metabolomics showed the products of the glycolysis pathway were significantly increased in UBTD1 overexpression cells. In vitro, we verified UBTD1 upregulating c-Myc protein and promoting CRC cell proliferation and migration via regulating c-Myc. UBTD1 promoted CRC cells’ glycolysis, evidenced by the increased lactate production and glucose uptake following UBTD1 overexpression. Mechanistically, UBTD1 prolonged the half-life of the c-Myc protein by binding to E3 ligase β-transducin repeat-containing protein (β-TrCP), thereby upregulated the expression of glycolysis rate-limiting enzyme hexokinase II (HK2), and enhanced glycolysis and promoted CRC progression. In conclusion, our study revealed that UBTD1 promotes CRC progression by upregulating glycolysis via the β-TrCP/c-Myc/HK2 pathway, suggesting its potential as a prognostic biomarker and therapeutic target in CRC.

AURKB promotes colorectal cancer progression by triggering the phosphorylation of histone H3 at serine 10 to activate CCNE1 expression.

Aurora kinase B (AURKB) initiates the phosphorylation of serine 10 on histone H3 (pH3S10), a crucial process for chromosome condensation and cytokinesis in mammalian mitosis. Nonetheless, the precise mechanisms through which AURKB regulates the cell cycle and contributes to tumorigenesis as an oncogenic factor in colorectal cancer (CRC) remain unclear. Here, we report that AURKB was highly expressed and positively correlated with Ki-67 expression in CRC. The abundant expression of AURKB promotes the growth of CRC cells and xenograft tumors in animal model. AURKB knockdown substantially suppressed CRC proliferation and triggered cell cycle arrest in G2/M phase. Interestingly, cyclin E1 (CCNE1) was discovered as a direct downstream target of AURKB and functioned synergistically with AURKB to promote CRC cell proliferation. Mechanically, AURKB activated CCNE1 expression by triggering pH3S10 in the promoter region of CCNE1. Furthermore, it was showed that the inhibitor specific for AURKB (AZD1152) can suppress CCNE1 expression in CRC cells and inhibit tumor cell growth. To conclude, this research demonstrates that AURKB accelerated the tumorigenesis of CRC through its potential to epigenetically activate CCNE1 expression, suggesting AURKB as a promising therapeutic target in CRC.

Establishment of a novel microfluidic co-culture system for simultaneous analysis of multiple indicators of gefitinib sensitivity in colorectal cancer cells.

The therapeutic effect of gefitinib on colorectal cancer (CRC) is unclear, but it has been reported that stromal cells in the tumor microenvironment may have an impact on drug sensitivity. Herein, we established a microfluidic co-culture system and explored the sensitivity of CRC cells co-cultured with cancer-associated fibroblasts (CAFs) to gefitinib. The system consisted of a multichannel chip and a Petri dish. The chambers in the chip and dish were designed to continuously supply nutrients for long-term cell survival and create chemokine gradients for driving cell invasion without any external equipment. Using this system, the proliferation and invasiveness of cells were simultaneously evaluated by quantifying the area of cells and the migration distance of cells. In addition, the system combined with live cell workstation could evaluate the dynamic drug response of co-cultured cells and track individual cell trajectories in real-time. When CRC cells were co-cultured with CAFs, CAFs promoted CRC cell proliferation and invasion and reduced the sensitivity of cells to gefitinib through the exosomes secreted by CAFs. Furthermore, the cells that migrated out of the chip were collected, and EMT-related markers were determined by immunofluorescent and western blot assays. The results demonstrated that CAFs affected the response of CRC cells to gefitinib by inducing EMT, providing new ideas for further research on the resistance mechanism of gefitinib. This suggests that targeting CAFs or exosomes might be a new approach to enhance CRC sensitivity to gefitinib, and our system could be a novel platform for investigating the crosstalk between tumor cells and CAFs and understanding multiple biological changes of the tumor cells in the tumor microenvironment.

EIF4A3-negatively driven circular RNA β-catenin (circβ-catenin) promotes colorectal cancer progression via miR-197-3p/CTNND1 regulatory axis.

Circβ-catenin, our first reported circRNA, has been reported to mediate tumorigenesis in various cancers. However, its biological functions and underlying mechanisms in colorectal cancer (CRC) remain unknown. The qRT-PCR examination was used to detect the expression of circβ-catenin, miR-197-3p, and CTNND1 in cells and human tissues. Western blot was conducted to detect the protein expression levels. The biological function of circβ-catenin was verified by MTT, colony formation, wound healing, and transwell assays. The in vivo effects of circβ-catenin were verified by nude mice xenograft and metastasis models. The regulatory network of circβ-catenin/miR-197-3p/CTNND1 was confirmed via dual-luciferase reporter and RIP assays. In the present study, circβ-catenin was found to promote CRC cell proliferation and metastasis in vitro and in vivo. Mechanistically, circβ-catenin served as miRNA decoy to directly bind to miR-197-3p, then antagonized the repression of the target gene CTNND1, and eventually promoted the malignant phenotype of CRC. More interestingly, the inverted repeated Alu pairs termed AluJb1/2 and AluY facilitated the biogenesis of circβ-catenin, which could be partially reversed by EIF4A3 binding to Alu element AluJb2. Our findings illustrated a novel mechanism of circβ-catenin in modulating CRC tumorigenesis and metastasis, which provides a potential therapeutic target for CRC patients.

TSC22D2 Regulates ACOT8 to Delay the Malignant Progression of Colorectal Cancer.

Colorectal cancer (CRC) is one of the cancers with high incidence and mortality rates worldwide. In China, there are approximately 400,000 new CRC cases each year, seriously endangering people's life and health. Transforming growth factor β-stimulated clone 22 domain family, member 2 (TSC22D2) is widely expression in cancers, but the role of TSC22D2 in CRC are still unknown. Real‑time quantitative PCR (qRT-PCR) and Western blot were applied to determine the TSC22D2 levels. CCK-8, colony formation and transwell assays were used to determine the proliferation and metastasis abilities of CRC cells in vitro. In vivo metastatic potential was assessed using a subcutaneously injected mouse model and. Western-blot and immunoprecipitation experiments were used to study the mechanism of TSC22D2‑mediated metastasis. We found TSC22D2 was deregulated in CRC tissues and cells and implied poor prognosis. Overexpression TSC22D2 significantly promoted CRC cells proliferation and tumorigenicity both in vitro and vivo, whereas knockdown TSC22D2 resulted in the opposite effects. Importantly using a co-immunoprecipitation (co-IP) assay combined with mass spectrometry analysis to identify TSC22D2-interacting acyl-coenzyme A thioesterases 8 (ACOT8), TSC22D2 maintained stability of ACOT8. Overexpression of TCC22D2 in CRC cells can promote the expression of ACOT8 and inhibit the proliferation and metastasis of CRC cells through EMT mechanism, highlighting the possibility of TSC22D2 as a potential target in CRC development. In summary, the present study revealed the inhibitory effect of TSC22D2 on the proliferation of colorectal cancer cells, suggesting that TSC22D2 may be an important tumor suppressor and a potential therapeutic target during colorectal carcinogenesis.

MIIP downregulation drives colorectal cancer progression through inducing peri-cancerous adipose tissue browning

BackgroundThe enrichment of peri-cancerous adipose tissue is a distinctive feature of colorectal cancer (CRC), accelerating disease progression and worsening prognosis. The communication between tumor cells and adjacent adipocytes plays a crucial role in CRC advancement. However, the precise regulatory mechanisms are largely unknown. This study aims to explore the mechanism of migration and invasion inhibitory protein (MIIP) downregulation in the remodeling of tumor cell-adipocyte communication and its role in promoting CRC.ResultsMIIP expression was found to be decreased in CRC tissues and closely associated with adjacent adipocyte browning. In an in vitro co-culture model, adipocytes treated with MIIP-downregulated tumor supernatant exhibited aggravated browning and lipolysis. This finding was further confirmed in subcutaneously allografted mice co-injected with adipocytes and MIIP-downregulated murine CRC cells. Mechanistically, MIIP interacted with the critical lipid mobilization factor AZGP1 and regulated AZGP1’s glycosylation status by interfering with its association with STT3A. MIIP downregulation promoted N-glycosylation and over-secretion of AZGP1 in tumor cells. Subsequently, AZGP1 induced adipocyte browning and lipolysis through the cAMP-PKA pathway, releasing free fatty acids (FFAs) into the microenvironment. These FFAs served as the primary energy source, promoting CRC cell proliferation, invasion, and apoptosis resistance, accompanied by metabolic reprogramming. In a tumor-bearing mouse model, inhibition of β-adrenergic receptor or FFA uptake, combined with oxaliplatin, significantly improved therapeutic efficacy in CRC with abnormal MIIP expression.ConclusionsOur data demonstrate that MIIP plays a regulatory role in the communication between CRC and neighboring adipose tissue by regulating AZGP1 N-glycosylation and secretion. MIIP reduction leads to AZGP1 oversecretion, resulting in adipose browning-induced CRC rapid progression and poor prognosis. Inhibition of β-adrenergic receptor or FFA uptake, combined with oxaliplatin, may represent a promising therapeutic strategy for CRC with aberrant MIIP expression.

Enterotoxigenic Bacteroides fragilis (ETBF) Enhances Colorectal Cancer Cell Proliferation and Metastasis Through HDAC3/miR-139-3p Pathway.

Enterotoxigenic Bacteroides fragilis (ETBF) is believed to promote the malignant process of colorectal cancer (CRC), but the underlying molecular mechanism still needs to be revealed. CRC cells (SW480 and HCT-116) were treated with ETBF strain. Cell proliferation, invasion and, migration were evaluated by cell counting kit 8 assay, EdU assay, colony formation assay, transwell assay, and wound healing assay. Protein expression was analyzed by western blot. MicroRNA (miR)-139-3p and histone deacetylase 3 (HDAC3) expression levels in tissues and cells were determined by qRT-PCR. Xenograft tumor model was conducted to evaluate the effect of miR-139-3p on CRC tumor growth. ETBF treatment could promote CRC cell proliferation, invasion and migration. MiR-139-3p expression was decreased by ETBF, and its overexpression reversed the effect of ETBF on CRC cell progression. HDAC3 negatively regulated miR-139-3p expression, and its overexpression facilitated CRC cell behaviors via reducing miR-139-3p expression. Moreover, HDAC3 expression was increased by ETBF, and its knockdown also abolished ETBF-mediated CRC cell progression. Additionally, miR-139-3p overexpression could reduce CRC tumor growth in vivo. ETBF aggravated CRC proliferation and metastasis via the regulation of HDAC3/miR-139-3p axis. The discovery of ETBF/HDAC3/miR-139-3p axis may provide a new direction for CRC treatment.

E3 ubiquitin ligase RNF148 functions as an oncogene in colorectal cancer by ubiquitination-mediated degradation of CHAC2.

We previously reported that RNF148 was involved in the ubiquitination-mediated degradation of CHAC2. However, its molecular mechanism was not determined. In this study, we investigated the role and mechanism of RNF148 in the progression of colorectal cancer (CRC), especially in the process of ubiquitination-mediated degradation of CHAC2. Our results revealed that RNF148 was upregulated in most CRC tissues, and its expression significantly correlated with the 3-year overall survival rate and most clinicopathological parameters of CRC patients. Furthermore, RNF148 served as an independent prognostic biomarker of CRC and promoted CRC cell proliferation and migration while inhibiting cell apoptosis and sensitivity to 5-FU. Mechanistically, RNF148 used its protease-associated domain to bind to the CHAC domain of CHAC2 and target it for degradation. In addition, we identified two phosphorylation and three ubiquitination residues of CHAC2 and identified Y118 and K102 as the critical phosphorylation and ubiquitination residues, respectively. We also identified CHAC2's and RNF148's interacting proteins and discovered their potential interaction network. In conclusion, our current study unveiled the role of RNF148 in CRC and the mechanism of RNF148 in the ubiquitination-mediated degradation of CHAC2, which shed light on providing potential prognostic biomarkers and molecular targets for CRC patients.

Systematic functional interrogation of genome-wide association studies locus 17p13.3 deciphered role and genetic control of FAM57A in colorectal cancer development.

Genome-wide association studies (GWAS) have identified over 150 risk loci linked to colorectal cancer (CRC), including the 17p13.3 locus with the tag single nucleotide polymorphism (SNP) rs12603526 in the Asian population. However, the specific causal gene and the functional regulatory mechanisms in this region remain unresolved, necessitating further investigation to elucidate the underlying mechanisms of CRC. We employed an RNA interference-based functional approach to identify genes critical for CRC cell proliferation at the GWAS locus 17p13.3. Bioinformatic fine-mapping analysis was conducted to prioritize causal variants. A large-scale study involving 7,013 cases and 7,329 controls from a Chinese population, along with another cohort of 5,158 cases and 20,632 controls from the UK Biobank, was performed to validate the association between the candidate variant and the gene. A series of biological experiments was conducted to explore the function of the candidate gene and its regulatory mechanisms. We identified FAM57A as a key oncogene that promotes CRC cell proliferation, and confirmed its carcinogenic role through in vitro proliferation assays. The variant rs526835 was prioritized as a causal candidate for CRC risk, located in a functional region with enhancer properties, and showed a significant quantitative association with FAM57A expression. The rs526835 [T] variant was associated with a 1.17-fold increase in CRC risk [95% confidence interval (95% CI): 1.11-1.23, P=1.23×10-9] in the large-scale Chinese cohort, which was further corroborated in the UK Biobank cohort. Mechanistically, we demonstrated that rs526835 enhances a promoter-enhancer interaction mediated by the transcription factor JUN, leading to increased expression of FAM57A. We reveal the underlying mechanisms of CRC predisposition at the GWAS locus 17p13.3. Additionally, our findings highlight the critical role of FAM57A in CRC pathogenesis and introduce a novel enhancer-promoter interaction between FAM57A and rs526835, which could inform future precision prevention and personalized cancer therapies.

SPTBN2 regulated by miR-214-3p inhibits the proliferation and migration of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common and lethal malignancies. According to our analysis in the GEPIA database, SPTBN2 was found to be significantly elevated in COAD patients.Western blot also verified this result, and SPTBN2 was highly expressed in two types of colorectal cancer cells, Caco2 and HCT-8. Therefore, we knocked down SPTBN2 to investigate its function in colorectal cancer, and the results of CCK-8 and Transwell assays showed that SPTBN2 deletion inhibited the proliferation, migration and invasion of CRC cells. In addition, we found that SPTBN2 may be a target of miR-214-3p through the staebase database. miR-214-3p inhibitors promote CRC cell proliferation, migration and invasion. And inhibition of SPTBN2 partially reversed the effect of miR-214-3p in CRC. Taken together, we demonstrated that SPTBN2 acts as an important target of miR-214-3p in CRC. Our study lays the foundation for the mechanism of CRC.

CircGPRC5A enhances colorectal cancer progress by stabilizing PPP1CA and inducing YAP dephosphorylation

BackgroundWith the advancements in bioinformatic technology, an increasing number of circular RNAs (circRNAs) have been discovered and their crucial roles in the development and progression of various malignancies have been confirmed through multiple pathways. However, the specific mechanisms involving protein-binding circRNAs in colorectal cancer (CRC) remain largely unexplored.MethodsDifferential circRNA expression was assessed using a human circRNA microarray in five CRC tissue and paired normal samples. CircGPRC5A expression was then confirmed in the CRC tissues and paired normal samples using qRT-PCR. The biological function of circGPRC5A in CRC were studied in vitro and in vivo. Western blotting, fluorescence in situ hybridization, immunofluorescence, RNA pulldown, mass spectrometry, immunoprecipitation, quantitative phosphoproteomics, and RNA-binding protein immunoprecipitation assays were used to study circGPRC5A.ResultsOur analysis revealed that circGPRC5A expression was higher in CRC tissues compared to normal tissues and was associated with tumor size, tumor stage and lymph node status. CircGPRC5A promoted CRC cell proliferation, migration, and metastasis in vitro and in vivo. CircGPRC5A could stabilize PPP1CA protein by inhibiting the binding between UBA1 and PPP1CA, and increasing YAP dephosphorylation.ConclusionsOur study revealed that circGPRC5A plays an essential function in CRC progression by stabilizing PPP1CA protein and enhancing YAP dephosphorylation. CircGPRC5A could act as a novel and potential target for CRC.

CircVMP1 promotes glycolysis and disease progression by upregulating HKDC1 in colorectal cancer.

Circular RNAs (circRNAs) have been reported to play important roles in cancers. Here, we characterized circVMP1 (hsa_circ_0006508), an important circRNA which promoted glycolysis and disease progression in colorectal cancer (CRC). In this study, we aimed to explore the mechanism by which circVMP1 regulated tumor glycolysis and its related pathways in promoting CRC cell proliferation and metastasis. The expression level of circVMP1 in CRC tissues and adjacent normal tissues was detected using quantitative PCR. In vitro and in vivo functional experiments were used to evaluate the effects of circVMP1 in the regulation of CRC cell proliferation and migration. Mitochondrial stress tests and glycolysis stress tests were conducted to detect the effect of circVMP1 on oxidative phosphorylation and glycolysis. Dual-luciferase reporter and RNA immunoprecipitation assays were used to evaluate the interaction between circVMP1, miR-3167, and HKDC1. We demonstrated that the level of circVMP1 was significantly upregulated in CRC tissues compared with normal tissues. In HCT116 and SW480 cells, overexpression of circVMP1 promoted proliferation, metastasis, and glycolysis. In vivo analysis indicated that circVMP1 accelerated the proliferation of xenograft tumors. As for the mechanism, overexpression of circVMP1 increased the levels of hexokinase domain component 1 (HKDC1) through competitive binding with miR-3167. Our study reported that circVMP1 was one of the tumor driver genes that promoted CRC malignant progression and glycolysis by upregulating HKDC1. CircVMP1/miR-3167/HKDC1 was a signaling axis that might be a target for CRC therapy.

TP53INP2 modulates the malignant progression of colorectal cancer by reducing the inactive form of β-catenin

TP53INP2 (tumor protein p53-inducible nuclear protein 2), known as an autophagy protein, is essential for regulating transcription and starvation-induced autophagy, which plays a crucial role in the oncogenesis and progression of various cancers. The present study aims to investigate the expression pattern, function and prognostic value of TP53INP2 in colorectal cancer (CRC). Here, we report that low expression of TP53INP2 correlates with poor survival in CRC patients. TP53INP2 was significantly downregulated in CRC tissues compared with adjacent tissues. As the malignancy of CRC progresses, the expression level of TP53INP2 gradually decreased. Knockdown of TP53INP2 promoted CRC cell proliferation and tumor growth in mice. Mechanistically, TP53INP2 deficiency decreased phosphorylation of β-catenin on S33, S37, and T41, resulting in increased accumulation of β-catenin and enhanced nuclear translocation and transcriptional activity. Moreover, we further demonstrated that TP53INP2 sequestered TIM50, thereby inhibiting its activation of β-catenin. Taken together, our findings indicate that the downregulation of TP53INP2 promotes CRC progression by activating β-catenin and suggest that TP53INP2 may be a candidate therapeutic target for CRC.

Circular RNA ZNF800 (hsa_circ_0082096) regulates cancer stem cell properties and tumor growth in colorectal cancer

BackgroundCancer stem cells form a rare cell population in tumors that contributes to metastasis, recurrence and chemoresistance in cancer patients. Circular RNAs (circRNAs) are post-transcriptional regulators of gene expression that sponge targeted microRNA (miRNAs) to affect a multitude of downstream cellular processes. We previously showed in an expression profiling study that circZNF800 (hsa_circ_0082096) was up-regulated in cancer stem cell-enriched spheroids derived from colorectal cancer (CRC) cell lines.MethodsSpheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo.ResultsCircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth.ConclusionsCircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.

Hsa_circRNA_001676 accelerates the proliferation, migration and stemness in colorectal cancer through regulating miR-556-3p/G3BP2 axis

Circular RNAs (circRNAs) play key roles in colorectal cancer (CRC) progression, but little is known about the biological functions of hsa_circRNA_001676 in CRC. Therefore, we explored the potential role of hsa_circRNA_001676 in CRC development. RT-qPCR was performed to determine hsa_circRNA_001676, miR-556-3p and Ras-GTPase-activating SH3 domain-binding-proteins 2 (G3BP2) levels in CRC tissues. Meanwhile, to evaluate the roles of hsa_circRNA_001676, miR-556-3p and G3BP2 on CRC, functional analysis of cell proliferation, migration and stemness were then performed. Our results showed that compared to normal tissues, hsa_circRNA_001676 and G3BP2 level was elevated, but miR-556-3p level was reduced in CRC tissues. Additionally, luciferase reporter results showed that hsa_circRNA_001676 was shown to target miR-556-3p, and G3BP2 was targeted by miR-556-3p. Hsa_circRNA_001676 or G3BP2 overexpression promoted CRC cell proliferation and migration. Conversely, miR-556-3p overexpression suppressed CRC cell proliferation and migration. Moreover, deficiency of hsa_circRNA_001676 or G3BP2 repressed the CRC cell proliferation, migration and stemness. Meanwhile, hsa_circRNA_001676 deficiency obviously reduced tumor growth and stemness in a CRC mouse xenograft model. Furthermore, hsa_circRNA_001676 deficiency notably reduced G3BP2 level, but elevated miR-556-3p level in tumor tissues from tumor-bearing mice. Mechanistically, hsa_circRNA_001676 targeted miR-556-3p to increase G3BP2 level, contributing to the progression of CRC. Collectively, hsa_circRNA_001676 was able to accelerate proliferation, migration and stemness in CRC through regulating miR-556-3p/G3BP2 axis, suggesting that hsa_circRNA_001676 may become a potential therapeutic target in treating CRC.