Axis In Colorectal Cancer Cells Research Articles

Quercetin's Dual Mode of Action to Counteract the Sp1-miR-27a Axis in Colorectal Cancer Cells.

Quercetin (Qc) inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The molecular mechanism of action has not been fully elucidated; however, interplay with some miRNAs has been reported, specifically with miR-27a, an onco-miRNA overexpressed in several malignancies. Here, we show that Qc reduces cell viability and induces apoptosis in HCT116 and HT-29 colon cancer cells, by upregulating negative modulators of proliferation pathways such as Sprouty2, PTEN and SFRP1. These are targets of miR-27a whose high expression is reduced by Qc. Moreover, miR-23a, and miR-24-2, the two other components of the unique gene cluster, and the pri-miRNA transcript are reduced, evoking a transcriptional regulation of the entire cluster by Sp1. Mechanistically, we show that Qc is rapidly internalized and localizes in the nucleus, where it likely interacts with Sp1, inducing its proteasomal degradation. Sp1 is further repressed by ZBTB10, an Sp1 competitor for DNA binding that is an miR-27a target and whose levels increase following Qc. SP1 mRNA is also reduced, supporting the regulation of its own gene transcription. Finally, Sp1 knockdown elicits the impaired transcription of the entire cluster and the upregulation of the miR-27a targets, phenocopying the effects of Qc. Through this dual mode of action, Qc counteracts the protumoral Sp1-miR-27a axis, opening the way for novel therapies based on its association as neoadjuvant with known anticancer treatments.

Non-canonical role of UCKL1 on ferroptosis defence in colorectal cancer.

Pyrimidine nucleotides fuel the growth of colorectal cancer (CRC), making their associated proteins potential targets for cancer intervention. Uridine-Cytidine Kinase Like-1(UCKL1) is an enzyme involved in the pyrimidine salvage pathway. It is highly expressed in multiple cancers. But the function and underlying mechanism of UCKL1 in CRC are yet to study. Large-scale genomic analysis was performed to search for potential CRC players related to pyrimidine metabolism. The function of UCKL1 in CRC were examined by RNA interference coupled with invitro and invivo assays. GSH/GSSG assay, NADP+assay, ROS, and Lipid peroxidation assays were performed to check the function of UCKL1 in ferroptosis. Metabolomics analyses, RNA sequencing, western blotting, and rescue assays were done to reveal the underlying mechanisms of UCKL1. Xenograft mouse model was used to examine the therapeutic potential of UCKL1 as a target in combination with other ferroptosis inducers. UCKL1 was identified to repress ferroptosis in CRC cells. It was highly expressed in CRC. It regulated CRC cells proliferation and migration. Downregulation of UCKL1 led to enhanced tumour lipid peroxidation. Intriguingly, UCKL1 reduction-mediated ferroptosis was not related to its role in catalyzing uridine monophosphate (UMP) and cytidine monophosphate (CMP) synthesis. Instead, UCKL1 stabilized Nrf2, which in turn promoted the expression of SLC7A11, a classical repressor of ferroptosis. Moreover, downregulation of UCKL1 sensitized CRC cells to GPX4 inhibitors invitro and invivo. Our study demonstrates that UCKL1 plays a non-canonical role in repressing ferroptosis through a UCKL1-Nrf2-SLC7A11 axis in CRC cells. Combinatorial strategy in targeting ferroptosis by depletion of UCKL1 and application of GPX4 inhibitors may serve as a new effective method for CRC treatment. This study was supported in part by fund from National Natural Science Foundation of China (Grant No. 31970674 to PY), by the Basic and Applied Basic Research Program of Guangdong Province (Grant No. 2023A1515030245 to KL), by the program of Guangdong Provincial Clinical Research Center for Digestive Diseases (2020B1111170004), and by National Key Clinical Discipline.

Deregulation of the miR-19b/PPP2R5E Signaling Axis Shows High Functional Impact in Colorectal Cancer Cells.

MicroRNA (miR)-19b is deregulated in colorectal cancer (CRC) and locally advanced rectal cancer (LARC), predicting worse outcome and disease progression in CRC patients, and acting as a promising prognostic marker of patient recurrence and pathological response to 5-fluorouracil (5-FU)-based neoadjuvant chemoradiotherapy in LARC. Moreover, there is a strong inverse correlation between miR-19b and PPP2R5E in LARC, and both predict the response to neoadjuvant therapy in LARC patients. However, the functional role of the miR-19b/PPP2R5E axis in CRC cells remains to be experimentally evaluated. Here, we confirm with luciferase assays that miR-19b is a direct negative regulator of PPP2R5E in CRC, which is concordant with the observed decreased PP2A activity levels after miR-19b overexpression. Furthermore, PPP2R5E downregulation plays a key role mediating miR-19b-induced oncogenic effects, increasing cell viability, colonosphere formation ability, and the migration of CRC cells. Lastly, we also confirm the role of miR-19b mediating 5-FU sensitivity of CRC cells through negative PPP2R5E regulation. Altogether, our findings demonstrate the functional relevance of the miR-19b/PPP2R5E signaling pathway in disease progression, and its potential therapeutic value determining the 5-FU response of CRC cells.

Ginsenoside Rh3 induces pyroptosis and ferroptosis through the Stat3/p53/NRF2 axis in colorectal cancer cells.

Ginsenoside Rh3 (GRh3) is a seminatural product obtained by chemical processing after isolation from Chinese herbal medicine that has strong antitumor activity against human tumors. However, its antitumor role remains to be elucidated. The aim of this study is to explore the mechanisms underlying the tumor suppressive activity of GRh3 from the perspective of pyroptosis and ferroptosis. GRh3 eliminates colorectal cancer (CRC) cells by activating gasdermin D (GSDMD)-dependent pyroptosis and suppressing solute carrier family 7 member 11 (SLC7A11), resulting in ferroptosis activation through the Stat3/p53/NRF2 axis. GRh3 suppresses nuclear factor erythroid 2-related factor 2 (NRF2) entry into the nucleus, leading to the decrease of heme oxygenase 1 (HO-1) expression, which in turn promotes NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and caspase-1 expression. Finally, caspase-1 activates GSDMD-dependent pyroptosis. Furthermore, GRh3 prevents NRF2 from entering the nucleus, which suppresses SLC7A11, causing the depletion of glutathione (GSH) and accumulation of iron, lipid reactive oxygen species (ROS) and malondialdehyde (MDA), and eventually leading to ferroptosis in CRC cells. In addition, GRh3 effectively inhibits the proliferation of CRC cells in vitro and in nude mouse models. Collectively, GRh3 triggers pyroptotic cell death and ferroptotic cell death in CRC cells via the Stat3/p53/NRF2 axis with minimal harm to normal cells, showing great anticancer potential.

Resveratrol Modulates Chemosensitisation to 5-FU via β1-Integrin/HIF-1α Axis in CRC Tumor Microenvironment.

Frequent development of resistance to chemotherapeutic agents such as 5-flourouracil (5-FU) complicates the treatment of advanced colorectal cancer (CRC). Resveratrol is able to utilize β1-integrin receptors, strongly expressed in CRC cells, to transmit and exert anti-carcinogenic signals, but whether it can also utilize these receptors to overcome 5-FU chemoresistance in CRC cells has not yet been investigated. Effects of β1-integrin knockdown on anti-cancer capabilities of resveratrol and 5-FU were investigated in HCT-116 and 5-FU-resistant HCT-116R CRC tumor microenvironment (TME) with 3D-alginate as well as monolayer cultures. Resveratrol increased CRC cell sensitivity to 5-FU by reducing TME-promoted vitality, proliferation, colony formation, invasion tendency and mesenchymal phenotype including pro-migration pseudopodia. Furthermore, resveratrol impaired CRC cells in favor of more effective utilization of 5-FU by down-regulating TME-induced inflammation (NF-kB), vascularisation (VEGF, HIF-1α) and cancer stem cell production (CD44, CD133, ALDH1), while up-regulating apoptosis (caspase-3) that was previously inhibited by TME. These anti-cancer mechanisms of resveratrol were largely abolished by antisense oligonucleotides against β1-integrin (β1-ASO) in both CRC cell lines, indicating the particular importance of β1-integrin receptors for the 5-FU-chemosensitising effect of resveratrol. Lastly, co-immunoprecipitation tests showed that resveratrol targets and modulates the TME-associated β1-integrin/HIF-1α signaling axis in CRC cells. Our results suggest for the first time the utility of the β1-integrin/HIF-1α signaling axis related to chemosensitization and overcoming chemoresistance to 5-FU in CRC cells by resveratrol, underlining its potential supportive applications in CRC treatment.

Transcriptomic and functional analyses reveal a tumour-promoting role for the IL-36 receptor in colon cancer and crosstalk between IL-36 signalling and the IL-17/ IL-23 axis.

The interleukin (IL)-36 cytokines are a sub-family of the IL-1 family which are becoming increasingly implicated in the pathogenesis of inflammatory diseases and malignancies. Initial studies of IL-36 signalling in tumorigenesis identified an immune-mediated anti-tumorigenic function for these cytokines. However, more recent studies have shown IL-36 cytokines also contribute to the pathogenesis of lung and colorectal cancer (CRC). The aim of this study was to investigate IL-36 expression in CRC using transcriptomic datasets and software such as several R packages, Cytoscape, GEO2R and AnalyzeR. Validation of results was completed by qRT-PCR on both cell lines anda patient cohort.Cellular proliferation was assessed by flow cytometry and resazurin reduction. We demonstrate that IL-36 gene expression increases with CRC development. Decreased tumoral IL-36 receptor expression was shown to be associated with improved patient outcome. Our differential gene expression analysis revealed a novel role for the IL-36/IL-17/IL-23 axis, with these findings validated using patient-derived samples and cell lines. IL-36γ, together with either IL-17a or IL-22, was able to synergistically induce different genes involved in the IL-17/IL-23 axis in CRC cells and additively induce colon cancer cell proliferation. Collectively, this data support a pro-tumorigenic role for IL-36 signalling in colon cancer, with the IL-17/IL-23 axis influential in IL-36-mediated colon tumorigenesis.

Circular RNA circ-CD44 regulates chemotherapy resistance by targeting the miR-330-5p/ABCC1 axis in colorectal cancer cells.

Colorectal cancer (CRC) is a common malignant tumor worldwide, ranking fourth for incidence. Recently, circular RNAs (circRNAs) have been demonstrated to play a key role in chemotherapy resistance to CRC treatment. Therefore, the role of circ-CD44 is investigated in CRC. The expression levels of circ-CD44, miR-330-5p, and ATP binding cassette subfamily C member 1 (ABCC1) were quantified by real-time quantitative polymerase chain reaction (RT-qPCR) assay. The sensitivity of CRC cells to oxaliplatin (OXA) was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay. Colony-forming experiment was performed to measure the colony-forming ability of CRC cells. The apoptosis, migration, and invasion of CRC cells were determined by flow cytometry and transwell assays. A xenograft experiment was established to clarify the functional role of circ-CD44 silencing in vivo. The interactional relationship among circ-CD44, miR-330-5p, and ABCC1 was confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. The protein expression of ABCC1 was quantified by western blot assay. Circ-CD44 was obviously upregulated in OXA-resistant colorectal cancer tissues and cells. Loss-of-function experiments revealed that inhibition of circ-CD44 suppressed proliferation, migration, and invasion while it increased OXA sensitivity and apoptosis in OXA-resistant colorectal cancer cells, which was overturned by suppression of miR-330-5p; besides, silencing of circ-CD44 also slowed the tumor growth in vivo. Additionally, overexpression of miR-330-5p inhibited chemotherapy resistance, proliferation, migration, and invasion while it induced apoptosis by targeting ABCC1. Mechanistically, circ-CD44 functioned as a miRNA sponge for miR-330-5p to upregulate the expression of ABCC1 and regulate chemotherapy resistance in CRC cells.

LncRNA PCGEM1 promotes colorectal cancer cell proliferation and migration in positive feedback loop through PCGEM1/miR-433–3p/CTCF axis

Background and objectivesProstate cancer gene expression marker 1 (PCGEM1) has been identified as an oncogenic long non-coding RNA (lncRNA) in diverse cancers, but it has never been linked with colorectal cancer (CRC). Former studies have shown the mutual regulation between lncRNAs and transcription factors (TFs) in cancer. CCCTC binding factor (CTCF) has been reported to transcriptionally activate lncRNAs in cancers. We predicted the binding of CTCF on PCGEM1 promoter through UCSC (https://genome.ucsc.edu/), but their relation has not been studied. We aimed to investigate whether and how PCGEM1 functioned in CRC cells and the interaction between PCGEM1 and CTCF. Methods and resultsThe impacts of PCGEM1 and CTCF inhibition on CRC cells were verified through loss-of-function experiments. Mechanism experiments were used to prove the binding between CTCF and PCGEM1 in CRC progression. PCGEM1 possessed a high expression level in CRC cells as well as tumors. CTCF transcriptionally activated PCGEM1 expression. Knockdown of PCGEM1 or CTCF impeded proliferation and migration and drove apoptosis of CRC cells. Moreover, PCGEM1 bound miR-433–3p to prevent miR-433–3p from targeting CTCF. ConclusionWe first revealed PCGEM1/miR-433–3p/CTCF positive feedback loop as an oncogenic axis in CRC cells, which potentially provides new clues for the advancement of CRC treatment.

Stabilizing and upregulating Axin with tankyrase inhibitor reverses 5-fluorouracil chemoresistance and proliferation by targeting the WNT/caveolin-1 axis in colorectal cancer cells.

Chemoresistance is a main obstacle for colorectal cancer treatment. In this study, we evaluated the effects and mechanisms of the WNT/β-catenin signaling pathway on the chemoresistance of SW480 and SW620 colorectal cancer cells. The activity of β-catenin was activated/inhibited by the small molecule compound GSK-3 inhibitor 6-bromo-indirubin-3'-oxime and the tankyrase inhibitor XAV939. The downstream target genes of the WNT/β-catenin signaling pathway were screened using a cDNA microarray and bioinformatics analysis. Apoptosis induced by 5-Fu, cell cycle distribution and expression levels of WNT/β-catenin/TCF12/caveolin-1 and multidrug resistance proteins were examed by flow cytometry and western blot after β-catenin activation/inhibition and caveolin-1 overexpression/interference. The effect and mechanism of XAV939 on proliferation and apoptosis induced by 5-Fu in xenograft tumors of nude mice were evaluated by immunohistochemistry and TUNEL staining. 6-Bromo-indirubin-3'-oxime treatment increased β-catenin expression by regulating GSK-3β phosphorylation, accompanied by upregulation of TCF12, caveolin-1, P-gp, and MRP2 and downregulation of apoptosis induced by 5-Fu. Conversely, XAV939 treatment decreased β-catenin expression by upregulating Axin, accompanied by downregulation of TCF12, Caveolin-1, P-gp, and MRP2 and upregulation of apoptosis induced by 5-Fu. The caveolin-1 gene was identified as an important downstream gene of the WNT/β-catenin signaling pathway. Caveolin-1 overexpression upregulated β-catenin expression, increased P-gp and MRP2 expression and decreased apoptosis induced by 5-Fu; conversely, caveolin-1 interference caused the opposite effects. In addition, in vivo experiments showed that XAV939 treatment reduced β-catenin expression, increased apoptosis induced by 5-Fu and repressed xenograft tumor growth. Our findings suggested that inhibition of WNT/β-catenin/TCF12/caveolin-1 provides a new promising therapeutic strategy for colorectal cancer treatment.

SOX11-dependent CATSPER1 expression controls colon cancer cell growth through regulation the PI3K/AKT signaling pathway.

Colorectal cancer (CRC) is one of the most common malignant tumors and the fourth leading cause of cancer death worldwide. Constitutive activation of the PI3K/AKT signaling pathway is a hallmark of colon tumor growth. CATSPER1 gene encodes a pore-forming and pH-sensing subunit of the CatSper Ca2+-permeable channel, a sperm-specific calcium channel essential for hyperactivated motility and male fertility. However, the function of CATSPER1 outside the male reproductive system is unclear. This study was designed to explore whether CatSper exerted its functional role in the progress of CRC, and investigate the possible mechanisms. Microarray data (GSE146587) from 6 patients diagnosed with stage III CRC post-surgery was analyzed by Limma R package. The Kaplan Meier plotter (KM plotter) database was used to assess the relevance of CATSPER1 mRNA expression to the overall survival (OS) rates in CRC. Western blot, real-time PCR and luciferase reporter assays were used to determine the SOX11-CATSPER1 axis in CRC cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing was used to generate CATSPER1 knockout (KO) CRC cells. The proliferation of CRC cells was determined by BrdU incorporation and colony formation assays. The effect of CATSPER1 on CRC tumor growth in vivo was investigated in a mice tumor xenograft model. Here, we show that CATSPER1 expression was significantly up-regulated in CRC and elevated CATSPER1 was associated with poor overall survival (OS). Moreover, the transcription factor SOX11 (SRY-related high-mobility-group (HMG) box 11) activated CATSPER1 transcription in CRC cells. Functionally, we showed that CATSPER1 promoted CRC cells proliferation both in vitro and in vivo. At the molecular level, we demonstrated that CATSPER1 might maintain CRC malignant process partly through the activation of the PI3K/AKT signaling pathway. Increased CATSPER1 expression facilitates CRC cells proliferation, suggesting that targeting CATSPER1 might represent a promising strategy for colon cancer treatment.

LncRNA LINC00525 activates HIF-1α through miR-338-3p / UBE2Q1 / β-catenin axis to regulate the Warburg effect in colorectal cancer

ABSTRACT Warburg effect is considered to be related to the malignancy of tumor cells under hypoxic conditions, but the underlying mechanism remains unknown. In this article, it has been reported that lncRNA LINC00525 is a hypoxia-responsive lncRNA and is essential for hypoxia-enhanced glycolysis. It was found that LINC00525 was up-regulated, and promoted cell proliferation in colorectal cancer in vitro and in vivo. In colorectal cancer cells, hypoxia increasedLINC00525 expression, whereas knocking down LINC00525 reduced hypoxia-enhanced glycolysis. For specific molecular mechanisms, it was found that LINC00525 promoted UBE2Q1 expression by binding miR-338-3p, and UBE2Q1-stabilized β-catenin enhances hypoxia-enhanced glycolysis by activating HIF-1α. In conclusion, these findings showed that LINC00525 was essential for hypoxia-enhanced glycolysis; its mechanism was related to activating HIF-1α through miR-338-3p/UBE2Q1/β-catenin axis in colorectal cancer cells.

Propofol suppresses colorectal cancer development by the circ-PABPN1/miR-638/SRSF1 axis

BackgroundPropofol has recently been attracted increasing attention for its anti-tumor property in cancers, including colorectal cancer (CRC). However, the anti-tumor molecular determinants of propofol largely remain to be elucidated. MethodsThe levels of circRNA poly(A) binding protein nuclear 1 (circ-PABPN1, hsa_circ_0031288), microRNA (miRNA)-638 and serine and arginine-rich factor 1 (SRSF1) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell viability, colony formation, apoptosis, invasion, and migration were detected by the Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, transwell, and wound-healing assays, respectively. Animal studies were used to evaluate the biological action of circ-PABPN1 in the propofol-mediated anti-CRC effect. Targeted relationships among circ-PABPN1, miR-638 and SRSF1 were validated by dual-luciferase reporter assays. ResultsOur data showed the anti-tumor activity of propofol in CRC, as evidenced by the repression in cell viability, colony formation, invasion, migration and the promotion in cell apoptosis in vitro, as well as the suppression in tumor growth in vivo. Circ-PABPN1 was overexpressed in CRC tissues and cells, and propofol down-regulated circ-PABPN1 in a dose-dependent manner. Moreover, circ-PABPN1 was a functional effector of propofol in suppressing CRC development in vitro and in vivo. Circ-PABPN1 directly targeted miR-638, and SRSF1 was a direct target of miR-638. Propofol repressed CRC development in vitro by up-regulating miR-638 or down-regulating SRSF1. Furthermore, propofol regulated SRSF1 expression by the circ-PABPN1/miR-638 axis in CRC cells. ConclusionOur current findings identified the circ-PABPN1/miR-638/SRSF1 axis as a novel anti-tumor mechanism of propofol in CRC, providing a new rationale for developing propofol as a promising therapeutic agent for CRC.

MiR193a-5p Mediated ZNF746 and c-Myc Signaling Axis Is Critically Involved in Morusin Induced Apoptosis in Colorectal Cancer Cells.

Novel target therapy is on the spotlight for effective cancer therapy. Hence, in the present study, the underlying apoptotic mechanism of Morusin was explored in association with miR193a-5p mediated ZNF746/c-Myc signaling axis in colorectal cancer cells (CRCs). Herein, Morusin reduced the viability and the number of colonies in HCT116 and SW480 CRCs. Additionally, Morusin increased sub-G1 population, cleavages of poly (ADP-ribose) polymerase (PARP) and caspase-3 and inhibited the expression of zinc finger protein 746 (ZNF746) and c-Myc in HCT116 and SW480 cells. Conversely, overexpression of ZNF746 suppressed the ability of Morusin to abrogate the expression of c-Myc in HCT116 cells, as ZNF746 enhanced the stability of c-Myc via their direct binding through nuclear colocalization in HCT116 cells by immunofluorescence and immunoprecipitation. Notably, Morusin upregulated miR193a-5p as a tumor suppressor, while miR193a-5p inhibitor masked the ability of Morusin to reduce the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells. To our knowledge, these findings provide the novel insight on miR193a-5p mediated inhibition of ZNF746/c-Myc signaling in Morusin induced apoptosis in CRCs.

Circ-ACAP2 facilitates the progression of colorectal cancer through mediating miR-143-3p/FZD4 axis.

Circular RNAs (circRNAs) play crucial roles in multiple cancers, including colorectal cancer (CRC). Here, we explored the role of circRNA ArfGAP with coiled-coil, ankyrin repeat and PH domains 2 (circ-ACAP2) in the progression and radioresistance of CRC. Quantitative real-time polymerase chain reaction (qPCR) and Western blot assay were used to detect RNA and protein expression, respectively. The proliferation, apoptosis, migration, invasion and radioresistance of CRC cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, transwell migration assay, transwell invasion assay and colony formation assay. The target interaction between microRNA-143-3p (miR-143-3p) and circ-ACAP2 or frizzled class receptor 4 (FZD4) was verified by dual-luciferase reporter assay. Murine xenograft model was established to explore the role of circ-ACAP2 in vivo. The expression of circ-ACAP2 was prominently enhanced in CRC tissues and cell lines. Circ-ACAP2 facilitated the proliferation, migration, invasion and radioresistance whereas inhibited the apoptosis of CRC cells. MiR-143-3p was a direct target of circ-ACAP2 in CRC cells. Circ-ACAP2 promoted the progression and radioresistance of CRC partly by sponging miR-143-3p. MiR-143-3p interacted with the 3' untranslated region (3'UTR) of FZD4 in CRC cells, and FZD4 overexpression partly reversed miR-143-3p-mediated effects in CRC cells. Wnt/β-catenin signalling was modulated by circ-ACAP2/miR-143-3p/FZD4 axis in CRC cells. Circ-ACAP2 contributed to the development and radioresistance of CRC partly through targeting miR-143-3p/FZD4 axis, which provided novel potential diagnostic and therapeutic targets for CRC.

CircRNA_0000392 promotes colorectal cancer progression through the miR-193a-5p/PIK3R3/AKT axis

BackgroundCircular RNAs (circRNAs), important members of the noncoding RNA family, have been recently revealed to play a role in the pathogenic progression of diseases, particularly in the malignant progression of cancer. With the application of high-throughput sequencing technology, a large number of circRNAs have been identified in tumor tissues, and some circRNAs have been demonstrated to act as oncogenes. In this study, we analyzed the circRNA expression profile in colorectal cancer (CRC) tissues and normal adjacent tissues by high-throughput sequencing. We focused on circRNA_0000392, a circRNA with significantly increased expression in CRCtissues, and further investigated its function in the progression of colorectal cancer.MethodsThe expression profile of circRNAs in 6 pairs of CRC tissues and normal adjacent tissues was analyzed by RNA sequencing. We verified the identified differentially expressed circRNAs in additional samples by qRT-PCR and selected circRNA_0000392 to evaluate its associations with clinicopathological features. Then, we knocked down circRNA_0000392 in CRC cells and investigated the in vitro and in vivo effects using functional experiments. Dual luciferase and RNA pull-down assays were performed to further explore the downstream potential molecular mechanisms.ResultsCircRNA_0000392 was significantly upregulated in CRC compared with normal adjacent tissues and cell lines. The expression level of circRNA_0000392 was positively correlated with the malignant progression of CRC. Functional studies revealed that reducing the expression of circRNA_0000392 could inhibit the proliferation and invasion of CRC both in vitro and in vivo. Mechanistically, circRNA_0000392 could act as a sponge of miR-193a-5p and regulate the expression of PIK3R3, affecting the activation of the AKT-mTOR pathway in CRC cells.ConclusionsCircRNA_0000392 functions as an oncogene through the miR-193a-5p/PIK3R3/Akt axis in CRC cells, suggesting that circRNA_0000392 is a potential therapeutic target for the treatment of colorectal cancer and a predictive marker for CRC patients.

Long non-coding RNA MALAT1 regulates oxaliplatin-resistance via miR-324-3p/ADAM17 axis in colorectal cancer cells

BackgroundColorectal cancer (CRC) is one of the most general malignant tumors. Accumulating evidence implied that long non-coding RNA Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) participated in the tumorigenesis of CRC. However, the effect of MALAT1 in drug-resistance needed to be further illustrated.MethodsLevels of MALAT1, microRNA (miR)-324-3p, and a disintegrin and metalloprotease metallopeptidase domain 17 (ADAM17) were detected using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assay. Cell Counting Kit 8 (CCK-8) was used to assess the half maximal inhibitory concentration (IC50) of oxaliplatin (Ox). Meanwhile, cell proliferation, migration and apoptosis were detected by CCK-8, transwell assay, and flow cytometry, respectively. The interaction between miR-324-3p and MALAT1 or ADAM17 was clarified by dual-luciferase reporter assay. Also, the effect of MALAT1 on tumor growth was detected in xenograft tumor mice treated with Ox.ResultsSignificant up regulation of MALAT1 and ADAM17, and decrease of miR-324-3p were observed in Ox-resistant CRC tissues and cells. MALAT1 deficiency enhanced the sensitivity of Ox-resistant CRC cells response to Ox, while miR-324-3p repression or ADAM17 acceleration could overturn this effect. Moreover, MALAT1 silencing repressed tumor growth in Ox-treated nude mice. Mechanically, MALAT1 exerted promotion effect on the resistance response to Ox via miR-324-3p/ADAM17 axis in Ox-resistant CRC cells.ConclusionMALAT1 modulated the sensitivity of Ox through ADAM17 in Ox-resistant CRC cells by sponging miR-324-3p, thus MALAT1 might serve as a novel insight for the therapy of CRC.

Long noncoding RNA XIST knockdown suppresses the growth of colorectal cancer cells via regulating microRNA-338-3p/PAX5 axis.

Colorectal cancer is one of the most common human cancers worldwide. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been reported as the regulators in cancers. The purpose of this study was to reveal the functional mechanisms of lncRNA x inactive specific transcript (XIST) and miR-338-3p in colorectal cancer cells. The transcription level and protein level of genes were assessed by quantitative real-time PCR (qRT-PCR) and western blot assay, respectively. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry analysis were used to determine cell proliferation ability and apoptosis rate, respectively. In addition, cell migratory ability and invasive ability were measured using transwell assay. Besides, the interaction between miR-338-3p and XIST or paired box 5 (PAX5) was predicted by starBase or TargetScan and then verified by the dual-luciferase reporter assay. XIST and PAX5 expression were increased, and miR-338-3p expression was decreased in colorectal cancer tissues and cells. XIST knockdown significantly repressed cell proliferation, migration and invasion, and accelerated apoptosis in colorectal cancer cells. Interestingly, XIST directly downregulated miR-338-3p expression to increase PAX5 level. As expected, XIST knockdown inhibited colorectal cancer cell growth by modulating miR-338-3p expression. Furthermore, miR-338-3p suppressed cell growth via downregulation of PAX5 level in colorectal cancer cells. Our results demonstrated that the downregulation of XIST inhibited cell proliferation, migration and invasion, and induced apoptosis through modulating miR-338-3p/PAX5 axis in colorectal cancer cells, providing potential target for the prevention and treatment of colorectal cancer.

Circ_0136666 Facilitates the Progression of Colorectal Cancer via miR-383/CREB1 Axis.

BackgroundThe changes in dietary patterns cause an increased incidence of colorectal cancer (CRC) globally. We aimed to explore the mechanism behind circular RNA circ_0136666 in the progression of CRC.Materials and MethodsThe expression of circ_0136666, miR-383 and cAMP response element binding protein 1 (CREB1) was detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, apoptosis and glycolysis were measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry and glucose or lactate detection kit, respectively. The combination between miR-383 and circ_0136666 or CREB1 in 293T cells was predicted by Circular RNA Interactome or Starbase software and confirmed by dual-luciferase reporter assay. Western blot assay was performed to detect the abundance of CREB1, hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA) in CRC cells. Murine xenograft model was established to verify the function of circ_0136666 in vivo.Resultscirc_0136666 was aberrantly up-regulated in CRC tissues and cells, and it promoted the proliferation and glycolysis and inhibited the apoptosis of CRC cells. circ_0136666 accelerated the progression of CRC through directly targeting and down-regulating miR-383. CREB1 could bind to miR-383 in 293T cells. The overexpression of CREB1 reversed the inhibitory effects of miR-383 accumulation on the proliferation and glycolysis and the promoting impact on the apoptosis of CRC cells. The enrichment of CREB1 was modulated by circ_0136666/miR-383 signaling in CRC cells. The glycolysis-related proteins (HK2 and LDHA) were modulated by circ_0136666/miR-383/CREB1 axis in CRC cells. circ_0136666 accelerated the growth of CRC tumors via circ_0136666/miR-383/CREB1 axis in vivo.Conclusioncirc_0136666 deteriorated CRC through miR-383/CREB1 axis. circ_0136666/miR-383/CREB1 axis might be an underlying therapeutic target for CRC therapy.

A novel protective role for microRNA-3135b in Golgi apparatus fragmentation induced by chemotherapy via GOLPH3/AKT1/mTOR axis in colorectal cancer cells

Chemotherapy activates a novel cytoplasmic DNA damage response resulting in Golgi apparatus fragmentation and cancer cell survival. This mechanism is regulated by Golgi phosphoprotein-3 (GOLPH3)/Myo18A/F-actin axis. Analyzing the functions of miR-3135b, a small non-coding RNA with unknown functions, we found that its forced overexpression attenuates the Golgi apparatus fragmentation induced by chemotherapeutic drugs in colorectal cancer (CRC) cells. First, we found that miR-3135b is downregulated in CRC cell lines and clinical tumors. Bioinformatic predictions showed that miR-3135b could be regulating protein-encoding genes involved in cell survival, resistance to chemotherapy, and Golgi dynamics. In agreement, ectopic transfection of miR-3135b in HCT-15 cancer cells significantly inhibited cell proliferation, sensitized cells to 5-fluoruracil (5-FU), and promoted late apoptosis and necrosis. Also, miR-3135b overexpression impaired the cell cycle progression in HCT-15 and SW-480 cancer cells. Because GOLPH3, a gene involved in maintenance of Golgi structure, was predicted as a potential target of miR-3135b, we studied their functional relationships in response to DNA damage induced by chemotherapy. Immunofluorescence and cellular ultrastructure experiments using antibodies against TGN38 protein, a trans-Golgi network marker, showed that 5-FU and doxorubicin treatments result in an apoptosis-independent stacks dispersal of the Golgi ribbon structure in both HCT-15 and SW-480 cells. Remarkably, these cellular effects were dramatically hindered by transfection of miR-3135b mimics. In addition, our functional studies confirmed that miR-3135b binds to the 3′-UTR of GOLPH3 proto-oncogene, and also reduces the levels of p-AKT1 (Ser473) and p-mTOR (Ser2448) signaling transducers, which are key in cell survival and autophagy activation. Moreover, we found that after treatment with 5-FU, TGN38 factor coimmunolocalizes with beclin-1 autophagic protein in discrete structures associated with the fragmented Golgi, suggesting that the activation of pro-survival autophagy is linked to loss of Golgi integrity. These cellular effects in autophagy and Golgi dispersal were reversed by miR-3135b. In summary, we provided experimental evidence suggesting for the first time a novel role for miR-3135b in the protection of chemotherapy-induced Golgi fragmentation via GOLPH3/AKT1/mTOR axis and protective autophagy in colorectal cancer cells.

Ceramide and palmitic acid inhibit macrophage-mediated epithelial\u2013mesenchymal transition in colorectal cancer

Accumulating evidence indicates that ceramide (Cer) and palmitic acid (PA) possess the ability to modulate switching of macrophage phenotypes and possess anti-tumorigenic effects; however, the underlying molecular mechanisms are largely unknown. The aim of the present study was to investigate whether Cer and PA could induce switching of macrophage polarization from the tumorigenic M2- towards the pro-inflammatory M1-phenotype, and whether this consequently altered the potential of colorectal cancer cells to undergo epithelial–mesenchymal transition (EMT), a hallmark of tumor progression. Our study showed that Cer- and PA-treated macrophages increased expression of the macrophage 1 (M1)-marker CD68 and secretion of IL-12 and attenuated expression of the macrophage 2 (M2)-marker CD163 and IL-10 secretion. Moreover, Cer and PA abolished M2 macrophage-induced EMT and migration of colorectal cancer cells. At the molecular level, this coincided with inhibition of SNAI1 and vimentin expression and upregulation of E-cadherin. Furthermore, Cer and PA attenuated expression levels of IL-10 in colorectal cancer cells co-cultured with M2 macrophages and downregulated STAT3 and NF-κB expression. For the first time, our findings suggest the presence of an IL-10-STAT3-NF-κB signaling axis in colorectal cancer cells co-cultured with M2 macrophages, mimicking the tumor microenvironment. Importantly, PA and Cer were powerful inhibitors of this signaling axis and, consequently, EMT of colorectal cancer cells. These results contribute to our understanding of the immunological mechanisms that underlie the anti-tumorigenic effects of lipids for future combination with drugs in the therapy of colorectal carcinoma.