Invasion Of HCT116 Cells Research Articles

Withametelin inhibits TGF-β induced Epithelial-to-Mesenchymal Transition and Programmed-Death Ligand-1 expression in vitro.

Withanolides are a group of naturally occurring plant-based small molecules known for their wide range of host cellular functions. The anticancer potential of withanolides has been explored in varying cancer cell lines in vitro. Based on our prior studies, among the tested withanolides, withametelin (WM) has shown significant cytotoxicity with the highest efficacy on HCT-116 colon cancer cells (IC50 0.719 ± 0.12μM). Treatment with WM reduced the TGF-β driven proliferation, colony-forming ability, migration, and invasiveness of HCT-116 cells in vitro. WM also downregulated the expression of mesenchymal markers such as N-CADHERIN, SNAIL, and SLUG in HCT-116 cells. At the molecular level, WM inhibited TGF-β induced phosphorylation of SMAD2/3 and reduced the expression of an immune checkpoint inhibitor programmed-death ligand-1 (PD-L1). Our study highlights the possible anticancer mechanisms of WM involving modulation of the TGF-β pathway and associated target gene expression, suggesting its potential utility in cancer therapy.

Meroterpenoids from Marine Sponge Hyrtios sp. and Their Anticancer Activity against Human Colorectal Cancer Cells.

Two new meroterpenoids, hyrtamide A (1) and hyrfarnediol A (2), along with two known ones, 3-farnesyl-4-hydroxybenzoic acid methyl ester (3) and dictyoceratin C (4), were isolated from a South China Sea sponge Hyrtios sp. Their structures were elucidated by NMR and MS data. Compounds 2-4 exhibited weak cytotoxicity against human colorectal cancer cells (HCT-116), showing IC50 values of 41.6, 45.0, and 37.3 μM, respectively. Furthermore, compounds 3 and 4 significantly suppressed the invasion of HCT-116 cells while also downregulating the expression of vascular endothelial growth factor receptor 1 (VEGFR-1) and vimentin proteins, which are key markers associated with angiogenesis and epithelial-mesenchymal transition (EMT). Our findings suggest that compounds 3 and 4 may exert their anti-invasive effects on tumor cells by inhibiting the expression of VEGFR-1 and impeding the process of EMT.

Cinobufotalin regulates the USP36/c-Myc axis to suppress malignant phenotypes of colon cancer cells in vitro and in vivo.

Ubiquitin-specific protease 36 (USP36) has been reported to exhibit oncogenic effects in various malignancies, but the function of USP36 in colon cancer progression remains indefinite. Herein, we aimed to determine the role and mechanism of USP36 in malignant phenotypes of colon cancer cells and explore the potential drug targeting USP36. Bioinformatics analyses indicated that USP36 is highly expressed and significantly related to tumor stages in colon cancer. Besides, USP36 was further up-regulated in oxaliplatin (Oxa)-resistant colon cancer cells. Colony formation, Edu staining, Transwell, wound healing, sphere formation, and CCK-8 assays were conducted and showed that the proliferation, Oxa-resistance, migration, stemness, and invasion of HCT116 cells were promoted after overexpressing USP36, while suppressed by USP36 knockdown. Mechanically, USP36 enhances c-Myc protein stabilization in HCT116 cells via deubiquitination. AutoDock tool and ubiquitin-AMC hydrolysis assay identified cinobufotalin (CBF), an anti-tumor drug, maybe a USP36 inhibitor by inhibiting its deubiquitination activity. CBF significantly prohibited proliferation, migration, invasion, and stemness of HCT116 cells and reversed Oxa-resistance, whereas enforced expression of USP36 blocked these effects. Moreover, in vivo analyses confirmed the oncogenic role of USP36 and the therapeutic potential of CBF in the malignancy of colon cancer. In conclusion, CBF may be a promising therapeutic agent for colon cancer due to its regulation of the USP36/c-Myc axis.

The activation of HAMP promotes colorectal cancer cell proliferation through zinc-mediated upregulation of SMAD4 expression.

Colorectal cancer (CRC) poses a significant challenge in digestive system diseases, and emerging evidence underscores the critical role of zinc metabolism in its progression. This study aimed to investigate the clinical implications of genes at the intersection of zinc metabolism and CRC. We downloaded CRC prognosis-related genes and zinc metabolism-related genes from public databases. Then, the overlapping genes were screened out, and bioinformatics analysis was performed to obtain the hub gene associated with CRC prognosis. Subsequently, in vitro assays were carried out to investigate the expression of this hub gene and its exact mechanism between zinc metabolism and CRC. HAMP was identified as the hub CRC prognostic gene from overlapping zinc metabolism-related and CRC prognostic genes. In vitro analysis showed HAMP was over-expressed in CRC, and its knockdown inhibited RKO and HCT-116 cell invasion and migration significantly. ZnSO4 induced HAMP up-regulation to promote cell proliferation, while TPEN decreased HAMP expression to inhibit cell proliferation. Importantly, we further found that ZnSO4 enhanced SMAD4 expression to augment HAMP promoter activity and promote cell proliferation in CRC. HAMP stands out as an independent prognostic factor in CRC, representing a potential therapeutic target. Its intricate regulation by zinc, particularly through the modulation of SMAD4, unveils a novel avenue for understanding CRC biology. This study provides valuable insights into the interplay between zinc metabolism, HAMP, and CRC, offering promising clinical indicators for CRC patients. The findings present a compelling case for further exploration and development of targeted therapeutic strategies in CRC management.

The Expression and Role of Aquaporin 4 in Colon Cancer.

Aquaporins (AQPs) were initially discovered as water channel proteins that facilitate transcellular water movements. Recent studies have shown that AQPs are expressed and play an oncogenic role in various cancers. However, the expression and role of Aquaporin 4 (AQP4) in colon cancer have not been investigated. This study aimed to examine the clinical and pathophysiologic significance of AQP4 in colon cancer. Immunohistochemistry (IHC) of AQP4 for 145 primary tumor samples obtained from patients with stage II or III colon cancer was performed, and the relationship between AQP4 expression and patients' prognoses was analyzed. Knockdown experiments with AQP4 small interfering RNA using human colon cancer cells were conducted to analyze the effects on cell invasiveness. IHC revealed that AQP4 was scarcely expressed in the noncancerous colonic mucosa. Of the 145 patients who enrolled in this study, 109 (75.2%) and 36 (24.8%) patients were classified as negative and positive for AQP4 expression, respectively. A high level of AQP4 expression is significantly associated with deeper tumors with lymph node metastasis and venous invasion. A 5-year progression-free survival rate of AQP4-positive patients was significantly worse than that of AQP-4 negative patients (70.7% vs. 87.0%, p=0.049). Furthermore, AQP4 knockdown significantly inhibited cell migration and invasion in HCT116 cells. AQP4 may be a novel biomarker and therapeutic target for colon cancer.

P1212 Effects of Enterococcus faecalis-derived Gelatinase E on HCT116 colorectal cancer cells

Abstract Background The leading cause of death in colorectal cancer (CRC) patients is metastasis to crucial organs, such as the lung, brain and liver. The tumour microenvironment (TME) influences metastasis heavily. One important component in the TME of CRC is the microbiota. Microbiota can affect several pathways related to the origin of metastasis and reorganization of the extracellular matrix. One of the possible contributors to early CRC metastasis is Enterococcus faecalis. E. faecalis produces Gelatinase E (GelE), a quorum-sensing protein with collagenolytic properties. The primary goal of our study was to determine the effect of GelE on CRC behaviour, and especially on migration and invasion in HCT-116 human CRC cells. Moreover, we aimed to uncover the underlying pathways by which GelE influences CRC cells. Methods Extracellular GelE was purified from E. faecalis strain V583 from the culture medium. HCT-116 cells were cultured and exposed to different concentrations of GelE. We performed a viability assay using CCK-8 to assess potential toxicity of GelE. To test effects of GelE on collagen structure, we measured stiffness of collagen-I. A transwell assay was used to assess the influence of GelE on invasive capacity of cells through a collagen-I matrix. GelE exposed cells were assessed for shape and motility markers using immunofluorescent staining for β-catenin (cell shape) and loss of E-cadherin (cell motility marker). For mechanistic insight of GelE exposure on HCT116, we performed proteomics analysis on the cells. Results GelE treatment did not affect relative cell viability of HCT116 cells up to a concentration of 100 μg/ml. Exposure to GelE caused a concentration-dependent increase in HCT-116 cell invasion through collagen-I. The immunostainings showed a downregulation of E-cadherin, indicating an induction of cell motility. Immunostaining for β-catenin showed a statistically significant effect on cell shape, becoming more elongated, an additional indication of cell motility. Proteomics showed possible mechanistic pathways that explain the increased motility including activation of the SMAD pathway, RhoGTPAse activation and activation the urokinase-plasminogen system. Reactome database analysis on the proteomics data also revealed an increase in cell-cycle related pathways. Conclusion Our results indicate that E. faecalis-derived GelE drives HCT-116 invasion with an elongated motility. Therefore, drugs inhibiting the activity of GelE could decrease the risk of metastasis in patients with a high E. faecalis V583 abundance in the TME.

Increased PKN2 and M2-Polarized Macrophages Promote HCT116 Cell Invasion.

Colorectal cancer is the third most common malignant tumor, with highly invasive and metastatic potential in the later stage. This study investigated the role of PKN2 overexpression and M2-polarized macrophages in dictating the malignant phenotype of colorectal cancer cells. HCT116 colorectal cancer cell line with PKN2 overexpression was generated to investigate the functional role of PKN2. THP-1 cells were polarized into M2-like macrophages, and the co-culture system of THP-1/M2 cells and HCT116 cells was established to examine the impacts of M2-polairzed macrophages on the malignant phenotype of colorectal cancer cells. PKN2 overexpression promoted cell proliferation, migration and invasion in HCT116 colorectal cancer cells, and reduced spontaneous cell death in the cell culture. Besides, the presence of M2-polarized THP-1 cells significantly enhanced the aggressive phenotype of HCT116 cells. Both PKN2 overexpression and M2-polarized THP-1 cells increased the expression of NF-κB p65 in HCT116 cells, indicating that enhanced NF-κB signaling may contribute to the augmented aggressiveness of HCT116 cells. These findings suggest PKN2 as an oncogenic factor in colorectal cancer and that M2-polarized THP-1 cells may promote the progression of colorectal cancer by activating NF-κB signaling.

A novel 4-(1,3,4-thiadiazole-2-ylthio)pyrimidine derivative inhibits cell proliferation by suppressing the MEK/ERK signaling pathway in colorectal cancer.

Colorectal cancer (CRC) is one of the most common types of malignant cancers worldwide. Although molecularly targeted therapies have significantly improved treatment outcomes, most of these target inhibitors are resistant. Novel inhibitors as potential anticancer drug candidates are still needed to be discovered. Therefore, in the present study, we synthesized a novel 4-(1,3,4-thiadiazole-2-ylthio)pyrimidine derivative (compound 4) using fragment- and structure-based techniques and then investigated the anticancer effect and underlying mechanism of anti-CRC. The results revealed that compound 4 significantly inhibited HCT116 cell proliferation with IC 50 values of 8.04 ± 0.94 µmol L-1 after 48 h and 5.52 ± 0.42 µmol L-1 after 72 h, respectively. Compound 4 also inhibited colony formation, migration, and invasion of HCT116 cells in a dose-dependent manner, as well as inducing cell apoptosis and arresting the cell cycle in the G2/M phase. In addition, compound 4 was able to inhibit the activation of the MEK/ERK signaling in HCT116 cells. And compound 4 yielded the same effects as the MEK inhibitor U0126 on cell apoptosis and MEK/ERK-related proteins. These findings suggested that compound 4 inhi bited cell proliferation and growth, and induced cell apoptosis, indicating its use as a novel and potent anticancer agent against CRC via the MEK/ERK signaling pathway.

Transcriptional expression, prognostic value and immune infiltration of SFRP family in colorectal cancer: a study based on comprehensive bioinformatics and in vitro analyses.

Secreted frizzled-related protein (SFRP) is a crucial regulator of Wnt signaling, involved in multiple biological processes including cell proliferation and metastasis. Despite the accumulation of evidence that indicated that SFRPs are differentially expressed and play a key role in various malignancies, the function of different SFRPs in colorectal cancer (CRC) remains insufficiently studied. Multicenter databases, including GEPIA, cBioPortal, UALCAN, Pathway Commons, STRING, TIMER, CCLE, and LinkedOmics, comprehensively analyzed differential expression, prognostic value, genetic alterations, signaling pathways, immune cell infiltration, and associated genes of the SFRP family in CRC patients. Colony formation, wound healing, and transwell assays were performed to further validate in vitro. SFRP family members were differentially expressed in CRC, with each member showing varying degrees of genetic alterations. Except for SFRP5, the remaining members show a significant correlation with immune cells. Interestingly, only SFRP2 significantly correlated with CRC prognosis and stage. Additionally, SFRP2 participated in a number of critical biological processes, including metastasis and cell proliferation. Moreover, cell function assays suggested the elimination of SFRP2 inhibits the proliferation, migration, and invasion of HCT116 cells. The differential expression of SFRP2 is closely associated with the prognosis of CRC patients. In addition, abnormal expression of SFRP2 has a significant impact on the progression of CRC, including proliferation, migration, and invasion. SFRP2 may become a novel prognostic factor for CRC.

Carboxypeptidase A6 suppresses the proliferation and invasion of colorectal cancer cells and is negatively regulated by miR-96–3p

BackgroundColorectal cancer (CRC) is a common malignant tumor, and this study aims to explore the role and the regulatory mechanism of carboxypeptidase A6 (CPA6) in CRC cells. MethodsSpecific shRNA targeting CPA6 mRNA was transfected into NCM460 and HT29 cells to down-regulate CPA expression, and expression plasmid was transfected into HCT116 cells to exogenously overexpress CPA6. The dual luciferase assay was used to detect the direct binding of miR-96–3p to CPA6 3′UTR. Phosphorylation and activation of Akt were detected using Western blot. Cells were treated with miR-96–3p mimics, Akt inhibitor (MK-2206) or agonist (SC79) for rescue experiments. The cell functions were evaluated using CCK-8, clone formation, transwell, and Western blot assays. Xenograft tumor assay was also used to analyze the effect of altered CPA6 expression on tumor growth. ResultsKnockdown of CPA6 promoted the proliferation, clone formation, migration, and invasion of NCM460 and HT29 cells in vitro, and the tumor growth of nude mouse xenograft tumor in vivo. Moreover, over-expression of CPA6 significantly inhibited the malignant proliferation and invasion of HCT116 cells in vitro, and the tumor growth of xenograft tumor in vivo. Furthermore, miR-96–3p could directly regulate CPA6 expression by targeting its 3′UTR, and miR-96–3p mimics rescued the inhibitory effects of CPA6 overexpression on the malignant proliferation and invasion of CRC cells. Finally, CPA6 knockdown enhanced Akt/mTOR phosphorylation and activation, while CPA6 overexpression inhibited Akt/mTOR activation. The regulatory effect of CPA6 on Akt/mTOR signaling was naturally regulated by miR-96–3p. Akt inhibitor or agonist rescued the effects of CPA6 knockdown or overexpression on proliferation and EMT of colon cancer cells. ConclusionCPA6 has a significant tumor suppressive effect on CRC by inhibiting the activation of Akt/mTOR signaling, and miR-96–3p negatively regulates the expression of CPA6.

N-methylsansalvamide elicits antitumor effects in colon cancer cells in vitro and in vivo by regulating proliferation, apoptosis, and metastatic capacity.

N-methylsansalvamide (MSSV), a cyclic pentadepsipeptide, was obtained from a strain of Fusarium solani f. radicicola. The current study investigated the anti-colorectal cancer effect of MSSV. MSSV exhibited the inhibition of the proliferation in HCT116 cells via induction of G0/G1 cell cycle arrest by downregulating CDK 2, CDK6, cyclin D, and cyclin E, and upregulating p21WAF1 and p27KIP1. Decreased phosphorylation of AKT was observed in MSSV-treated cells. Moreover, MSSV treatment induced caspase-mediated apoptosis through elevating the level of cleaved caspase 3, cleaved PARP, cleaved caspase 9, and pro-apoptotic Bax. MSSV revealed the declined MMP-9 level mediated by reduction in the binding activity of AP-1, Sp-1, and NF-κB motifs, which led to the migration and invasion of HCT116 cells. In vitro metabolism with rat liver S9 fractions was performed to examine the effect of MSSV metabolites. The metabolic process enhanced the inhibitory effect of MSSV on the HCT116 cell proliferation via decline of cyclin D1 expression and AKT phosphorylation. Finally, oral administration of MSSV inhibited the tumor growth of HCT116 xenograft mice. These results suggest that MSSV is a potential anti-tumor agent in colorectal cancer treatment.

Raw Lacquer Extract from Toxicodendron vernicifluum in Combination with ONC201 Enhances the Inhibitory Effects on Colorectal Cancer Cell Activity.

The purpose of the present research was to explore the therapeutic impact of raw lacquer extract from Toxicodendron vernicifluum on colorectal cancer cells and to investigate the outcome of raw lacquer extract and ONC201 co-treatment on the activity of colorectal cancer cells. The cells of HCT116 were treated with raw lacquer extract, ONC201, or co-treatment. Subsequently, MTT, trypan blue staining, colony formation, annexin V/propidium iodide staining, wound healing, and transwell assays were performed to assess the effects of raw lacquer extract, ONC201 and the synthesis effect of co-treatment on cell activity, survival, proliferation, apoptosis, migration, and invasion in HCT116 cells. Western blotting and immunostaining assay were also performed to detect the expressions of tumor necrosis factor-related apoptosis-inducing ligand, death receptor-5, cleaved caspase-8, p-mTOR/mTOR, and p-S6K/S6K in cells. The results showed that ONC201 and raw lacquer extract had effective anti-cancer effects on HCT116 cells. ONC201 and raw lacquer extract treatment on colorectal cancer cells inhibited cell viability and growth, as well as induced cell apoptosis and cell death of HCT116. The migration and invasion of HCT116 cells were also inhibited. Significantly, raw lacquer extract and ONC201 cotreatment further enhanced the anti-colorectal cancer cell activity in HCT116 cells. Western blotting and immunostaining assay showed that raw lacquer extract in combination with ONC201 induced tumor necrosis factor-related apoptosis-inducing ligand/death receptor-5 expression activation, inhibited the expression of cleaved caspase-8/procaspase-8, and reduced the expression of p-mTOR/mTOR and p-S6K/S6K. These results indicated that raw lacquer extract in combination with ONC201 enhanced the inhibitory effects on colorectal cancer cell activity.

Expression and prognostic value of PRDX family in colon adenocarcinoma by integrating comprehensive analysis and in vitro and in vivo validation.

The peroxiredoxin family, a crucial regulator of redox reactions, is strongly associated with various tumorigenesis. However, the role of peroxiredoxin4 (PRDX4) in colon adenocarcinoma (COAD) remains poorly understood. Multicenter databases, including GEPIA, HPA, UALCAN, cBioPortal, cancerSEA, STRING, CCLE, and LinkedOmics, comprehensively analyzed transcriptional expression, prognostic value, genetic alterations, signaling pathways, and associated genes of the PRDXs in COAD patients. Colony formation, transwell, flow cytometry, sphere formation, and xenograft assays were performed to validate further in vitro and in vivo. Members of the PRDX family were differentially expressed in COAD, with each member showing varying degrees of genetic alterations. Intriguingly, only PRDX4 significantly correlated with COAD prognosis and stage. The single-cell sequencing suggested that PRDX4 is positively correlated with proliferation, apoptosis, and invasion, whereas negatively correlated with stemness. Moreover, PRDX4 involved in a series of critical biological processes, such as cell growth. Furthermore, in vivo and in vitro analyses indicated that knocking down PRDX4 inhibits the proliferation and invasion of HCT116 cells while promoting apoptosis and stemness. We identified PRDX4 expression as a novel potential prognostic marker in COAD.

Role of green synthesized platinum nanoparticles in cytotoxicity, oxidative stress, and apoptosis of human colon cancer cells (HCT-116)

Progresses in the medicinal application of nanocompounds were accepted for the treatment of cancer. Nanoparticles-based therapy is of benefit for effective biodistribution and specific targeting. The current study investigated the anticancer effect of green synthesized platinum nanoparticles (PtNPs) against colon cancer cells (HCT-116). Flow cytometry and ELISA techniques were employed for detecting apoptotic and oxidative stress markers. Furthermore, PtNPs-lycopene (PtNPs-LP) on cell migration and invasion of HCT-116 cells was also examined. The PtNPs-LP was capable of diminishing cell proliferation and viability of HCT-116 cells in a dose-dependent mode. After treatment with PtNPs-LP, a significant increase in pro-apoptotic Bax and caspase-3 and a decrease in anti-apoptotic Bcl-2 was observed in treated cells that subsequently released cytochrome C into its cytoplasm, initiating cell death. Moreover, PtNPs-LP induced excessive generation of reactive oxygen species (ROS) and oxidative stress in cancer cells. In conclusion, PtNPs-LP exerts an antitumor effect against colon cancer cells via mediating important mechanisms such as cytotoxicity, apoptosis, and oxidative stress.

Supercritical fluid extract of Angelica sinensis promotes the anti-colorectal cancer effect of oxaliplatin

Oxaliplatin-based chemotherapy regimens are recommended for patients with advanced colorectal cancer (CRC). However, oxaliplatin (OXA) can cause toxic side effects at the recommended dosage. Therefore, it is necessary to find new drug candidates that can synergize with OXA and thereby lower the OXA dose while still maintaining its efficacy. Angelica sinensis is a common drug in traditional Chinese medicine and has demonstrated a significant anti-CRC effect in modern pharmacological studies. The active ingredients in Angelica sinensis can be effectively extracted by a supercritical fluid extract. In this study, the supercritical fluid extract of Angelica sinensis (A-SFE) was obtained by a stable extraction process and was chemically characterized by GC/MS. The anti-cancer effect of A-SFE when applied individually was explored in vitro through MTT, scratch, and Transwell assay. The effect of A-SFE on CRC cells under the influence of tumor-associated macrophages (TAMs) was explored by a co-culture model. The results showed that A-SFE could inhibit the viability, metastasis, and invasion of HCT116 cells, especially under the influence of TAMs. When 20-100μg/ml of A-SFE and 8-64μg/ml of OXA were used in combination in HCT116 cells, synergistic or additive effects were shown in different concentration combinations. The CT26 syngeneic mouse model was used to explore the anti-CRC effect of OXA combined with A-SFE in vivo. The tumor volume, expression levels of Ki67, MMP9, and CD206 in the OXA + A-SFE group were less than those in the OXA group. In conclusion, A-SFE has the potential to become an adjuvant drug for OXA in the treatment of CRC, which provides new strategies for anti-colorectal cancer research.

Inhibitor of DNA binding 2 knockdown inhibits the growth and liver metastasis of colorectal cancer

BackgroundLiver metastasis of colorectal cancer (CRC) remains high mortality and the mechanism is still unknown. Here we investigated the effects of inhibitor of DNA binding 2 (Id2) on growth and liver metastasis of CRC. MethodsqPCR and western blotting were used to demonstrate mRNA and protein expressions in Id2-knockdown HCT116 cells. Cell growth was observed by cell proliferation assay, colony formation assay and flow cytometry. Cell migration and invasion were observed with wound healing assay and transwell migration and invasion assay. The effects of Id2 knockdown on tumor growth and liver metastasis in vivo were evaluated respectively with subcutaneous tumor model and colorectal liver metastasis model by injecting HCT116 cells into the mesentery triangle of cecum in mice. ResultsId2 overexpression was found in CRC cell lines. Id2 knockdown resulted in a reduction in the proliferation, colony formation, migration and invasion of HCT116 cells. The suppression of cell proliferation was accompanied by the cell cycle arrest in the G0/G1 phase with down-regulation of Cyclin D1, Cyclin E, p-Cdk2/3, Cdk6, p-p27 and up-regulation of p21 and p27. Id2 knockdown reversed epithelial-mesenchymal transition (EMT) through increasing E-Cadherin and inhibiting N-Cadherin, Vimentin, β-catenin, Snail and Slug. Id2 was also found to inhibit CRC metastasis via MMP2, MMP9 and TIMP-1. Furthermore, Id2 knockdown suppressed CRC liver metastasis in vivo. ConclusionId2 promotes CRC growth through activation of the PI3K/AKT signaling pathway, and triggers EMT to enhance CRC migration and invasion.

Activating transcription factor 3-activated long noncoding RNA forkhead box P4-antisense RNA 1 aggravates colorectal cancer progression by regulating microRNA-423-5p/nucleus accumbens associated 1 axis

ABSTRACT Long noncoding RNAs (lncRNAs) have vital roles in the progression of colorectal cancer (CRC). Forkhead box P4-antisense RNA 1 (FOXP4-AS1) showed a potential unfavorable prognostic factor for CRC, while its underlying mechanism remains elusive. Thus, the goal of this research is to determine mechanism of FOXP4-AS1 in CRC occurrence and development. Herein, a Dual-luciferase reporter assay was performed to assess the regulation of miR-423-5p to nucleus accumbens-associated protein 1 (NACC1) and activating transcription factor 3 (ATF3) to FOXP4-AS1 promoter. Hematoxylin-eosin (H&E) staining was performed to detect the pathological changes of tumor tissues. Flow cytometry, cell counting kit 8, Transwell, and wound healing assays were conducted to assess apoptosis, proliferation, migration, and invasion of CRC cells, respectively. The results showed that FOXP4-AS1 was highly expressed in CRC cell lines and tissues. CRC progression was promoted by the overexpression of FOXP4-AS1 in HTC116 cells and animal models. Furthermore, FOXP4-AS1 served as a molecular sponge for miR-423-5p, and NACC1 is a direct target of miR-423-5p. MiR-423-5p silencing or overexpression of NACC1 increased proliferation, migration, and invasion of HCT116 cells while suppressing apoptosis. We also found that the upregulation of FOXP4-AS1 was activated by ATF3 in CRC cells. Collectively, our results demonstrated that ATF3-activated FOXP4-AS1 aggravates CRC progression by regulating miR-423-5p/NACC1 axis, indicating a new target for CRC treatment.

The mechanism of FZXJJZ decoction suppresses colorectal liver metastasis via the VDR/TGF-β/Snail1 signaling pathways based on network pharmacology-TCGA data-transcriptomics analysis

Ethnopharmacological relevanceFuzheng Xiaojijinzhan (FZXJJZF) decoction is an effective prescription for treating colorectal cancer liver metastasis (LMCRC). Aim of the studyTo elucidate the pharmacological mechanism of the FZXJJZF decoction therapy on LMCRC. Materials and methodsFirstly, a network pharmacological approach was used to characterize the underlying targets of FZXJJZF on LMCRC. Secondly, LMCRC-related genes are obtained from the public database TCGA, and those genes are further screened and clustered through Mfuzz, an R package tool. Then, targets of FZXJJZF predicted by network pharmacology were overlapped with LMCRC related genes screened by Mfuzz. Meanwhile, FZJZXJF intervened in LMCRC model,epithelial-to-mesenchymal transition (EMT), and migration and invasion of HCT-116 cells. Thirdly, the transcriptomics data of FZJZXJF inhibited HCT-116 cells of EMT cells were overlapped with EMT database data to narrow the possible range of targets. Based on this, the potential targets and signal pathways of FZJZXJF were speculated by combining the transcriptomics data with the targets from network pharmacology-TCGA. Finally, the anti-cancer mechanism of FZXJJZF on LMCRC was verified in vitro by Real-Time PCR and Western Blot in vitro. ResultsBy network pharmacological analysis, 282 ingredients and 429 potential targets of FZXJJZF were predicted. The 9268 LMCRC-related genes in the TCGA database were classified into 10 clusters by the Mfuzz. The two clustering genes with the most similar clustering trends were overlapped with 429 potential targets, and 32 genes were found, such as CD34, TRPV3, PGR, VDR, etc. In vivo experiments, FZJZXJF inhibited the tumor size in LMCRC models, and the EMT, migration, and invasion of HCT-116 also be inhibited. Intersecting transcriptomics dates with 32 target genes, it is speculated that the VDR-TGF-β signaling pathway may be an effective mechanism of FZXJJZF. Additionally, it is shown that FZXJJZF up-regulated the expression levels of VDR and E-cadherin and down-regulated the expression levels of TGF-β and Snail1 in vitro. These results confirmed that FZXJJZF plays an effective role in LMCRC mainly by inhibiting EMT phenotype via the VDR-TGF-β signaling pathway. ConclusionsCollectively, this study reveals the anti-LMCRC effect of FZXJJZF and its potential therapeutic mechanism from the perspective of potential targets and potential pathways.

CircRNA_0084927 promotes colorectal cancer progression by regulating miRNA-20b-3p/glutathione S-transferase mu 5 axis.

BACKGROUNDColorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related death worldwide. The 5-year survival rate of patients with early-stage CRC could reach 90%, but it is very low in patients with advanced-stage CRC. Recent studies have shown that circular RNAs play important roles in regulating the migration and invasion of CRC cells.AIMTo elucidate the role of circRNA_0084927 (circ_0084927) in the migration and invasion of CRC cells and its underlying mechanism.METHODSClinical tissue samples and cells were collected, and the expression of circ_0084927 was detected by quantitative polymerase chain reaction (qPCR). The diagnostic performance of circ_0084927 was assessed by receiver operating characteristic curve analysis. The role of circ_0084927 in CRC cell proliferation, migration, and invasion was determined using cell counting kit-8 assay, wound healing assay, and transwell assay, respectively. The regulatory relationship among circ_0084927, miRNA-20b-3p (miR-20b-3p), and glutathione S-transferase mu 5 (GSTM5) was identified using databases, luciferase reporter assay, qPCR, and Western blot analysis. AKT-mTOR signaling was also verified after circ_0084927 knockdown or miR-20b-3p mimic treatment.RESULTSThe expression of circ_0084927 was significantly increased in CRC tissues and cells, and it was higher in advanced-stage CRC compared with early-stage CRC. The area under the curve (AUC) of circ_0084927 was 0.806 [95% confidence interval (CI): 0.683-0.896]. In addition, the AUC was 0.874 (95%CI: 0.738-0.956) in patients with advanced-stage CRC and 0.713 (95%CI: 0.555-0.840) in those with early-stage CRC. Knockdown of circ_0084927 inhibited the migration and invasion of HCT116 cells. Moreover, circ_0084927 was found to act as a sponge of miR-20b-3p. MiR-20b-3p activation reduced the circ_0084927 level, whereas miR-20b-3p inhibition increased the circ_0084927 level. But the effect was not found after circ_0084927 mutation. In addition, miR-20b-3p expression in CRC patients was also reduced and negatively correlated with circ_0084927 expression. The function of circ_0084927 in HCT116 cells with circ_0084927 knockdown was rescued by miR-20b-3p. Moreover, GSTM5 expression was significantly decreased after overexpressing miR-20b-3p or inhibiting circ_0084927, but its expression was rescued when circ_0084927 and miR-20b-3p were both inhibited. Finally, AKT-mTOR signaling was markedly regulated by circ_0084927 and miR-20b-3p.CONCLUSIONThe expression of circ_0084927 is significantly increased in CRC and higher in advanced-stage CRC than in early-stage CRC. Moreover, circ_0084927 potentially regulates CRC cell migration and invasion via the miR-20b-3p/GSTM5/ AKT/mTOR pathway.

MicroRNA-let-7 Targets HMGA2 to Regulate the Proliferation, Migration, and Invasion of Colon Cancer Cell HCT116.

Objective To investigate the effect of microRNA-let-7 (miR-let-7) on the proliferation, migration, and invasion of colon cancer cell HCT116 in vitro and its regulatory mechanism on downstream HMGA2. Methods It was planned to synthesize miR-let-7 overexpression (mimics) and interference expression (inhibitor) and transiently transfect colon cancer cell HCT116, detect the expression levels of miR-let-7 and HMGA2 in the cells after transfection and the targeted regulation effect of miR-let-7 on HMGA2, then detect the effect of upregulation/downregulation of miR-let-7 on HMGA2, and detect the proliferation, migration, and invasion of HCT116 cells. Results The expression of miR-let-7 was downregulated, and the expression of HMGA2 was upregulated in HCT116. The expression of miR-let-7 increased significantly after HCT116 was transfected with miR-let-7 mimics. The expression of miR-let-7 decreased significantly after HCT116 was transfected with miR-let-7 inhibitor. The bioinformatics websites predicted that miR-let-7 has a binding site with HMGA2, and the dual-luciferase reporter gene experiment detected that miR-let-7 has a targeting relationship with HMGA2. The expression of HMGA2 decreased after HCT116 was transfected with miR-let-7 mimics; the expression of HMGA2 increased after HCT116 was transfected with miR-let-7 inhibitor. After upregulating miR-let-7, the proliferation, migration, and invasion ability of HCT116 was weakened. After miR-let-7 was inhibited, the proliferation, migration, and invasion ability of HCT116 was enhanced. Conclusion Abnormal expression of miR-let-7 is an important factor affecting the proliferation, migration, and invasion of HCT116 cells, and it can promote or inhibit the biological behavior of cancer cells by targeting the expression of HMGA2. This study provides ideas for the drug development of new gene targets.