Colon Epithelial Cell Line NCM460 Research Articles

Ginsenoside Rh2 suppresses ferroptosis in ulcerative colitis by targeting specific protein 1 by upregulating microRNA-125a-5p

BackgroundWorldwide, ulcerative colitis (UC) is becoming increasingly fast growing. Ginsenoside Rh2 has been reported to alleviate UC. However, the latent biological mechanism of Rh2 in the treatment of UC remains uncertain. In this study, the goal was to determine the therapeutic effect of Rh2 on dextran sulfate sodium (DSS)-induced UC.MethodsA DSS-induced UC mouse model was established and divided into 7 groups for Rh2 gavage and/or miR-125a-5p lentivirus injection (n = 10 per group). Colonic specimens were collected for phenotypic and pathological analysis. miR-125a-5p and specific protein 1 (SP1) expression, inflammation-related factors IL-6 and IL-10, and apoptosis were detected in mice. Human normal colon epithelial cell line NCM460 was treated with H2O2 and ferric chloride hexahydrate to construct an in vitro cell model of colitis and induce ferroptosis. Independent sample t-test was used to compare cell proliferation, cell entry, apoptosis, and oxidative stress between the two groups. One way analysis of variance combined with the least significant difference t test was used for comparison between groups. Multiple time points were compared by repeated measurement analysis of variance.ResultsDSS-induced UC mice had significantly decreased body weight, increased disease activity index, decreased colon length, and decreased miR-125a-5p expression (all P < 0.05). In the DSS-induced mouse model, the expression of miR-125a-5p rebounded and ferroptosis was inhibited after Rh2 treatment (all P < 0.05). Inhibition of miR-125a-5p or upregulation of SP1 expression counteracted the protective effects of Rh2 on UC mice and ferroptosis cell models (all P < 0.05).ConclusionsRh2 mitigated DSS-induced colitis in mice and restrained ferroptosis by targeting miR-125a-5p. Downregulating miR-125a-5p or elevating SP1 could counteract the protective impacts of Rh2 on ferroptotic cells. The findings convey that Rh2 has a latent application value in the treatment of UC.Graphical

3,4,5-O-tricaffeoylquinic acid alleviates ionizing radiation-induced injury in vitro and in vivo through regulating ROS/JNK/p38 signaling.

Ionizing radiation (IR) brings many health problems to humans, causing damage to the digestive system, hematopoietic system, and immune system. Natural compounds derived from plants have attracted widespread attention due to their low toxicity. Here, we found that 3,4,5-O-tricaffeoylquinic acid (tCQA) extracted from natural plant Azolla imbricata could significantly alleviate the systemic damage in mice caused by IR. In order to further explore the molecular mechanism of the radioprotective effect of tCQA, in vitro experiments confirmed that tCQA could attenuate the cytotoxic effect of IR on the colonic epithelial cell line NCM460 and alleviate the IR-induced mitochondrial dysfunction characterized by the decrease of mitochondrial transmembrane potential, ROS production, and caspase-dependent apoptosis. In addition, the generation of ROS induced by H2 O2 could also be reversed by tCQA. Then, Western blot demonstrated that tCQA could reverse the MAPK signaling pathway activated by IR. However, the inhibitory effect of tCQA on JNK and P38 levels activated by the JNK agonist anisomycin is not obvious; meanwhile, tCQA could inhibit the activation of JNK/P38 induced by H2 O2 , which suggests that tCQA might inhibit the JNK/P38 signaling pathway by reducing ROS. In short, tCQA inhibits the generation of ROS caused by IR, and then regulates the activity of caspase in the mitochondrial pathway by inhibiting the JNK/P38 signaling pathway, thereby alleviating the apoptosis of NCM460. This research provides an experimental basis for the development of new types of radioprotective agents for medical diagnosis and radiotherapy.

A Novel Prognostic Prediction Model for Colorectal Cancer Based on Nine Autophagy-Related Long Noncoding RNAs.

IntroductionColorectal cancer (CRC) is the most common gastrointestinal cancer and has a low overall survival rate. Tumor–node–metastasis staging alone is insufficient to predict patient prognosis. Autophagy and long noncoding RNAs play important roles in regulating the biological behavior of CRC. Therefore, establishing an autophagy-related lncRNA (ARlncRNA)-based bioinformatics model is important for predicting survival and facilitating clinical treatment.MethodsCRC data were retrieved from The Cancer Genome Atlas. The database was randomly divided into train set and validation set; then, univariate and multivariate Cox regression analyses were performed to screen prognosis-related ARlncRNAs for prediction model construction. Interactive network and Sankey diagrams of ARlncRNAs and messenger RNAs were plotted. We analyzed the survival rate of high- and low-risk patients and plotted survival curves and determined whether the risk score was an independent predictor of CRC. Receiver operating characteristic curves were used to evaluate model sensitivity and specificity. Then, the expression level of lncRNA was detected by quantitative real-time polymerase chain reaction, and the location of lncRNA was observed by fluorescence in situ hybridization. Additionally, the protein expression was detected by Western blot.ResultsA prognostic prediction model of CRC was built based on nine ARlncRNAs (NKILA, LINC00174, AC008760.1, LINC02041, PCAT6, AC156455.1, LINC01503, LINC00957, and CD27-AS1). The 5-year overall survival rate was significantly lower in the high-risk group than in the low-risk group among train set, validation set, and all patients (all p < 0.001). The model had high sensitivity and accuracy in predicting the 1-year overall survival rate (area under the curve = 0.717). The prediction model risk score was an independent predictor of CRC. LINC00174 and NKILA were expressed in the nucleus and cytoplasm of normal colonic epithelial cell line NCM460 and colorectal cancer cell lines HT29. Additionally, LINC00174 and NKILA were overexpressed in HT29 compared with NCM460. After autophagy activation, LINCC00174 expression was significantly downregulated both in NCM460 and HT29, while NKILA expression was significantly increased.ConclusionThe new ARlncRNA-based model predicts CRC patient prognosis and provides new research ideas regarding potential mechanisms regulating the biological behavior of CRC. ARlncRNAs may play important roles in personalized cancer treatment.

Abstract 2840: HK1 induces epithelial-mesenchymal transition and promotes metastasis of colorectal cancer

Abstract Hexokinase 1 (HK1) has been implicated in tumorigenesis. However, its role in tumor metastasis and progression has not been evaluated. The aim of this study is to evaluate the role of HK1 in the metastasis in CRC. Methods: The expression level of HK1 was detected using quantitative RT-PCR and western blot in 9 CRC cell lines and 1 human colon epithelial cell line NCM460 (Figure 1A). HK1 was overexpressed via lentiviral vector system and silenced using specific small interference RNA (si-HK1) in DLD1 and HCT8. Then wound-healing, migration assay and invasion assay were carried out to investigate the function of HK1 in CRC cells. RT-PCR and Western blot was performed to detect the change of Snail2 following the up-regulated expression of HK1. Results: HK1 expression was overexpressed by HK1-cDNA and was inhibited by si-HK1 in DLD1 and HCT8 (Figure 1B and 1C). Ectopic expression of HK1 promoted cell migration and invasion in CRC cells and promoted lung metastasis in mouse models (Figure 1D). Furthermore, HK1 induced migration and invasion in CRC cells was found to activate expression of Snail2 and in turn, induce epithelial-mesenchymal transition (EMT) and cancer progression (Figure 1E). Conclusions: High expression of HK1 could activate expression of Snail2 and in turn, induce EMT. Our findings suggest that HK1 may be a potential prognostic biomarker and therapeutic target for CRC invasion and metastasis. Keywords: Colorectal Cancer, HK1, Migration, Invasion Figure 1. A. The expression level of HK1 in 10 cell lines. B, C. HK1 expression was overexpressed by HK1-cDNA and was inhibited by si-HK1 in DLD1 and HCT8. *P&amp;lt; 0.05. D. Overexpression of HK1 in HCT8 increased the capacity of cell migration and invasion in vitro and in vivo. E. Expression levels of Snail2 and EMT were induced after HK1 overexpression. Citation Format: Xiaobin Zheng, Huashan Liu, Xiaoli Zhong, Xuanhui Liu, Zerong Cai, Yufeng Chen, Xiaosheng He, Ping Lan, Xiaojian Wu. HK1 induces epithelial-mesenchymal transition and promotes metastasis of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2840.

Exosomes derived from colon cancer cells and plasma of colon cancer patients promote migration of SW480 cells through Akt/mTOR pathway

ObjectiveTo investigate the effect of exosomes derived from colon cancer (CC) cells and plasma of CC patients on migration of SW480 cells. MethodsThe exosomes derived from culture medium of human colon epithelial cell line NCM460 and CC cell line SW620 were isolated by ultracentrifugation. The exosomes derived from plasma of CC patients and healthy controls were isolated by size exclusion chromatography (SEC). The particle size and morphology of exosomes were identified by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) respectively, and exosomal markers were detected by Western blotting. The uptake of fluorescent DiI labeled exosomes by SW480 cells was observed by confocal microscopy. Transwell assay was used to detect the effect of exosomes on the migration of SW480 cells. The expression level of associated proteins in signaling pathway were analyzed by Western blotting. Rapamycin, an inhibitor of mTOR, was used to study the role of mTOR signaling pathway on exosomes mediated migration of SW480 cells. ResultsThe results of NTA and TEM showed that the particle size of the isolated exosomes was about 120 nm, which were small vesicles with membrane structure. The expressions of exosomal markers Alix, TSG101 and CD63 could be detected. The exosomes were evidenced by a red fluorescent signal inside the cytoplasm of SW480 recipient cells, and could promote the migration of SW480 cells, which is associated with Akt/mTOR signaling pathway. Compared with the control group, plasma exosomes derived from CC patients could significantly promote the migration of SW480 cells. Inhibition the activity of mTOR signaling could attenuate the migration of SW480 cells. ConclusionsExosomes derived from CC cells and plasma of CC patients could promote the migration of SW480 cells, which is associated with Akt/mTOR signaling pathway.

Downregulation of miR‑143 modulates KRAS expression in colorectal carcinoma cells.

MicroRNAs (miRs) are a class of non‑coding small RNAs that have been demonstrated to be involved in the pathogenesis of human cancer. There is even evidence that microRNAs can act as oncogenes or tumor suppressors. Although microRNA expression profiles have been characterized in colorectal carcinoma, their precise physiological functions are largely unknown. It has become clear that the activated KRAS Proto‑Oncogene, GTPase (KRAS) oncogene plays an important role in colorectal carcinogenesis. In the present study, it was found that the level of mature miR‑143 was lower in colorectal carcinoma tissues compared with that observed in normal adjacent tissues. A lack of miR‑143 was detected in human colorectal carcinoma cell lines, SW480, LoVo and HT‑29, compared to the high expression observed in normal colon epithelial cell line NCM460. pcDNA3.1‑pri‑miR‑143 and its mutant were successfully constructed and transfected into colorectal carcinoma cells. Increased accumulation of mature miR‑143 was observed in the pcDNA3.1‑pri‑miR‑143‑transfected cells. In SW480 cells, transfection of pcDNA3.1‑pri‑miR‑143 resulted in a 35 and 47% reduction in cell growth after incubation for 4 and 5 days, respectively, compared with transfection of the pcDNA3.1‑pri‑miR‑143 mutant; while in LoVo cells, transfection of pcDNA3.1‑pri‑miR‑143 resulted in a 33 and 46% reduction in cell growth respectively. In contrast, transfection of pcDNA3.1‑pri‑miR‑143 had no significantly effects on HT‑29 cell growth. We also found that transfection of pcDNA3.1‑pri‑miR‑143 had no effect on levels of KRAS mRNA, but resulted in a 58% decrease in the KRAS protein level in the transfected SW480 cells, while an approximate 54 and 43% KRAS protein reduction in LoVo and HT‑29 cells, respectively, compared with the pcDNA3.1‑pri‑miR‑143 mutant. Two fragments containing the putative complementary site were cloned into the pGL3 vector, constructing the luciferase reporter pGL3‑KRAS‑CS1 and pGL3‑KRAS‑CS2. Cotransfection of pcDNA3.1‑pri‑miR‑143 with pGL3‑KRAS‑CS1 and pGL3‑KRAS‑CS2 respectively resulted in 4.6‑ and 3.3‑fold inhibition of luciferase activity in the SW480 cells, while a 4.0‑ and 3.2‑fold inhibition of luciferase activity in the LoVo cells, 3.7‑ and 3.1‑fold inhibition in the HT‑29 cells. Differences in pGL3‑KRAS‑CS1 and pGL3‑KRAS‑CS2 activity were not significant. Our results revealed that increased accumulation of miR‑143 is likely to modulate levels of KRAS protein expression at the post‑transcriptional level by interacting specifically with the complementary site, and consequently inhibiting proliferation of the transfected cells.

Alpinoid c analog inhibits angiogenesis and induces apoptosis in COLO205 cell line

Diarylheptanoids display an array of biological and pharmacological properties. We previously reported the synthesis of a diarylheptanoid Alpinoid c and a series of its derivatives, evaluated their cytotoxicity against various human cancer cells. We found some of these derivatives were significantly more potent than Alpinoid c in preventing the proliferation of various cancer cell lines. Among these, (S, E)-1-(3, 4 dimethoxyphenyl)-6-hydroxy-7-phenylhept-4-en-3-one (DPHP) showed most potent cytotoxicity against COLO205 cells, however, the mechanism by which DPHP prevents the growth of these colon cancer cells remains unknown. In the current study, we investigated the molecular mechanism of DPHP on colon cancer cells. DPHP inhibited the proliferation of COLO205 (IC50 7.01 ± 0.62 μM) and A549 (IC50 20.03 ± 3.11 μM) cells more specifically than normal human colon epithelial cell line NCM460 (IC50 55.6 ± 4.02 μM). In COLO205 cells, DPHP induced cell shrinkage, membrane blebbing, chromatin condensation, phosphatidylserine externalization, and an accumulation of cells at sub-G1 phase. Further analysis these cells treated with DPHP revealed a decrease in mitochondrial membrane potential, an increase in Bax/Bcl2 ratio, the release of cytochrome c, activation of caspases −9, −3/7, and cleavage of the poly-ADP-ribose polymerase. DPHP treatment resulted in inhibition of hypoxia induced VEGF downstream signaling pathway in COLO205 cells is concurrent with inhibition of angiogenesis in CAM. Based on these data we suggest that DPHP significantly induced apoptosis possibly via intrinsic mitochondrial apoptosis pathway and inhibited angiogenesis. Our study suggests DPHP could be a therapeutic agent in treating colon cancer.

Overexpression of lncRNA TUSC7 reduces cell migration and invasion in colorectal cancer.

Colorectal cancer (CRC) is a highly heterogeneous disease worldwide. Long non‑coding RNA (lncRNA) tumor suppressor candidate 7 (TUSC7) plays a crucial role in the development of several cancers. However, the role of TUSC7 in the tumorigenesis of CRC has not been explored. The TUSC7‑overexpressing CRC cell lines SW480 and CaCo‑2 were generated to investigate the effects of TUSC7 on the growth, migration, invasion and epithelial‑mesenchymal transition (EMT) of CRC cells. CCK‑8, wound‑healing and Transwell assays were used to evaluate CRC cell proliferation, migration and invasion. The mRNA and protein expression of TUSC7 were detected by quantitative real‑time PCR and immunoblotting, respectively. In the present study, we observed that the expression of TUSC7 was decreased in CRC cells compared to the expression in the normal colon epithelial cell line NCM460. Moreover, overexpression of TUSC7 inhibited CRC cell proliferation, metastasis, invasion and EMT. These findings indicated that TUSC7 is involved in CRC development.

Analysis of promoter methylation and epigenetic regulation of miR-32 in colorectal cancer cells.

MicroRNA-32 (miR-32) is upregulated in colorectal cancer (CRC) tissues; its overexpression leads to increased cell proliferation, migration and invasion, as well as reduced apoptosis of CRC cells, at least partly by inhibiting the target gene phosphatase and tensin homolog. However, the mechanisms of its upregulation have remained elusive. In the present study, the effects of methylation and acetylation on the expression of miR-32 were investigated. The promoter methylation status of miR-32 in the CRC cell lines HT-29 and HCT-116 and the normal colonic epithelial cell line NCM460 was investigated by bisulfate sequencing polymerase chain reaction (BSP). The potential role of methylation and histone acetylation in the regulation of miR-32 expression in CRC cells was investigated using the demethylation reagent 5-aza-2'-deoxycytidine (5-Aza-dC), the histone deacetylase inhibitor trichostatin A (TSA) and transfection of DNA methyltransferase 1 (DNMT1) overexpression plasmid. BSP revealed that CpG sites in the miR-32 promoter region of CRC and normal colonic epithelial cells were all hypomethylated, with methylation rates of 0.12, 1.14 and 0.64% in HCT-116, HT-29 and NCM460 cells, respectively. Treatment with 5-Aza-dC and/or TSA and transfection with DNMT1 plasmid did not significantly alter the expression of miR-32. Therefore, the present results suggest that methylation and histone acetylation do not affect miR-32 expression in CRC cells.

MiR-451a suppressing BAP31 can inhibit proliferation and increase apoptosis through inducing ER stress in colorectal cancer

The global morbidity and mortality of colorectal cancer (CRC) are ranked the third among gastrointestinal tumors in the world. MiR-451a is associated with several types of cancer, including CRC. However, the roles and mechanisms of miR-451a in CRC have not been elucidated. BAP31 is a predicted target gene of miR-451a in our suppression subtractive hybridization library. Its relationship with miR-451a and function in CRC are unclear. We hypothesized that miR-451a could induce apoptosis through suppressing BAP31 in CRC. Immunohistochemistry and real-time PCR were used to measure BAP31 expressions in CRC tissues and pericarcinous tissues from 57 CRC patients and CRC cell lines. Dual-luciferase reporter assay was used to detect the binding of miR-451a to BAP31. The expression of BAP31 protein in CRC tissues was significantly higher than that in pericarcinous tissues, which was correlated with distant metastasis and advanced clinical stages of CRC patients. The expression of BAP31 was higher in HCT116, HT29, SW620, and DLD cells than that in the normal colonic epithelial cell line NCM460. The expression of BAP31 was absolutely down-regulated when over-expressing miR-451a in HCT116 and SW620 cells compared with control cells. Mir-451a inhibited the expression of BAP31 by binding to its 5’-UTR. Over-expressing miR-451a or silencing BAP31 suppressed the proliferation and apoptosis of CRC cells by increasing the expressions of endoplasmic reticulum stress (ERS)-associated proteins, including GRP78/BIP, BAX, and PERK/elF2α/ATF4/CHOP, which resulted in increased ERS, cytoplasmic calcium ion flowing, and apoptosis of CRC cells. These changes resulting from over-expressing miR-451a were reversed by over-expressing BAP31 with mutated miR-451a-binding sites. Over-expressing miR-451a or silencing BAP31 inhibited tumor growth by inducing ERS. The present study demonstrated that miR-451a can inhibit proliferation and increase apoptosis through inducing ERS by binding to the 5’-UTR of BAP31 in CRC.

The role of Parvimonas micra in intestinal tumorigenesis in germ-free and conventional APCmin/+ mice.

531 Background: Our in-house meta-analysis of fecal shotgun metagenomic sequences from colorectal cancer (CRC) and control subjects from four cohorts of various ethnicities identified a higher abundance of Parvimonas micra in CRC. We aimed to investigate the effect of P. micra in colon tumor formation and development. Methods: We collected 309 fecal samples and 259 colon biopsies from patients with CRC, advanced adenoma and healthy subjects. P. micra strain was isolated from the feces of a CRC patient. APC min/+ mice and germ-free (GF) mice were orally gavaged with P. micra, or Esherichia coli. Colon epithelial cell line NCM460 and cancer cell lines HT-29 and Caco-2 were exposed to P. micra or E. coli conditional medium. Results: P. micra was significantly enriched both in the feces (n = 207, p &lt; 0.01) and tissue biopsies (n = 99, p &lt; 0.01) of CRC patients compared with controls (n = 102 for fecal samples, n = 160 for tissues biopsies). APCmin/+ mice gavaged with P. micra exibited significantly higher tumor burden ( p &lt; 0.01) and tumor load ( p &lt; 0.01), compared to mice gavaged with either E. coli or non-bacterial control. Consistently, cell proliferation was significantly higher in the colon tissues of P. micra gavaged GF mice relative to control mice evidenced by increased Ki-67 positive cells ( p &lt; 0.05) and PCNA protein expression ( p &lt; 0.01) at weeks 20 and 32. In line with this, colon cell lines NCM460, HT-29 and Caco-2 exposed to P. micra conditional medium significantly increased proliferation than control group (all p &lt; 0.05). Flow cytometry analyses showed that Th2 and Th17 cells were markedly increased, while Th1 were reduced in the lamina propria of the colon tissues of P. micra gavaged mice compared to control mice (all p &lt; 0.01) . Consistently, P. micra colonization in GF mice was associated with increased expression of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-12 (all p &lt; 0.01). Conclusions: The abundance of P. micra was significantly increased in the feces and tissue biopsies of CRC patients. P. micra promotes intestinal carcinogenesis in APC min/+ mice and increase cell proliferation in GF mice. The tumor promoting effect of P. micra is associated with altered immune responses and enhanced inflammation in the gut.

The expression profile and clinical application potential of hsa_circ_0000711 in colorectal cancer.

IntroductionCircular RNAs, as a class of long-time-ignored non-coding RNA, have been revealed as multifunctional RNAs in recent years, especially in the cancer research. However, the mechanism of most circular RNAs and their clinical application values in human cancers remain unknown, including in colorectal cancer (CRC).MethodsIn this study, we focused on the expression pattern and clinical values of hsa_circ_0000711 in CRC. The expression level of hsa_circ_0000711 in 101 paired CRC tissues and 3 CRC cell lines (HCT116, COLO205, and H-T29), as well as human normal colon epithelial cell line NCM460, were measured by quantitative real-time polymerase chain reaction.ResultsOur results revealed that the expression level of hsa_circ_0000711 was significantly downregulated in CRC tissues (P=9.35E-16) and CRC cell lines (P<0.01). In addition, the area under the receiver characteristic curves was 0.81. The sensitivity and specificity were 0.91 and 0.58, respectively. Meanwhile, our study showed that low expression of hsa_circ_0000711 could serve as an independent prediction biomarker associated with poor overall survival of CRC patients (hazard ratio =2.409; 95% CI: 1.276–4.547; P=0.004).ConclusionAll of these results indicated that hsa_circ_0000711 may play a crucial role in CRC carcinogenesis and could be a potential effective biomarker for the diagnosis and prognosis of CRC.

Roseburia intestinalis inhibits interleukin‑17 excretion and promotes regulatory T cells differentiation in colitis.

Roseburia intestinalis (R. intestinalis) is one of the dominant intestinal bacterial microbiota and is decreased in patients with inflammatory bowel disease (IBD). It helps protect colonic mucosa against the development of inflammation and subsequent IBD, however its underlying mechanisms are unclear. The aim of the present study was to evaluate the anti-inflammatory properties of R. intestinalis in vitro and in an animal model of IBD. The effects of R. intestinalis on disease activity index (DAI) scores, intestinal pathology, the expression of interleukin (IL)-17 and the frequency of CD4+CD25+Foxp3+ regulatory T cells (Treg) were evaluated in vivo in a model of 2,4,6-trinitrobenzenesulfonic acid solution (TNBS)-induced colitis. Compared with the control group, TNBS-treated mice had significantly higher secretion of IL-17, higher DAI scores, a lower ratio of Treg, reduced colon lengths and higher histological scores for colon inflammation. The administration of R. intestinalis significantly downregulated the expression of IL-17, increased the ratio of Treg and ameliorated the high DAI scores and the pathological signs of inflammation in the colon compared with mice treated with TNBS alone. Gene expression profiling was also used to detect the expression of IL-17 in human IBD and healthy control specimens. To extend these findings to an in vitro model of inflammation the human colon epithelial cell line NCM460 was stimulated with lipopolysaccharide (LPS) to induce inflammation and co-cultured with R. intestinalis and changes in IL-17 expression were evaluated. R. intestinalis inhibited the LPS-induced secretion of IL-17 by NCM460 cells. In conclusion, these results demonstrate that R. intestinalis inhibits IL-17 secretion and promotes Treg differentiation in colitis, suggesting that R. intestinalis could be of potential use in the treatment of IBD.

MiR-186-5p upregulation inhibits proliferation, metastasis and epithelial-to-mesenchymal transition of colorectal cancer cell by targeting ZEB1

MicroRNA-186-5p (miR-186-5p) is upregulated and exhibits as a crucial oncogene in various human tumors. However, the functions and underlying mechanisms of this microRNA on colorectal cancer remain largely unknown. Here, we report that miR-186-5p share a lower expression in colorectal cancer cell lines (HT116, H29, SW620 and LoVo) than in normal colonic epithelial cell line NCM460. MiR-186-5p overexpression inhibits proliferation, metastasis and epithelial-to-mesenchymal transition (EMT) of colorectal cancer cell line LoVo. Zinc Finger E-Box Binding Homeobox 1 (ZEB1), an EMT related marker, is predicted as a target of miR-186-5p. Luciferase reporter assay, qRT-PCR and western blot analysis showed that miR-186-5p directly targeted the 3′-untranslated regions (3′UTR) of ZEB1 messenger RNA. Further functional experiments indicated that overexpression of miR-186-5p suppress the proliferation and metastasis ability of LoVo, which was consistent with the inhibitory effects by knockdown of ZEB1. Additionally, overexpression of ZEB1 could significantly reverse the miR-186-5p mimics initiated suppression impact of proliferation, metastasis and EMT on LoVo. In summary, miRNA-186-5p affects the proliferation, metastasis and EMT process of colorectal cancer cell by inhibition of ZEB1. Hence, it may serve as a promising therapeutic target for colorectal cancer.

Abstract 4849: ELMO1 promotes metastasis in colorectal cancer cells via activation of MAPK/ERK signaling pathway

Abstract Background and Aim: Engulfment and cell motility 1 (ELMO1) has been linked to the invasive phenotype of various types of cancer cells such as glioma cells, rhabdomyosarcoma cells, ovarian cancer cells and so on. However, the biological functions of ELMO1 in colorectal cancer (CRC) cells remains unclear. The aim of this study is to evaluate the role of ELMO1 in the metastasis in CRC. Methods: The expression level of ELMO1 was detected using quantitative RT-PCR and western blot in 9 CRC cell lines and 1 human colon epithelial cell line NCM460. ELMO1 was overexpressed via lentiviral vector system and silenced using specific small interference RNA (si-ELMO1) in HCT116 and SW620. Then wound-healing, migration assay and invasion assay were carried out to investigate the function of ELMO1 in CRC cells. Western blot was performed to detect the change of signaling molecule extracellular signal-regulated kinase 1/2 (ERK1/2) following the down-regulated or up-regulated expression of ELMO1. Results: ELMO1 expression was inhibited by si-ELMO1 and was overexpressed by ELMO1-cDNA in HCT116 and SW620. Ectopic expression of ELMO1 in HCT116 and SW620 was shown to promote migration and invasion, while inhibited ELMO1 expression suppressed the capacity of cell migration and invasion. Furthermore, ELMO1 induced migration and invasion in CRC cells was found to be associated with the activation of MAPK/ERK pathway. Conclusions: High expression of ELMO1 was found to be associated with the metastasis via the activation of MAPK/ERK signaling pathway. This finding provides a molecular basis for the role of ELMO1 in the progression of CRC, which may suggest a novel target for the treatment of CRC. Citation Format: Xiao-bin Zheng, Chi Zhou, Hai-chun Cheng, Tuo Hu, Hua-shan Liu, Xuan-hui Liu, Xian-rui Wu, Feng-wei Wang, Yu-feng Chen, Jian-ping Wang, Xiao-sheng He, Ping Lan. ELMO1 promotes metastasis in colorectal cancer cells via activation of MAPK/ERK signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4849. doi:10.1158/1538-7445.AM2017-4849

Decrease of miR-622 expression suppresses migration and invasion by targeting regulation of DYRK2 in colorectal cancer cells.

BackgroundMore and more evidence indicates that microRNAs are present and involved in many tumor-related diseases. The function of microRNA-622 (miR-622) in colorectal cancer (CRC) remains controversial. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) has been reported as a tumor suppressor gene in different cancers. The detailed regulation mechanism of DYRK2 in CRC remains unclear.MethodsmiR-622 and DYRK2 expression levels were detected at tissue and cellular level respectively by using real time polymerase chain reaction (PCR), Western blot, and immunohistochemical staining. Pearson’s correlation analysis was used to evaluate the correlation between miR-622 and DYRK2. Transwell assay was applied to measure the effect of miR-622 on migration and invasion of SW1116 and SW480. We used dual luciferase reporter assay to confirm the targeted binding effect of miR-622 and DYRK2 3′-untranslated region (3′UTR). An antisense experiment was executed to further confirm the role miR-622 had played with regard to migration and invasion by targeting regulation of DYRK2 pathway in CRC cells.ResultsIn our research, we found that the expression of miR-622 was elevated in CRC tissues and cell lines compared to that of nonCRC tissues and the normal human colon epithelial cell line NCM460. Correspondingly, the expression of DYRK2 in CRC tissues and cell lines showed a contrary tendency. The different expression level of DYRK2 was closely correlated with clinicopathological characteristics of CRC patients. We demonstrated that down-regulation of miR-622 could inhibit the ability of migration and invasion of CRC cell lines SW1116 and SW480. Also, we confirmed that DYRK2 was negatively regulated by miR-622 via a specific targeted binding site within the 3′UTR. We finally verified that the migration and invasion ability of CRC cells in the conducted DYRK2 3′UTR defect plasmid transfection group were lower compared to miR-622 and cotransfection group.ConclusionThe findings of this study indicate that a decrease of miR-622 expression could suppress migration and invasion by targeting regulation of DYRK2 and miR-622/DYRK2 could be a potential molecular treating target of CRC.

Peptostreptococcus anaerobius Induces Intracellular Cholesterol Biosynthesis in Colon Cells to Induce Proliferation and Causes Dysplasia in Mice

Stool samples from patients with colorectal cancer (CRC) have a higher abundance of Peptostreptococcus anaerobius than stool from individuals without CRC, based on metagenome sequencing. We investigated whether P anaerobius contributes to colon tumor formation in mice and its possible mechanisms of carcinogenesis. We performed quantitative polymerase chain reaction analyses to measure P anaerobius in 112 stool samples and 255 colon biopsies from patients with CRC or advanced adenoma and from healthy individuals (controls) undergoing colonoscopy examination at hospitals in Hong Kong and Beijing. C57BL/6 mice were given broad-spectrum antibiotics, followed by a single dose of azoxymethane, to induce colon tumor formation. Three days later, mice were given P anaerobius or Esherichia coli MG1655 (control bacteria), via gavage, for 6 weeks. Some mice were also given the nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin. Intestine tissues were collected and analyzed histologically. The colon epithelial cell line NCM460 and colon cancer cell lines HT-29 and Caco-2 were exposed to P anaerobius or control bacteria; cells were analyzed by immunoblot, proliferation, and bacterial attachment analyses and compared in gene expression profiling studies. Gene expression was knocked down in these cell lines with small interfering RNAs. P anaerobius was significantly enriched in stool samples from patients with CRC and in biopsies from patients with colorectal adenoma or CRC compared with controls. Mice depleted of bacteria and exposed to azoxymethane and P anaerobius had a higher incidence of intestinal dysplasia (63%) compared with mice not given the bacteria (8.3%; P < .01). P anaerobius mainly colonized the colon compared with the rest of the intestine. Colon cells exposed to P anaerobius had significantly higher levels of proliferation than control cells. We found genes that regulate cholesterol biosynthesis, Toll-like receptor (TLR) signaling, and AMP-activated protein kinase signaling to be significantly up-regulated in cells exposed to P anaerobius. Total cholesterol levels were significantly increased in colon cell lines exposed to P anaerobius via activation of sterol regulatory element-binding protein 2. P anaerobius interacted with TLR2 and TLR4 to increase intracellular levels of reactive oxidative species, which promoted cholesterol synthesis and cell proliferation. Depletion of reactive oxidative species by knockdown of TLR2 or TLR4, or incubation of cells with an antioxidant, prevented P anaerobius from inducing cholesterol biosynthesis and proliferation. Levels of P anaerobius are increased in human colon tumor tissues and adenomas compared with non-tumor tissues; this bacteria increases colon dysplasia in a mouse model of CRC. Panaerobius interacts with TLR2 and TLR4 on colon cells to increase levels of reactive oxidative species, which promotes cholesterol synthesis and cell proliferation.

Phenotype transformation of immortalized NCM460 colon epithelial cell line by TGF-β1 is associated with chromosome instability.

Transforming growth factor-β1 (TGF-β1) within tumor microenvironment has a pivotal function in cancer initiation and tumorigenesis, and hence this study was to observe the malignant transformation induced by TGF-β1 in an immortalized colon epithelial cell line NCM460 for better understanding the mechanisms of colon carcinogenesis. Immortalized colon epithelial cell line NCM460 was used as the model of this study, and was treated with different concentrations of TGF-β1 for different time. Then, immunofluorescence was performed to observe the change of phenotype hallmarks including adherent junction protein E-cadherin, cytoskeleton protein vimentin, and tight junction marker ZO-1, western blotting analysis was performed to detect the expression of the above three markers and two transcription factors (Snail and Slug) involved in the transformation by TGF-β1. In addition, chromosome instability (CHI) including analysis of DNA-ploid was detected by flow cytometry. Our results revealed significant loss or reduction of ZO-1 and E-cadherin, and robust emergence of vimentin in the cell line NCM460 after a 15-, 20-, and 25-day treatment with 10ng/ml TGF-β1. Interestingly, 20 and 25days after stimulation with 5ng/ml TGF-β1, expression of E-cadherin and ZO-1 revealed a pattern roughly similar to that of 10ng/ml TGF-β1, especially, both expressions was vanished and vimentin expression was dramatically increased at days 25 after TGF-β1 stimulation. After a stimulation with 10ng/ml TGF-β1 for 15, 20, and 25days, the levels of Snail and Slug expression in the cells were significantly up-regulated, compared with the cells treated with TGF-β1 inhibitor LY364947, PBS or balnk control (P<0.01). Our results found that many abnormal mitotic patterns including lagging chromosomes and anaphase bridges in NCM460 cells were induced by TGF-β1 after its stimulation for 15, 20, and 25days. Very few mitotic cells with treatment of PBS for 15, 20 and 25days were non-diploid whose DNA content was greater or less than 4N, but these cells were significantly increased after exposure to TGF-β1 for 15, 20, and 25days, which was associated with the induction of hypo-diploid, hyper-diploid, and poly-diploid (P<0.05).These data indicate that TGF-β1 induces a phenotypic transformation of normal colon epithelium similar to its pro-tumoral behaviors in TME, involving in alteration of chromosome stability.

A serine protease extracted from Trichosanthes kirilowii induces apoptosis via the PI3K/AKT-mediated mitochondrial pathway in human colorectal adenocarcinoma cells.

Trichosanthes kirilowii exhibits various biological functions including anti-inflammatory, antidiabetic and anticancer activities. In this study, a novel protein with anti-cancer activity, named as TKP, was purified from Trichosanthes kirilowii fruit by cell-base screening. Mass spectrometry and protease assays revealed that TKP is a serine protease. TKP decreased cell viability in a concentration-and time-dependent manner in human colorectal adenocarcinoma cells. Notably, TKP presented very little effect on human colon epithelial cell line NCM460. Furthermore, TKP inhibited colorectal adenocarcinoma cells to aggregate into viable colony clusters and induced cell apoptosis. The loss of mitochondrial membrane potential, upregulation of cytochrome c and Bax, downregulation of Bcl-2, and activation of caspase-9 and -3 were observed, indicating mitochondria-dependent apoptosis induced by TKP. We found that TKP activated MAPK/ERK and suppressed PI3K/AKT signaling. Inhibition of MAPK/ERK signaling by employing inhibitor PD98059 did not antagonize the effects of TKP. Treatment with PI3K inhibitor LY294003 increased the impact of TKP on mitochondria-related apoptosis proteins (cytochrome c, Bcl-2, Bax, caspase-9 and caspase-3) and cell apoptosis. On the contrary, PI3K/AKT activator insulin-like growth factor-1 reversed the effects of TKP. Taken together, our results demonstrated that TKP has potential anti-colorectal cancer activity by inducing apoptosis, which was regulated by the PI3K/AKT-mediated mitochondria-dependent pathway.

Abstract 2322: β-catenin-dependent TCF/LEF transcriptional regulation of phosphodiesterase expression in colon cancer cells

Abstract Cyclic nucleotide phosphodiesterases (PDE) regulate cyclic nucleotides, cAMP and cGMP, blocking signaling by hydrolyzing the 3′,5′-phosphodiester bond resulting in their degradation to inactive forms. We have previously reported that certain PDE isozymes (e.g. PDE5) are elevated in several types of cancer, which can disrupt cyclic nucleotide signaling to suppress apoptosis. The inhibition of PDE5 by agents such as sulindac sulfide (SS) results in increased cGMP levels and reduced β-catenin/TCF/LEF mediated transcription leading to the selective inhibition of colon cancer cell proliferation and induction of apoptosis. Currently, little is known regarding the transcriptional regulation of PDE5 in cancer cells. We therefore analyzed the promoter regions of PDE5, as well as PDE10, for transcription factor binding sites using the Matinspector Genomatix Software. Matinspector analysis predicted that both PDE5 and PDE10 promoters contain binding sites for TCF/LEF-1, an important transcriptional factor that is regulated by Wnt/β-catenin signaling, which plays an important role in colorectal cancer. To determine if Wnt/β-catenin signaling can regulate PDE5 and PDE10 expression, human HCT116 colon tumor cells and normal colonic epithelial cell line NCM-460 were treated with the GSK-3β inhibitor, lithium chloride (LiCl) to inhibit β-catenin degradation and stimulate TCF/LEF transcriptional activity. TCF/LEF transcriptional activity was measured by mRNA levels of genes (e.g. survivin, cyclin D1 and c-myc) that are known to be regulated by Wnt/β-catenin signaling. Survivin, cyclin D1 and c-myc mRNA were confirmed to be significantly increased after LiCl treatment in comparison to control cells treated with NaCl. Translocation of β-catenin to the nucleus was also evident after treatment with LiCl. Levels of PDE5 and 10 mRNA were found to be significantly elevated after LiCl treatment in comparison to NaCl treated cells. Inhibitors such as SS, the novel derivative SS-D1 and a tadalafil (a selective PDE5 inhibitor) derivative TA-D1 demonstrated selective inhibition of colon cancer cell growth in comparison to normal colonocytes. The potential mechanism of these selective inhibitors may be through the inhibition of Wnt/β-catenin signaling and thus the inhibition of β-catenin/TCF/LEF mediated transcription. This study demonstrates a novel regulatory mechanism of PDE5 and PDE10 expression by the β-catenin-dependent TCF/LEF transcription factor in colon cancer cells and identifies a potential mechanism for novel chemopreventive agents. Defining the role of this regulatory mechanism in cancer progression may provide valuable insight into targeting PDE5 and/or PDE10 by small molecules for cancer chemoprevention. Citation Format: Alexandra M. Fajardo, Bernard D. Gary, Kevin J. Lee, Adam B. Keeton, Bing Zhu, Xi Chen, Ashraf H. Abadi, Gary A. Piazza. β-catenin-dependent TCF/LEF transcriptional regulation of phosphodiesterase expression in colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2322. doi:10.1158/1538-7445.AM2014-2322