Normal Colorectal Epithelial Cells Research Articles

Bioinformatics analysis and identification of cuproptosis-related long non-coding RNAs in colorectal cancer.

Identifying precise biomarkers for colorectal cancer (CRC) detection and management remains challenging. Here, we developed an innovative prognostic model for CRC using cuproptosis-related long non-coding RNAs (lncRNAs). In this retrospective study, CRC patient transcriptomic and clinical data were sourced from The Cancer Genome Atlas database. Cuproptosis-related lncRNAs were identified and used to develop a prognostic model, which helped categorize patients into high- and low-risk groups. The model was validated through survival analysis, risk curves, independent prognostic analysis, receiver operating characteristic curve analysis, decision curves, and nomograms. In addition, we performed various immune-related analyses. LncRNA expression levels were examined in normal human colorectal epithelial cells (FHC) and CRC cells (HCT-116) using quantitative polymerase chain reaction (qPCR). Six cuproptosis-related lncRNAs were identified: ZKSCAN2-DT, AL161729.4, AC016394.1, AC007128.2, AL137782.1, and AC099850.3. The prognostic model distinguished between high-/low-risk populations, demonstrating excellent predictive ability for survival outcomes. Immunocorrelation analysis showed significant differences in immune cell infiltration and functions, immune checkpoint expression, and m6A methylation-related genes. The qPCR results showed significant upregulation of ZKSCAN2-DT, AL161729.4, AC016394.1, AC007128.2 in HCT-116 cells, while AL137782.1 and AC099850.3 expression patterns were significantly downregulated. Cuproptosis-related lncRNAs can potentially serve as reliable diagnostic and prognostic biomarkers for CRC.

Expression and Prognosis of Differential Gene Troponin T1 Between Right and Left Colon Cancers.

Colorectal cancer (CRC) is one of the most common digestive tract tumors in humans. At present, many scholars believe that the primary site of the tumor has a direct and profound impact on its curative effect. There are significant differences in the expression of many genes, tumor microenvironment, and prognosis between the left and right colon. However, there is a lack of detailed studies on whether the differentially expressed genes in the left and right colon significantly impact the prognosis of patients with CRC. Troponin T1 ( TNNT1 ) is an important gene that affects the prognosis difference between left and right colon cancer screening from "The Cancer Genome Atlas" database. By analyzing the differential gene expression data and clinical data of the left and right hemicolons in the database, the online prognostic database was used to screen the key molecules that significantly affect the tumor immune microenvironment and patient prognosis and to predict their functions and pathways. Quantitative reverse transcription-polymerase chain reaction was used to verify the expression difference of TNNT1 in CRC cell lines SW480 and HCT116, and normal human colorectal epithelial cell line FHC. The relationship between TNNT1 expression in 88 pairs of CRC samples and clinical information and pathologic parameters of patients with CRC was analyzed to judge the impact of TNNT1 expression on patient survival. Database analysis showed that TNNT1 was significantly overexpressed in CRC, and TNNT1 was one of the main differential genes between left colon cancer (LCC) and right colon cancer (RCC). The expression of TNNT1 was significantly increased in RCC, which could lead to poor prognosis of patients. Quantitative reverse transcription-polymerase chain reaction indicated that the expression of TNNT1 was significantly up-regulated in CRC cell lines SW480 and HCT116. Eighty-eight immunohistochemistry (IHC) of CRC tissues and adjacent tissues suggested that the expression of TNNT1 in CRC was significantly higher than that in normal adjacent tissues. By analyzing the clinical information and pathologic indicators matched with these clinical samples, we found that high TNNT1 expression in the primary tumor location (right colon) and high N stage (N2, N3) were unfavorable factors affecting the prognosis of patients with CRC. Multivariate Cox regression analysis suggested that high expression of TNNT1 may be an independent risk factor for the prognosis of patients with CRC. As one of the main differential genes between LCC and RCC, TNNT1 is representative to some extent. Its high expression may be one of the reasons why the prognosis of patients with RCC is worse than that of patients with LCC.

Targeting PTGES/PGE2 axis enhances sensitivity of colorectal cancer cells to 5-fluorouracil.

Insensitivity and resistance to 5-fluorouracil (5FU) remain as major hurdles for effective and durable 5FU-based chemotherapy in colorectal cancer (CRC) patients. In this study, we identified prostaglandin E synthase (PTGES)/prostaglandin E2 (PGE2) axis as an important regulator for 5FU sensitivity in CRC cells. We found that PTGES expression and PGE2 production are elevated in CRC cells in comparison to normal colorectal epithelial cells. Depletion of PTGES significantly enhanced the inhibitory effect of 5FU on CRC cell viability that was fully reverted by exogenous supplement of PGE2. Inhibition of PTGES enzymatic function, by either inducing loss-of-function mutant or treatment with selective inhibitors, phenocopied the PTGES depletion in terms of 5FU sensitization. Mechanistically, PTGES/PGE2 axis modulates glycolysis in CRC cells, thereby regulating the 5FU sensitivity. Importantly, high PTGES expression is correlated with poor prognosis in 5FU-treated CRC patients. Thus, our study defines PTGES/PGE2 axis as a novel therapeutic target for enhancing the efficacy of 5FU-based chemotherapy in CRC.

Inhibition of the bone morphogenetic protein pathway suppresses tumor growth through downregulation of epidermal growth factor receptor in MEK/ERK-dependent colorectal cancer.

The bone morphogenetic protein (BMP) pathway promotes differentiation and induces apoptosis in normal colorectal epithelial cells. However, its role in colorectal cancer (CRC) is controversial, where it can act as context-dependent tumor promoter or tumor suppressor. Here we have found that CRC cells reside in a BMP-rich environment based on curation of two publicly available RNA-sequencing databases. Suppression of BMP using a specific BMP inhibitor, LDN193189, suppresses the growth of select CRC organoids. Colorectal cancer organoids treated with LDN193189 showed a decrease in epidermal growth factor receptor, which was mediated by protein degradation induced by leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) expression. Among 18 molecularly characterized CRC organoids, suppression of growth by BMP inhibition correlated with induction of LRIG1 gene expression. Notably, knockdown of LRIG1 in organoids diminished the growth-suppressive effect of LDN193189. Furthermore, in CRC organoids, which are susceptible to growth suppression by LDN193189, simultaneous treatment with LDN193189 and trametinib, an FDA-approved MEK inhibitor, resulted in cooperative growth inhibition both in vitro and in vivo. Taken together, the simultaneous inhibition of BMP and MEK could be a novel treatment option in CRC cases, and evaluating in vitro growth suppression and LRIG1 induction by BMP inhibition using patient-derived organoids could offer functional biomarkers for predicting potential responders to this regimen.

Integrative single-cell multiomics analyses dissect molecular signatures of intratumoral heterogeneities and differentiation states of human gastric cancer.

Human gastric cancer is a highly lethal disease, but the underlying multiomic molecular signatures remain largely unclear. Here, we performed multi-regional sampling, parallel single-cell multiomics sequencing and integrated analyses of human gastric cancer. We identified common transcriptomic alterations of gastric cancer cells, such as aberrant down-regulation of genes associated with normal stomach function and up-regulation of KRT7, PI3, S100A4, etc. Surprisingly, aberrant and prevalent up-regulation of genes highly expressed in normal colorectal epithelial cells were also identified in cancer cells, which may be partially regulated by promoter chromatin accessibility and DNA methylation levels. We revealed the single-cell DNA methylome landscape of gastric cancer, and identified candidate DNA methylation biomarkers, such as hypermethylated promoters of TMEM240 and HAGLROS, and hypomethylated promoters of TRPM2-AS and HRH1. Additionally, the relationships between genetic lineages, DNA methylation and transcriptomic clusters were systematically revealed at single-cell level. We showed that DNA methylation heterogeneities were mainly among different genetic lineages of cancer cells. Moreover, we found that DNA methylation levels of cancer cells with poorer differentiation states tend to be higher than those of cancer cells with better differentiation states in the primary tumor within the same patient, although still lower than in normal gastric epithelial cells. Cancer cells with poorer differentiation states also prevalently down-regulated MUC1 expression and immune-related pathways, and had poor infiltration of CD8+ T cells. Our study dissected the molecular signatures of intratumoral heterogeneities and differentiation states of human gastric cancer using integrative single-cell multiomics analyses.

Bromodomain-containing protein 9 activates proliferation and epithelial-mesenchymal transition of colorectal cancer via the estrogen pathway in vivo and in vitro.

Bromodomain-containing protein 9 (BRD9) has been reported to be upregulated in multiple malignancies and facilitate cancer progression. However, there is a paucity of data relating to its expression and biological role in colorectal cancer (CRC). Therefore, this current study examined the prognostic role of BRD9 in CRC and the underlying mechanisms involved. Real-time polymerase chain reaction (PCR) and Western blotting were used to examine the expression of BRD9 in paired fresh CRC and para-tumor tissues from colectomy patients (n=31). Immunohistochemistry (IHC) was performed to assess BRD9 expression in 524 paraffin-embedded archived CRC samples. The clinical variables are including age, sex, carcinoembryonic antigen (CEA), location of tumor, T stage, N stage, and TNM classification. The effect of BRD9 on the prognosis of CRC patients was explored by Kaplan-Meier and Cox regression analyses. Cell counting kit 8 (CCK-8), clone formation assay, transwell assay, and flow cytometry were used to determine CRC cell proliferation, migration, invasion, and apoptosis, respectively. Xenograft models in nude mice were established to investigate the role of the BRD9 in vivo. BRD9 mRNA and protein expression levels were significantly upregulated in CRC cells compared to normal colorectal epithelial cells (P<0.001). IHC analysis of 524 paraffin-embedded archived CRC tissues showed that high BRD9 expression was significantly associated with TNM classifications, CEA, and lymphatic invasion (P<0.01). Univariate and multivariate analyses indicated that BRD9 [hazard ratio (HR): 3.04, 95% confidence interval (CI): 1.78-5.20; P<0.01] expression and sex (HR: 6.39, 95% CI: 3.94-10.37; P<0.01) were independent prognostic factors for overall survival in the entire cohort. Overexpressing BRD9 promoted CRC cell proliferation, while silencing BRD9 inhibited the proliferation of CRC cells. Furthermore, we showed that BRD9 silencing significantly inhibited epithelial-mesenchymal transition (EMT) via the estrogen pathway. Finally, we demonstrated that silencing BRD9 significantly inhibited the proliferation and tumorigenicity of SW480 and HCT116 cells in vitro and in vivo in nude mice (P<0.05). This study demonstrated that BRD9 high could be an independent prognostic risk factor for CRC. Furthermore, the BRD9/estrogen pathway may contribute to the proliferation of CRC cells and EMT, suggesting that BRD9 may be a novel molecular target in the therapeutic treatment of CRC.

Effects of Long Noncoding RNA AK093407 on the Biological Behavior of Colon Cancer Cells and the Underlying Mechanism.

The incidence of colorectal cancer is steadily increasing, and the detection of related molecular targets is critical for its diagnosis and treatment. Long noncoding RNA (lncRNA) can play a regulatory role before and after genome transcription, and epigenetic regulation is involved in the process of tumorigenesis and tumor development. In this study, qRT-PCR was performed to evaluate the expression of AK093407 in colon cancer and colon para-carcinoma tissues and HCT-15 and HCT-116 cells. SiRNA was transfected into HCT-15 and HCT-116 cells to knock down lncRNA-AK093407. Then, MTT assay was used to test cell proliferation, and flow cytometry was used to test apoptosis and cell cycle. The protein expression of caspase-3, caspase-8, caspase-9, bax, bcl-2, cyclin-A1, cyclin-B1, cyclin-D1, cyclin- E1, p21, p27, and p-Stat3 was determined by Western blot. The results showed that the expression of AK093407 in human colon cancer tissue was higher than in para-carcinoma tissue. The amount of AK093407 in HCT-15 and HCT-116 cells was higher than that in normal colorectal epithelial NM460 cells. When AK093407 was silenced, the proliferation of HCT-15 and HCT-116 cells decreased, the apoptosis rate increased, the cell cycle was arrested in the G1/S phase, the expression of caspase-3, caspase-8, caspase-9, bax, cyclin-A1, cyclin- B1, p21, p27 increased, and the expression of bcl-2, cyclin-D1, cyclin-E1, p-Stat3 decreased. These results showed that knockdown of AK093407 could inhibit colon cancer cell proliferation, induce apoptosis and cell cycle arrest, influence the expression of vital factors in mitochondrial apoptosis pathway and cell cycle regulatory pathway, and may negatively regulate JAK/STAT3 through down-regulating p-Stat3.

Inhibition of DNAJC12 Inhibited Tumorigenesis of Rectal Cancer via Downregulating HSPA4 Expression.

Background Dysregulation of DnaJ heat shock protein family (HSP40) member C12 (DNAJC12) is implicated in the malignancy progression of multiple cancers. The current study aimed to determine the biology function and mechanism of DNAJC12 in rectal cancer (RC). Methods RC tissues, adjacent tissues, RC cell lines, and normal colorectal epithelial cell lines were collected to analyze DNAJC12 expression. The abilities of DNAJC12 on proliferation, migration, and apoptosis of RC cells were detected by CCK-8, wound healing, and flow cytometry assays. Co-IP assays were carried out to confirm the association between DNAJC12 and HSPA4. The effect of DNAJC12 on tumor growth was detected by using the xenograft model of nude mice. Results Elevation of DNAJC12 was uncovered in RC tissues and cell lines. DNAJC12 upregulation facilitated RC cell proliferation and migration and induced apoptosis, while DNAJC12 interference showed the opposite results. Besides, HSAP4 served as a potential binding protein for DNAJC12. Rescue experiments revealed that elevated of HSAP4 restored the impact of DNAJC12 silencing on the cell functions. Finally, DNAJC12 silencing hampered tumor growth of RC in vivo. Conclusion In summary, this study highlighted a key player of DNAJC12 in modulating the malignant biological progression of RC via DNAJC12/HSPA4 axis, displaying a potential therapeutic target for RC.

Freeze-dried curcumin-loaded poly(lactic-co-glycolic acid) nanoparticles restrain colorectal cancer progression by targeting the notch pathway to regulate epithelial-mesenchymal transition

Despite advances in technology and treatment, colorectal cancer (CRC) remains a primary cause of cancer death worldwide, causing nearly 694,000 deaths annually with a high mortality among metastatic cases. Therefore, there is urgency to develop effective strategies to control metastasis or restrain its initiating stage and epithelial-mesenchymal transition (EMT). Activation of Notch1 is noted to positively induce cancer cell growth when inducing EMT. Curcumin is a polyphenol with anticancer activity, which works by activating various molecular pathways in cancer cells. Herein, we established curcumin-Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (Cur-NPs) and investigated their physicochemical properties in CRC to provide a novel insight into treatment of CRC. Cur-NPs were established by emulsion solvent evaporation. CRC cells (HCT116, HT29) were co-cultured with 0, 1, 2.5, 5, and 10 μM Cur-NPs, respectively. After treatment, clone formation assay determined CRC cell viability, migration and invasion, whilst Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) assessed cell apoptosis. Besides, EMT markers levels in each group were detected to evaluate the impact of Cur-NPs on the EMT process. The transmission electron microscopy (TEM) pictures depicted classical features of Cur-NPs, with encapsulation efficiency of Cur-NPs 70.685±2.597%, and drug loading rate of 2.831±0.108%. The CRC cells absorbed the Cur-NPs within 30 min, and the absorption was as early as 10 min, while fluorescence reached climax at 30 min. The advent of Cur-NPs at different concentration greatly suppressed CRC cell viability and hindered their clone formation in HCT116 and HT29 cells, but elevated apoptotic rate of cancer cells with concentration of Cur-NPs proportional to the rate, while the apoptosis of normal colorectal epithelial cells was not affected. 10 μM Cur-NPs group had the highest apoptosis rate and poorest invasion and migration ability. Moreover, upon treatment with Cur-NPs, E-cadherin expression increased, but the proteins in the Notch pathway declined dramatically. Cur-NPs suppressed CRC cell progression through regulating the Notch signaling pathway and decelerating the EMT process of CRC cells.

Discovery of novel conjugates of quinoline and thiazolidinone urea as potential anti-colorectal cancer agent

Based on the obtained SARs, further structural optimisation of compound BC2021-104511-15i was conducted in this investigation, and totally ten novel quinoline derivates were designed, synthesised and optimised for biological activity. Among them, compound 10a displayed significant in vitro anticancer activity against COLO 205 cells with an IC50 value of 0.11 μM which was over 90-fold more potent than that of Regorafenib (IC50>10.0 μM) and Fruquintinib (IC50>10.0 μM). Furthermore, compound 10a exhibited over 90-fold selectivity towards COLO 205 relative to human normal colorectal mucosa epithelial cell FHC cells. Flow cytometry study demonstrated that compound 10a could induce apoptosis in COLO 205 cells, however, it could not induce cell cycle arrest in COLO 205 cells. The results of preliminary kinase profile study showed that compound 10a was a potential HGFR and MST1R dual inhibitor, with IC50 values of 0.11 μM and 0.045 μM, respectively.

Abstract PL02-01: The pathogenesis of cancer susceptibility due to inherited DNA repair defects

Abstract The landscapes of somatic mutation in the genomes of normal human cells have, until recently, been poorly described compared with those of cancer cells. However, advances in technology are now changing this picture and are enabling exploration of the earliest stages of cancer development. In this talk, the patterns of somatic mutation found in normal colorectal epithelial cells are outlined and serve as a backdrop to understanding the pathogenesis of colorectal cancer susceptibility syndromes. Multiple mutational signatures are present in normal colorectal epithelial cells indicating that multiple mutational processes are operative in them during the course of life. Some of these mutational signatures are ubiquitous, present in every colorectal epithelial cell, with mutations generated at constant rates throughout life. Others are sporadic, found only in some cells or in some people, with mutations generated in bursts. Some are of endogenous origins whereas others are caused by exogenous exposures. Several inherited colorectal cancer predisposition syndromes are known to be mediated by DNA repair deficiencies that ultimately generate elevated somatic mutation rates. The results presented here show that in some of these syndromes all normal colorectal cells, and probably all cells in the body, have markedly elevated somatic mutation rates throughout life with mutational signatures not found in healthy individuals. Conversely, in other syndromes normal cells have normal mutation rates with standard mutational signatures but a small minority of cells acquire elevated mutation rates during the course of life. In both scenarios only a small proportion of cells with elevated mutation rates ever become adenomas or carcinomas. The results elucidate the different ways in which elevated somatic mutation rates due to DNA repair deficiencies lead to cancer and potentially offer a new modality contributing to clinical management of colorectal cancer susceptibility. They also provide a new perspective on the role of somatic mutations in ageing. Citation Format: Michael R. Stratton. The pathogenesis of cancer susceptibility due to inherited DNA repair defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr PL02-01.

Tumor-derived immunoglobulin like transcript 5 induces suppressive immunocyte infiltration in colorectal cancer.

Infiltration of immunosuppressive cells in the tumor microenvironment (TME) induced colorectal cancer (CRC) progression and its resistance to immunotherapy. Identification of tumor‐specific factors to modulate inhibitory immunocyte infiltration would provide alternative and novel targets for CRC immunotherapy. Immunoglobulin‐like transcript (ILT) 5 is a negative regulator of myeloid cell activation. However, its expression and functional role in solid tumors is still unknown. Using human CRC tissues and cell lines, we found that ILT5 was highly expressed in CRC cells compared with normal colorectal epithelial cells. Enriched ILT5 in tumor cells was correlated with advanced tumor stages and poor patient survival. Our subsequent in vitro and in vivo studies revealed that tumor‐derived ILT5 inhibited the infiltration of T cells, especially that of CD8+ T cells in the TME, creating suppressive T‐cell contexture. Furthermore, ILT5 directed M2‐like polarization of tumor‐associated macrophages (TAMs). Inhibition of tumor‐derived ILT5 restored the immunosuppressive T‐cell and TAM contexture, and restricted CRC progression. Our findings identified ILT5 expression in solid tumor cells for the first time and raised ILT5 as a potential immunotarget and prognostic predictor in CRC.

Curcumin-Coated Poly(lactic-co-glycolic acid) Nanoparticles Affect Colorectal Cancer Cells Growth by Regulating Notch Signaling Pathway

Colorectal cancer (CRC) is a common malignant tumor worldwide, and its incidence increases significantly, causing nearly 694,000 deaths annually worldwide. Previous data have shown that Notch signaling is involved in regulating tumor cell proliferation. Curcumin is a polyphenol that has anticancer activity by activating various molecular pathways in cancer cells. In this study, curcumin was combined in PLGA nanomembrane to explore the therapeutic effect of curcumin coated PLGA nanoparticles (CUR-NPs) on CRC, in providing novel ideas for CRC management. CUR-NPs were in this study manufactured, and different concentrations of CUR-NPs were used to treat CRC cells and normal epithelial cells. Anticancer effect of CUR-NPs was evaluated by analyzing the effects of different concentrations of CUR-NPs on invasion, migration and apoptosis of cells. Finally, the relationship between CUR-NPs and Notch signaling pathway was analyzed to determine its mechanism. Results from this study showed that, the CUR-NPs suppressed motility, migration and invasion of CRC cells by inhibiting Notch signaling pathway, and enhanced apoptosis. In addition, it was found that the CUR-NPs were easily absorbed by cancer cells, and inhibited cancer cell viability, colony formation, and EMT processes and induced apoptosis of cancer cells without affecting the normal colorectal epithelial cells. Our study suggests that the CUR-NPs suppressed the CRC cells’ growth and motility apoptosis by inhibiting the Notch signaling without affecting normal colorectal epithelial cells, which provides novel ideas in CRC management.

A "conversion-deterioration-double mutation" theory for the evolution and progression of colorectal cancer.

In this study, based on some clinical phenomena and recently published knowledge, we proposed our “conversion‐deterioration‐double mutation” theory, which provides a possible unifying explanation for the evolutionary process of colorectal cancer cells in the human body. In this theory, we proposed that there is a partial interconversion and a jump conversion relationship among normal colorectal epithelial cells, colorectal cancer cells, stem cells, and cancer stem cells (conversion). This conversion leads to tumor heterogeneity. We also proposed that well‐differentiated cancer cells converted from cancer stem cells have a more aggressive pattern than primary cancer cells (deterioration). The deterioration of primary cancer cells leads to differences in treatment responses and prognosis. Finally, we speculate a double mutation theory, indicating that for metastasis to occur, both mutations of cancer cells and mutations of target organs are needed and should match and meet. All these three points constitute the “conversion‐deterioration‐double mutation” theory.

Induction of DNA Damage in Mouse Colorectum by Administration of Colibactin-producing Escherichia coli, Isolated from a Patient With Colorectal Cancer.

Among colorectal cancer-associated intestinal microbiota, colibactin-producing (clb+) bacteria are attracting attention. We aimed to clarify the interaction between clb+ Escherichia coli and normal colorectal epithelial cells in vivo and in vitro. Five-week-old female Balb/c mice were divided in an untreated group, a group treated with clb+ E. coli isolated from a Japanese patient with colorectal cancer (E. coli-50), and a group treated with non colibactin-producing E. coli (E. coli-50/ΔclbP). Mice were sacrificed at 18 weeks of treatment. Treatment with clb+ E. coli increased positivity for H2A histone family member X phosphorylated at Ser-139 (γH2AX) in epithelial cells of the luminal surface of the mouse rectum but this did not occur in the E. coli-50/ΔclbP and untreated groups. In an in vitro setting, the ratio of apoptotic cells was increased and cell counts were reduced by treatment with clb+ E. coli more than in untreated cells and normal rat colorectal epithelial cells. E. coli-50 induced DNA damage in the mouse rectum, possibly by direct interaction between clb+ E. coli and normal colorectal epithelial cells. Our findings imply that regulation of clb+ E. coli infection may be a useful strategy for colorectal cancer control.

Long Non-Coding RNA CCAT2 Activates RAB14 and Acts as an Oncogene in Colorectal Cancer.

Here, we investigated the clinicopathological and prognostic potential of the long noncoding RNA Colon Cancer-Associated Transcript 2 (CCAT2) in human colorectal cancer (CRC). We used qPCR to quantify CCAT2 levels in 44 pairs of CRC tissues and adjacent nontumor and healthy colon mucosa tissues, and in several CRC cell lines (SW620, SW480, HT-29, LOVO, HCT116 and DLD-1) and normal human colorectal epithelial cells (HFC). We assessed the effects of CCAT2 overexpression or knockdown on the proliferation, migration and invasion by SW620 and LOVO cells using CCK-8, transwell, and wound−healing assays, respectively. We also investigated the potential interaction between CCAT2 and TAF15 through RNA pull down and rescue experiments. Lastly, we evaluated the expression of the cell cycle progression markers and GSK3β signaling pathway proteins using Western blotting. Our results showed that CCAT2 was upregulated in CRC tissues and cell lines as com-pared to controls. Ectopic expression of CCAT2 promoted CRC cell proliferation, migration and invasion, likely through direct interaction with TAF15, transcriptional activation of RAB14, and activation of the AKT/GSK3β signaling pathway. In vivo, CCAT2 promoted CRC cell growth and metastasis in nude mice. Taken together, these results highlight the actions of CCAT2 as a CRC oncogene.

Overproduction of Gastrointestinal 5-HT Promotes Colitis-Associated Colorectal Cancer Progression via Enhancing NLRP3 Inflammasome Activation.

Chronic inflammation is a key driver for colitis-associated colorectal cancer. 5-hydroxytryptamine (5-HT), a neurotransmitter, has been reported to promote inflammation in the gastrointestinal tract. However, the mechanism behind this remains unclear. In this study, we found that 5-HT levels, as well as the expression of tryptophan hydroxylase 1 (TPH1), the 5-HT biosynthesis rate-limiting enzyme, were significantly upregulated in colorectal tumor tissues from patients with colorectal cancer, colorectal cancer mouse models, and colorectal cancer cell lines when compared with normal colorectal tissues or epithelial cell lines. Colorectal cancer cell-originated 5-HT enhanced NLRP3 inflammasome activation in THP-1 cells and immortalized bone marrow-derived macrophages (iBMDM) via its ion channel receptor, HTR3A. Mechanistically, HTR3A activation led to Ca2+ influx, followed by CaMKIIα phosphorylation (Thr286) and activation, which then induced NLRP3 phosphorylation at Ser198 (mouse: Ser194) and inflammasome assembling. The NLRP3 inflammasome mediated IL1β maturation, and release upregulated 5-HT biosynthesis in colorectal cancer cells by inducing TPH1 transcription, revealing a positive feedback loop between 5-HT and NLRP3 signaling. Silencing TPH1 or HTR3A by short hairpin RNA slowed down tumor growth in an established CT26 and iBMDM coimplanted subcutaneous allograft colorectal cancer mouse model, whereas treatment with TPH1 inhibitor 4-chloro-DL-phenylalanine or HTR3A antagonist tropisetron alleviated tumor progression in an azoxymethane/dextran sodium sulfate-induced colorectal cancer mouse model. Addressing the positive feedback loop between 5-HT and NLRP3 signaling could provide potential therapeutic targets for colorectal cancer.

Effect of the ACY‑1 gene on HER2 and TRAIL expression in rectal carcinoma

The incidence of rectal carcinoma (RC) is increasing and the age at onset of the disease is reducing. Therefore, elucidating the pathogenesis of RC is beneficial for early diagnosis and improving the prognosis. Aminoacylase-1 (ACY-1) is abnormally expressed in various malignant tumor tissues. Furthermore, the human epidermal growth factor receptor-2 (HER2) gene is involved in tumor metastasis and invasion, while tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumor cell apoptosis. The aim of the present study was to investigate the effect of the ACY-1 gene on the expression of HER2 and TRAIL in RC. Cancerous and adjacent tissues from RC patients were collected. ACY-1 expression was analyzed by immunohistochemistry. The rectal cancer cell lines HT29 and SW620, and normal colorectal mucosal epithelial fetal human cells were cultured in vitro. ACY-1 gene and protein expression levels were tested by reverse transcription-quantitative PCR and western blotting. ACY-1 small interfering RNA (siRNA) was transfected into HT29 and SW620 cells. Cell proliferation was detected by thiazolyl blue MTT assay. Caspase-3 activity was assessed using a commercial kit. HER2 and TRAIL expression levels were determined by western blotting. ACY-1 expression was significantly increased in cancer tissue compared with adjacent tissue (P<0.05). ACY-1 expression was elevated in HT29 and SW620 cells compared with normal colorectal mucosal epithelial cells (P<0.05). ACY-1 siRNA transfected into HT29 cells downregulated its expression, inhibited cell proliferation, enhanced caspase-3 activity, reduced HER2 expression and upregulated TRAIL expression (P<0.05). ACY-1 expression was found to be increased in rectal cancer tissue. Therefore, targeting the ACY-1 gene may regulate HER2 and TRAIL expression levels, and may reduce the occurrence and inhibit the development of rectal cancer.

MiR-579-3p Protects Intestinal Mucosal Epithelial Cells from Hypoxia-Reoxygenation Injury by Targeting Cyclin-Dependent Kinase Inhibitor 1B

Intestinal ischemia/reperfusion (I/R) injury is a common tissue and organ injury during surgery. This study explores miR-579-3p’s effect on the hypoxia-reoxygenation injury of intestinal mucosal epithelial cells via cyclin-dependent kinase inhibitor 1B (CDKN1B). Fetal human cells (FHC) cells, which are human normal colorectal mucosal epithelial cells, were cultured in vitro to establish a hypoxia-reoxygenation (H/R) cell model. Nano-based qRT-PCR and Western blot detected miR-579-3p and CDKN1B expressions in HCCLM3 cells treated with H/R. CCK-8 method and flow cytometry measured miR-579-3p and CDKN1B expressions on cell activity and death after H/R treatment. Dual-Luciferase reporter experiment and Western blot analyzed the relationship between miR-579-3p and CDKN1B. After the FHC cells were treated with H/R, miR-579-3p expression was decreased, whereas CDKN1B expression was increased (P &lt; 0.05). FHC cells’ activity was decreased, and its apoptosis rate was upregulated; also, TNF-α and IL-6 protein levels were significantly enhanced (P &lt; 0.05). Nevertheless, the activity of FHC cells treated with H/R after miR-579-3p overexpression was significantly increased, while the apoptosis rate was upregulation, and TNF-α level, IL-6 levels were reduced (P &lt; 0.05). The effect of inhibiting CDKN1B expression was the same as that of overexpression miR-579-3p. After CDKN1B overexpression, the H/R-treated FHC cells’ viability was reduced, while the apoptosis rate was elevated, and TNF-α and IL-6 levels were elevated (P &lt; 0.05). Compared to miR-579-3p overexpression, FHC cell activity in treated with H/R after the overexpression of miR-579-3p+CDKN1B was reduced. At the same time, the apoptosis rate and the level of TNF-α and IL-6 protein were elevated (P &lt; 0.05). In summary, MiR-579-3p’s targeting of CDKN1B protects FHC cells from H/R injury by alleviating H/R-induced apoptosis and inflammation.

Wnt7a Promotes the Occurrence and Development of Colorectal Adenocarcinoma.

ObjectiveThe expression of Wnt7a in colorectal cancer tissues and cell lines was analyzed, and the effect of Wnt7a on the proliferation of colorectal cancer cells was studied, so as to confirm the relationship between Wnt7a and the occurrence and development of colorectal cancer.Methods(1) Immunohistochemical method was used to compare the expression of Wnt7a in different tissues and its relationship with the clinicopathology of colorectal adenocarcinoma. (2) The expression levels of Wnt7a in colorectal cancer cell lines HT-29 and HCT 116 were detected by qRT-PCR. (3) The down-regulated Wnt7A expression vector was constructed, and the down-regulated Wnt7A expression cell line was established. The regeneration ability of cancer cells was detected by stem cell ball formation assay, and the influence of plate cloning assay on the proliferation ability of colorectal cancer cells was detected.Results(1) The positive rates of Wnt7a in normal colorectal mucosa, colorectal adenoma and colorectal adenocarcinoma tissues gradually increased,Wnt7a are closely related to the degree of colorectal adenocarcinoma differentiation, lymph node metastasis and Duke stage. (2) The expression level of Wnt7a in colorectal cancer cells was higher than that in normal colorectal epithelial cells. (3) The down-regulation of Wnt7A reduced the proliferation ability of colorectal cancer cells.ConclusionsWnt7a promotes the occurrence and development of colorectal adenocarcinoma.