Proliferation Of HCT116 Cells Research Articles

39P - The regulation of INK4 locus by long non-coding RNAs

BackgroundINK4 locus is located on human chromosome 9p21 region and encodes three tumor suppressor genes, p15, p16 and ARF. So far, we revealed that a long non-coding RNA (lncRNA), ANRIL, transcribed from INK4 locus represses the transcription of p15 and p16 genes. ANRIL associates with polycomb protein complexes and recruits them on INK4 locus, leading to the transcriptional repression. The depletion of ANRIL inhibits the proliferation of cancer cells such as non-small cell lung cancer and colorectal cancer, indicating that ANRIL functions to promote cancer cells proliferation. Recently, we found a novel lncRNA induced by oncogenic Ras signal (named LION: lncRNA induced by oncogenic Ras signal). In this study, we showed that LION is involved in the transcriptional regulation of INK4 locus and cell proliferation. MethodsHuman non-small cell lung cancer cell H1299 and colorectal cancer cell HCT116 were transfected with siRNA oligonucleotides against LION. The expression analysis of INK4 locus genes was performed by quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). Cell cycle analysis was performed by using Muse cell analyzer. ResultsRT-PCR analysis showed that LION is highly expressed in several cancer cells such as non-small cell lung cancer, cervical cancer and colorectal cancer compared with normal lung fibroblasts. Silencing LION by siRNA oligonucleotides inhibits the proliferation of H1299, HCT116 and HeLa cells. Q-RT-PCR analysis showed that silencing LION increases the p15 and p16 mRNA, suggesting that LION is involved in the transcriptional repression of INK4 locus. Cell cycle analysis showed that silencing LION causes G2/M phase arrest in cell cycle, suggesting that LION functions to promote G2/M transition. ConclusionsLION is involved in the promotion of cancer cells proliferation such as H1299 and HCT116 cells via regulating p15, p16 and other genes related to G2/M phase control. Legal entity responsible for the studyThe authors. FundingJSPS KAKENHI. DisclosureAll authors have declared no conflicts of interest.

MiR-372-3p promotes tumor progression by targeting LATS2 in colorectal cancer.

Many studies suggest that microRNAs can promote the malignant development of tumors. MiRNA-372-3p (miR-372-3p) has been proved to be associated with a variety of cancers. However, the role of miR-372-3p in colorectal cancer (CRC) is unclear. We analyzed the expression of miR-372-3p in CRC tissues and several CRC cell lines by quantitative Real Time-PCR. The relationship between miR-372-3p and clinical pathology was also analyzed in CRC patients. Kaplan-Meier analysis and Cox multivariate analysis were used to evaluate the prognostic significance of miR-372-3p in CRC. Next, we investigated the biological function of miR-372-3p, including cell proliferation, migration, and invasion and analyzed its potential molecular mechanism in vivo and in vitro. Our data showed that the expression of miR-372-3p was dramatically increased in CRC tissues compared with normal tissues. Moreover, the high expression of miR-372-3p was significantly correlated with tumor size and differentiation. Kaplan-Meier analysis showed that the high miR-372-3p expression group patients had a significantly shorter recurrence-free survival (RFS) and disease-specific survival (DSS) than those with the low miR-372-3p group. The analysis of the prognostic factors revealed that miR-372-3p was an independent prognostic factor for RFS and DSS in CRC patients. The knockdown of miR-372-3p inhibited the proliferation, migration, and invasion in HCT116 and SW480 cells. Interestingly, the overexpression of LATS2 partially reversed the miR-372-3p -mediated cell proliferation, migration, and invasion of CRC. Besides, the Hippo signaling pathway was demonstrated to be activated by decreasing of miR-372-3p in CRC. Thus, our study revealed that miR-372-3p is involved in CRC progression by inhibiting the Hippo signaling pathway through its target LATS2. MiR-372-3p and its target genes with signaling pathways are new hope for precise treatment of CRC. The upregulation of miR-372-3p was involved in the process of CRC progression by inhibiting the Hippo signaling pathway through inhibition of LATS2. We showed that miR-372-3p and its target genes with signaling pathways are a novel hope for precise treatment of CRC.

The up-regulated lncRNA DLX6-AS1 in colorectal cancer promotes cell proliferation, invasion and migration via modulating PI3K/AKT/mTOR pathway.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths around the world. Recently, using the high-throughput techniques, long non-coding RNAs (lncRNAs) have been shown to play an important role in CRC progression. In the present study, we aimed to determine lncRNA DLX6 Antisense RNA 1 (DLX6-AS1) in CRC tissues and cell lines and to investigate the molecular mechanisms of DLX6-AS1 in CRC progression. Quantitative real-time PCR was performed to detect gene expression; cell counting kit-8, colony formation, cell invasion, and migration assays were performed to determine cell proliferation, invasion, and migration, respectively; caspase-3 activity assay kit was used to detect caspase-3 activity; in vivo tumor growth was evaluated in a nude mice xenograft model. DLX6-AS1 was up-regulated in 60 CRC tissues when compared to normal adjacent colorectal tissues, and high expression of DLX6-AS1 was correlated with advanced T stage and distant metastasis in CRC patients. The up-regulation of DLX6-AS1 was further confirmed in CRC cell lines. The gain-of-function assays showed that DLX6-AS1 overexpression promoted HCT116 cell proliferation, invasion, and migration, but inhibited cell apoptosis; while the loss-of-function assays showed that DLX6-AS1 knockdown exerted the opposite effects in SW480 cells. In vivo studies revealed that DLX6-AS1 knockdown suppressed tumor growth in the nude mice xenograft model. In addition, DLX6-AS1 overexpression caused an increase in the phosphorylated phosphoinositide 3-kinase (p-PI3K), p-AKT and p-mammalian target of rapamycin (mTOR) protein levels, and DLX6-AS1 knockdown had the opposite effects. Blockade of PI3K/AKT/mTOR signalling pathway by using mTOR inhibitor partially abolished the enhanced effects of DLX6-AS1 overexpression on CRC cell proliferation and metastasis. In summary, our data indicated that DLX6-AS1 promoted CRC cell proliferation, invasion, and migration but inhibited cell apoptosis via targeting PI3K/AKT/mTOR signalling pathway, suggesting the key role of DLX6-AS1 in CRC progression.

Talin1 knockdown prohibits the proliferation and migration of colorectal cancer cells via the EMT signaling pathway.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second highest cause of cancer-associated death worldwide. Talin1 activates integrins, which mediate cell adhesion, proliferation, tumorigenesis and metastasis. The aim of the present study was to determine talin1 expression levels in colorectal cancer (CRC) and investigate the role of talin1 in CRC proliferation and invasion in vitro and in vivo. Talin1 protein expression levels were detected in human CRC and adjacent normal tissues by immunohistochemistry. Talin1 short hairpin RNA and control vectors were designed and stably transfected into HCT116 CRC cells. Cell proliferation was determined by MTT assay. Cell migratory and invasive capabilities were detected by wound-healing and Matrigel invasion assays. The expression of proteins in the epithelial-to-mesenchymal transition signaling pathway was determined by western blotting and reverse transcription-quantitative PCR. The effect of talin1 on tumor growth was explored in vivo using BALB/c nude mice. Immunohistochemical analysis of CRC and adjacent normal tissue revealed that talin1 expression was upregulated in CRC. Talin1 knockdown significantly reduced the proliferation, migration and invasive ability of HCT116 cells compared with the control. Protein levels of phosphorylated STAT3 and vimentin were significantly lower in talin1-knockdown HCT116 cell lines compared with the control, whereas protein levels of E-cadherin were increased. Interleukin-6 mRNA levels were significantly increased in patients' blood samples compared with blood samples from healthy controls, as well as in CRC compared with adjacent normal tissue. In vivo experiments demonstrated that talin1 knockdown reduced CRC tumor growth and weight in nude mice. In conclusion, Talin1 knockdown may prevent the proliferation and migration of CRC cells by downregulating various factors involved in the epithelial-to-mesenchymal transition process, such as phosphorylated STAT3 and vimentin; therefore, talin1 may provide a novel therapeutic target for CRC.

In Silico Design of MDM2-Targeting Peptides from a Naturally Occurring Constrained Peptide.

Naturally occurring constrained peptides are frequently used as scaffolds for bioactive peptide grating due to their high stability. Here, we used in silico methods to design several constrained peptides comprising a scorpion toxin scaffold, a MDM2 binding epitope, and a cluster of positively charged residues. The designed peptides displayed varied binding affinity to MDM2 despite differing by only one or two residues. One of the peptides, SC426, had nanomolar binding affinity (KD =6.6±2.6 nm) to MDM2, and exhibited stronger inhibitory activity on the proliferation of HCT116 cells (p53-wild type) and SW480 cells (p53-mutant) than that of nutlin-3a. Binding mode analysis of the designed peptide at MDM2 suggests that the conserved "FWL" epitope was buried in the hydrophobic binding pocket, and the residues located at the periphery of the binding site contributed to the high binding affinity of SC426. Overall, in silico design of miniproteins with therapeutic potential through epitope grafting to the naturally occurring constrained peptide is an effective strategy.

Identification of benzo[d]pyrrolo[2,1-b]thiazole derivatives as CENP-E inhibitors

Kinesin centromere-associated protein E (CENP-E) has emerged as a potential target for the development of anticancer drugs due to its involvement in the mitotic progression of the cell cycle. Although several CENP-E inhibitors have been reported, more knowledge of chemical structures and inhibitory mechanisms is necessary for developing CENP-E inhibitors. Here, we describe the identification of new CENP-E inhibitors. Screening of a small-molecule chemical library identified benzo[d]pyrrolo[2,1-b]thiazole derivatives, including 1, as compounds with inhibitory activity against the microtubule-stimulated ATPase of the CENP-E motor domain. Among the mitotic kinesins examined, 1 selectively inhibited the kinesin ATPase activity of CENP-E. In a steady-state ATPase assay, 1 exhibited ATP-competitive behavior, which was different from the CENP-E inhibitor GSK923295. Compound 1 inhibited the proliferation of tumor-derived HeLa and HCT116 cells more efficiently than that of non-cancerous WI-38 cells. The inhibition of cell proliferation was attributed to the ability of 1 to induce apoptotic cell death. The compound showed antimitotic activity, which caused cell cycle arrest at mitosis via interference with proper chromosome alignment. We identified 1 and its derivatives as the lead compounds that target CENP-E, thus providing a new opportunity for the development of anticancer agents targeting kinesins.

A novel compound, ferulic acid-bound resveratrol, induces the tumor suppressor gene p15 and inhibits the three-dimensional proliferation of colorectal cancer cells.

Resveratrol, a phytoalexin present in grapes and other edible foods, has been reported to have beneficial effects against various diseases including cancer. We previously reported that resveratrol and its derivative, caffeic acid-adducted resveratrol, selectively inhibit the three-dimensional (3D) proliferation of a human colorectal cancer cell line, HCT116 with activating KRAS mutation. Herein, we demonstrated that a novel compound, ferulic acid-bound resveratrol, also represses the 3D proliferation of HCT116 cells. We observed that resveratrol conjugated to two ferulic acids represses the 3D proliferation of HCT116 cells more strongly than resveratrol and resveratrol conjugated to one ferulic acid. Resveratrol conjugated to two ferulic acids also inhibited the 3D proliferation of MCF7 human breast cancer cells. We further uncovered that the resveratrol derivative increases the mRNA level of the tumor suppressor p15, a CDK inhibitor that functions as a brake of cell proliferation in HCT116 cells. These results imply that the resveratrol derivative represses 3D proliferation via increasing p15 expression in HCT116 cells.

Sophocarpine Inhibits Tumorgenesis of Colorectal Cancer via Downregulation of MEK/ERK/VEGF Pathway.

Colorectal cancer (CRC) is one of the most common malignant tumors and the third leading cause of cancer-related deaths in the world. It was reported that sophocarpine could attenuate the progression of CRC in mice. However, the mechanisms by which sophocarpine regulate the proliferation and migration in CRC remain unclear. Thus, this study aimed to investigate anti-tumor mechanisms of sophocarpine in CRC cells. CCK-8 assay, wound healing assay and transwell migration were used to detect cell proliferation and migration, respectively. In addition, Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to further detect protein expressions and cytokines in vitro. The results revealed that sophocarpine significantly inhibited proliferation in HCT116 and SW620 cells, respectively. Meanwhile, sophocarpine inhibited CRC cells migration via downregulation of the levels of N-cadherin, matrix metalloproteinase (MMP)-9, phosphorylated extracellular signal-regulated kinase (p-ERK), p-mitogen-activated protein kinase kinase (MEK), vascular endothelial growth factor (VEGF)-A, VEGF-C and VEGF-D. Moreover, overexpression of MEK reversed the anti-migration effects of sophocarpine on CRC cells via upregulation of VEGF-A/C/D. Our findings indicated that sophocarpine could inhibit CRC cells migration via downregulation of MEK/ERK/VEGF pathway. Thus, sophocarpine may act as a potential agent for the treatment of CRC.

PDZRN4-mediated colon cancer cell proliferation and dissemination is regulated by miR-221-3p.

BackgroundSuppression of PDZRN4 expression in colon cancer tissues may be associated with elevated levels of the microRNA 221 (miR-211). To uncover potential targets for treatment, the present study investigated PDZRN4 in colon cancer development, and explored the role of miR-221-3p in the regulation of PDZRN4.MethodsRNA expression arrays were searched in the NCBI database, and PDZRN4 (PDZ domain containing ring finger 4) was selected as a potential downregulated gene in colon cancer. PDZRN4 mRNA and protein in colon cancer and matched normal tissues were analyzed. The proliferation and dissemination of HCT116 cells overexpressing PDZRN4 was assessed via functional assays. Bioinformatics analysis and luciferase reporter assay were applied to determine the regulatory link between miR-221-3p and PDZRN4 mRNA.ResultsThere was significantly less PDZRN4 mRNA and PDZRN4 protein in colon cancer tissue compared with normal tissues. HCT116 cells overexpressing PDZRN4 were less able to disseminate relative to the control. Expression of PDZRN4 was directly inhibited by miR-221-3p. Knockout of miR-211-3p was associated with attenuated proliferation and dissemination of HCT116 cells.ConclusionsPDZRN4 may function as a tumor suppressor and is downregulated in colon cancer tissues, possibly due to dysregulation via miR-221-3p. This study provides new insight into colon cancer development.

CircLMNB1 promotes colorectal cancer by regulating cell proliferation, apoptosis and epithelial-mesenchymal transition.

ObjectiveThe aberrant expression of circular RNAs (circRNAs) is a frequent occurrence in various cancers. However, the functions and roles played by most circRNAs in colorectal cancer (CRC) remain largely unknown.Materials and methodsLevels of circLMNB1 expression were evaluated by qRT-PCR and FISH assays. The influence of circLMNB1 knockdown on LoVo and HCT116 cell proliferation, cycling, apoptosis, migration, and invasion were assessed by the CCK-8, assay, Edu assay, flow cytometry, Hoechst staining, and the Transwell assay, respectively. The relative levels of EMT- and apoptosis-related proteins were determined by Western blotting.ResultsCircLMNB1 expression was significantly upregulated in CRC tissues and cells. Knockdown of circLMNB1 by siRNA in LoVo cells suppressed cell proliferation, migration and invasion, and facilitated cell cycle arrest and apoptosis In addition, we proved that knockdown of circLMNB1 upregulated E-cadherin, Bax and caspase-3 expression, and downregulated MMP2, MMP-9, and N-cadherin expression in LoVo cells. Further results showed that overexpression of circLMNB1 enhanced the malignant characteristics of HCT116 cells.ConclusionOur findings revealed that blocking of circLMNB1 could inhibit CRC development, and help to explain the underlying mechanism by which circLMNB1 knockdown inhibits the metastasis of CRC. Finally, this study suggests circLMNB1 as a novel biomarker for CRC.

Antitumoral activity of Ficus carica L. on colorectal cancer cell lines

In traditional medicine, Ficus carica (also known as fig) latex is recognized as a remedy with various therapeutic effects. In the present study we investigated the antitumor activity of Ficus carica extracts and latex. We evaluated the effects of increasing concentrations of Ficus carica extracts and latex on HCT-116 and HT-29 human colorectal cell proliferation using MTT assay and apoptosis induction by evaluating PARP cleavage by Western blot analysis. Peel, pulp, leaves, whole fruit and latex extracts of Ficus carica exerted significant antiproliferative effects on HCT-116 (IC50 values 239, 343, 177, 299, 206 µg/ml) and HT-29 cells (IC50 values 207, 249, 230, 261, 182 µg/ml) after 48h of treatment. Furthermore, treatment with different extracts of Ficus carica induced apoptosis in both HT-29 and HCT-116 cancer cells. Leaves and latex extracts of Ficus carica showed the strongest antiproliferative activities. Overall, our results showed that these natural products are strong apoptosis inducers which suggest their use of for therapeutic purposes.

Sesamolin exerts anti-proliferative and apoptotic effect on human colorectal cancer cells via inhibition of JAK2/STAT3 signaling pathway

Colorectal cancer (CRC) is a common malignant tumor that seriously threatens human health and quality of life. At present, the search for safe and more effective treatment for CRC has become necessary. The present study investigated the anti-proliferative and apoptotic effects of sesamolin on human colorectal cancer (HCT116) cells, and the underlying mechanism. Cell proliferation was determined using MTT assay, while the expressions of JAK2, STAT3 and p-STA3 were determined using Western blotting. The levels of expression of matrix metalloproteinases-1, 2 and 9 (MMP1, MMP2 and MMP9) were determined using real-time quantitative polymerase chain reaction (qRT-PCR). The degree of migration and invasion of the cells was assessed using wound healing assay. The results of MTT assay showed that sesamolin significantly and time- and dose-dependently inhibited the proliferation of HCT116 cells (p < 0.05). Treatment of HCT116 cells with sesamolin significantly inhibited their migratory ability (p < 0.05). The expressions of p-JAK2 and p-STAT3 were significantly down-regulated 48 h after 20 µM of JAK2 specific inhibitor (AG490) was added to HCT116 cells (p < 0.05). The expression of p-STAT3 was also significantly and dose-dependently down-regulated 6 h after treatment of HCT116 cells with sesamolin (p < 0.05). Sesamolin and AG490 had synergistic effect and their combination significantly down-regulated the expression of p-STAT3, when compared with sesamolin alone (p < 0.05). Treatment of HCT116 cells with sesamolin significantly and dose-dependently reduced the levels of IL-6-induced expressions of MMP-1, MMP-2 and MMP-9 (p < 0.05). These results suggest that sesamolin induces apoptosis in HCT116 cells and prevents cell invasion via inhibition of the JAK2/STAT3 signaling pathway.

MicroRNA-33a-5p suppresses colorectal cancer cell growth by inhibiting MTHFD2.

Colorectal cancer (CRC) is one of the most common malignancies with high levels of invasiveness, drug resistance and mortality, but the internal mechanisms of CRC are largely unknown. MicroRNAs (miRs) have been reported to be involved in the development of CRC, and numerous studies have demonstrated that the abnormal expression of miR-33a-5p might be associated with CRC. However, the function and downstream mechanism of miR-33a-5p in colorectal cancer (CRC) remains unclear. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme involved in folic acid metabolism, interestingly was confirmed to be one of the target genes of miR-33a-5p in the present study. We first confirmed that miR-33a-5p in CRC tissues and cell lines were downregulated (P<0.05), and that the proliferation, clone formation capacities, G1/S progression, and migration capacities of the two CRC cell lines HCT116 cells and HT29 were suppressed by miR-33a-5p overexpression in vitro (P<0.05). Ctrl HCT116 and miR-33a-5p-overexpressing HCT116 were injected into nude mice. In vivo tumour formation was significantly suppressed by miR-33a-5p overexpression (P<0.05) as well as the proliferation marker Ki67 (P<0.05). Additionally, MTHFD2 protein expression was significantly enhanced in CRC tissues. From bioinformatics predictions and a luciferase report analysis, MTHFD2 was confirmed to be one of the target genes of miR-33a-5p. In contrast to the role of miR-33a-5p overexpression, MTHFD2 overexpression promoted the proliferation and migration of HCT116 and HT29 cells (P<0.05), which confirmed that MTHFD2 was a functional target gene of miR-33a-5p. In conclusion, miR-33a-5p inhibits the growth and migration of CRC by targeting MTHFD2.

MiR-27a-3p regulates proliferation and apoptosis of colon cancer cells by potentially targeting BTG1.

microRNA (miR/miRNA)-27a-3p has been reported to be abnormally expressed in various types of cancer, including colorectal cancer (CRC). B-cell translocation gene 1 (BTG1) has also been implicated with CRC. However, the association between miR-27a-3p and BTG1 in CRC, to the best of our knowledge, has not been investigated. In order to assess whether miR-27a-3p is associated with CRC, reverse transcription-quantitative PCR was performed on 20 paired CRC and paracancerous tissues for miRNA analysis. For the screening and validation of miR-27a-3p expression in colon cancer, several colon cancer cell lines (HCT-116, HCT8, SW480, HT29, LOVO and Caco2) and the normal colorectal epithelial cell line NCM460 were examined. The highest expression levels of miR-27a-3p were detected in the HCT-116, which was selected for further experimentation. The HCT-116 cells were divided into control, miR-27a-3p mimic and inhibitor groups, and cell proliferation was tested using an MTT assay. Additionally, miR-27a-3p inhibitor/mimic or BTG1 plasmid were transfected into the HCT-116 cells, and flow cytometry was performed to analyze cell cycle distributions. TUNEL analysis was performed to detect apoptosis. Protein levels of factors in the downstream signaling pathway mediated by miR-27a-3p [ERK/mitogen-activated extracellular signal-regulated kinase (MEK)] were detected. miR-27a-3p was revealed to be overexpressed in human CRC tissues and colon cancer cell lines. Knockdown of miR-27a-3p suppressed proliferation of HCT-116 cells and apoptosis was increased. It further markedly upregulated expression levels of BTG1 and inhibited activation of proteins of the ERK/MEK signaling pathway. In addition, overexpression of BTG1 in HCT-116 cells triggered G1/S phase cell cycle arrest and increased apoptosis via the ERK/MEK signaling pathway. In conclusion, the present study demonstrated that the effects of miR-27a-3p on colon cancer cell proliferation and apoptosis were similar to those of the tumor suppressor gene BTG1. The miR-27a-3p/BTG1 axis may have potential implications for diagnostic and therapeutic approaches in CRC.

Role and mechanism of circular RNA-vimentin in the proliferation and apoptosis of colorectal cancer cells

Objective To investigate the role and mechanism of circular RNA-vimentin (circ-VIM) in the proliferation and apoptosis of colorectal cancer cells. Methods From December 2016 to December 2017, at Department of General Surgery of The First Affiliated Hospital of Zhengzhou University, the clinical data of 100 patients who underwent radical resection of colorectal cancer and were confirmed by pathological examination after operation were collected. The tumor tissues and corresponding paracancerous tissues (negative control) were also collected. The expression of circ-VIM in the colorectal cancer tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation of HCT-116 and HT29 colorectal cancer cells was detected by cell counting kit-8 assay. The ratio of apoptosis of HCT-116 and HT29 cells was measured by annexin Ⅴ/propidium iodide double staining assay. The mitochondrial membrane potential of HCT-116 and HT29 cells was examined by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl-imidacarbocyanine iodide (JC-1) assay. The expression changes of protein kinase B and mammalian target of rapamycin were tested by Western blotting. The target miRNA of circ-VIM was predicted by miRDB software. T-test and chi-square test were performed for statistical analysis. Results The expression of circ-VIM in colorectal cancer tissues was 2.387±0.536, which was higher than that in corresponding paracancerous tissues (1.110±0.134), and the difference was statistically significant (t=23.096, P<0.01). And the expression levels of circ-VIM were significantly different in patients with different tumor size, TNM stage and lymph node metastasis (all P<0.05). The proliferation of HCT-116 cells and HT29 cells in lenti-circ-VIM group was 0.737±0.023 and 0.835±0.025, respectively, which were both higher than those in control group (0.449±0.020 and 0.531±0.019), and the differences were statistically significant (t=20.706 and -15.374, both P<0.01). The proliferation of HCT-116 cells and HT29 cells in lenti-circ-VIM-shRNA group was 0.236±0.027 and 0.243±0.019, which were lower than those in control group, and the differences were statistically significant (t=24.557 and -23.197, both P<0.01). The ratio of apoptosis of HCT-116 cells and HT29 cells in lenti-circ-VIM-shRNA group was (18.00±1.82)% and (20.80±0.61)%, which was higher than those in control group ((6.64±2.01)% and (7.35±1.36)%), and the differences were statistically significant (t=8.826 and 17.454, both P<0.01). The fluorescence intensity ratio of JC-1 aggregate and JC-1 monomer of HCT-116 cells and HT29 cells in lenti-circ-VIM-shRNA group was 2.21±0.12 and 1.40±0.11, which was lower than those in control group (14.54±1.00 and 9.24±1.18), and the differences were statistically significant (t=-19.558 and -15.685, both P<0.01), which indicated mitochondrial membrane potential decreased. After treated with lenti-circ-VIM-shRNA, the expression of phosphorylated protein kinase B, phosphorylated mammalian target of rapamycin, B-cell lymphoma-2 and mitochondrial cytochrome C at protein level were all down-regulated, however the expression of cytoplasmic cytochrome C, B-cell lymphoma-2 associated X protein and cleaved caspase-3 at protein level were all up-regulated. When the expression of circ-VIM was up-regulated, the expression of miRNA-147b, miRNA-4447 and miRNA-3656 was down-regulated. When the expression of circ-VIM was down-regulated, the expression of miRNA-147b, miRNA-4447 and miRNA-3656 was up-regulated. Conclusion The expression of circ-VIM in colorectal cancer is abnormally increased, which is involved in the proliferation and apoptosis of colorectal cancer cells. Key words: Colorectal neoplasms; Cell proliferation; Apoptosis; circ-VIM

Abstract 1595: Functional genomic analysis of 18q21.1 locus identifies potential functional variants and genes for colorectal cancer

Abstract Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in the world. Genome-wide association studies have identified approximately 50 genetic risk loci for CRC. We have previously identified SNP rs7229639, located in the second intronic region of the SMAD7 gene at locus 18q21.1, in relation to CRC risk in Asians. In this study, we performed functional genomic analysis to identify potential functional variants and target genes for this association. Functional annotation analysis using epigenetic data from ENCODE and Roadmap projects showed functional evidence of promoter and/or enhancer activities for four highly correlated potential functional SNPs, including rs7229639, rs77544449, rs112540150 and rs57668004. Using in vitro luciferase reporter assays in colon cancer HCT116 and PKO cells, we showed that the risk alleles of rs7229639 and rs77544449 increase promoter or enhancer activities, compared to reference alleles. The risk alleles of both SNPs also affect the ability of interacting with nuclear proteins in the electrophoretic mobility shift assays, compared to reference alleles. To identify candidate target genes for this risk locus, we performed cis-expression quantitative trait loci (eQTL) analysis using transcriptome data from the Genotype-Tissue Expression (GTEx) project, The Cancer Genome Atlas (TCGA) and Colonmics. We identified putative target gene LIPG, located 500 kb downstream of rs7229639, in all three datasets. No associations were observed for SMAD7, or eight other genes in the region, via eQTL analysis. To investigate the biological functions of LIPG gene in colorectal cancer, we knock-downed LIPG gene expression using siRNA in HCT116 and PKO cells, which resulted in a significant decrease in cell proliferation, colony formation, migration and invasion in both HCT116 and PKO cells. These results suggest that LIPG gene plays a significant role in colorectal tumorigenesis via disruption of cell behaviors. Taken together, results from our study suggest that the association between the rs7229639/18q21.1 locus and CRC risk may be mediated through SNPs that regulate expression of the LIPG gene. Citation Format: Jifeng Wang, Xingyi Guo, Shimian Qu, Joshua A. Bauer, Jirong Long, Wei Zheng, Qiuyin Cai. Functional genomic analysis of 18q21.1 locus identifies potential functional variants and genes for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1595.

Inhibitor of DNA binding 1 (Id1) mediates stemness of colorectal cancer cells through the Id1-c-Myc-PLAC8 axis via the Wnt/β-catenin and Shh signaling pathways.

BackgroundInhibitor of DNA binding 1 (Id1) is upregulated in multiple cancers, and Id1overexpression correlates with cancer aggressiveness and poor clinical outcomes in cancer patients. However, its roles in cancer stem-like cells (CSCs) and epithelial-mesenchymal transition (EMT) are still elusive.PurposeThis study aimed to examine the role of Id1 on the mediation of CRC stemness and explore the underlying mechanisms.MethodsId1 and CD133 expression was detected by qPCR assay and immunohistochemistry (IHC) in normal mucosal and primary colorectal cancer (CRC) specimens. Id1 was stably knocked down (KD) in human CRC cell lines. Spheres forming assay and tumorigenic assay were performed to evaluate self-renewal capacity and tumor initiation. Expression of CSC- and EMT-related markers and TCF/LEF activity were assessed in HCT116 cells after Id1 KD.ResultsqPCR assay showed higher Id1 and CD133 expression in CRC specimens than in normal mucosal specimens (P<0.05). IHC detected high cytoplasmic Id1 expression in 35 CRC specimens (46.7%), and high CD133 expression in 22 CRC specimens (29.3%) and negative expression in 18 normal mucosal specimens. High Id1 expression positively correlated with poor differentiation (P=0.034), and CD133 expression correlated with T category in CRC patients (P=0.002). Spearman correlation analysis revealed a positive correlation between Id1 and CD133 expression in CRC patients (P<0.05). Id1 KD resulted in suppression of proliferation, cell-colony formation, self-renewal capability and CSC-like features in HCT116 cells, and impaired the tumor-initiating capability in CRC cells. In addition, Id1 maintained the stemness of CRC cells via the Id1-c-Myc-PLAC8 axis through activating the Wnt/β-catenin and Shh signaling pathways.ConclusionsId1 expression significantly correlates with CD133 expression in CRC patients, and Id1 KD impairs CSC-like capacity and reverses EMT traits, partially via the Wnt/β-catenin signaling. Id1 may be a promising therapeutic target against colon CSCs.

Abstract 4266: Endoplasmic reticulum (ER), a potential therapeutic target for mutant p53 colorectal cancer

Abstract Introduction: Inactivating p53 mutations contribute to tumor progression and treatment-resistance, resulting in poor patient survival in colorectal cancer patients (CRC). The goal of this study is to identify novel small molecules that therapeutically target mutant p53 in CRC. Methods: We analyzed the effects of small molecule (ASR458) on p53-wild type (p53-wt; HCT116) and mutant p53 (p53-mut; SW620) colon cancer cells by phenotypic, molecular and in vivo assays. Results: ASR458 treatment significantly inhibited the proliferation of both HCT116 and SW620 cells at nM concentrations. In p53-wt HCT116 cells, ASR458 caused induction of p53 that resulted in caspase-mediated cell death in both in vitro and in vivo models. On the contrary, ASR458 treatment induced ER-stress signaling (i.e., phosphorylation of ERK and eIF2-α) in p53-mut SW620 cells, which triggered ATF4 activation and subsequent induction of cascade of autophagy events (Atg family proteins, LC3B and Lamp1), causing autophagy-mediated cell death. Silencing ER stress marker ATF-4, a key regulator of autophagy, caused resistance to ASR458 and abrogated autophagy signaling in SW620 cells. This suggested that induction of ER-stress is critical for the cytotoxic effects of ASR458 in p53-mut CRC. Preliminary knockdown studies indicate that silencing of ER markers causes resistance to ASR458 in vitro, further confirming ER-stress as the mechanism of ASR458 action in p53-mut CRC. ASR458 significantly inhibited the growth of SW620 tumors in xenograft. Tissue analysis confirmed the ER-stress signaling observed in vitro. Conclusion: Our results demonstrate ASR458 as a potential therapeutic agent with distinct targets in p53-wt and p53-mut CRC. This study also suggests that ATF4 mediated autophagy in unmanaged ER stress can reduce CRC pathogenesis. Further investigation into the pharmacokinetics and pharmacodynamics of ASR458 should help clinical translation of this agent. Citation Format: Ashish Tyagi, Balaji chandrasekaran, Venkatesh Kolluru, Becca V. Baby, Alatassi Houda, Srinivasa Ramisetti, Arun Sharma, Murali Ankem, Chendil Damodaran. Endoplasmic reticulum (ER), a potential therapeutic target for mutant p53 colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4266.

&lt;p&gt;Jatrorrhizine inhibits colorectal carcinoma proliferation and metastasis through Wnt/β-catenin signaling pathway and epithelial–mesenchymal transition&lt;/p&gt;

PurposeJatrorrhizine (JAT) is a natural protoberberine alkaloid, possesses detoxification, bactericidal and hypoglycemic activities. However, its anti-cancer mechanism is not clear. This study aimed to investigate the mechanism of JAT through which inhibits colorectal cancer in HCT-116 and HT-29 cells.MethodsMTT assay and colony formation assay were used to check the cell proliferation ability. Cell apoptosis and cell cycle were measured by Hoechst 33342 staining and flow cytometry, respectively. Cell migration and invasion were detected by scratch wound healing assay and trans-well assay, respectively. Further, expression of related proteins was examined via Western blotting and the in vivo anti-cancer effect of JAT was confirmed by nude mice xenograft model.ResultsThe research showed that JAT inhibited the proliferation of HCT-116 and HT-29 cells with IC50 values of 6.75±0.29 μM and 5.29±0.13 μM, respectively, for 72 hrs. It has also showed a time dependently, cell cycle arrested in S phase, promoted cell apoptosis and suppressed cell migration and invasion. In addition, JAT inhibited Wnt signaling pathway by reducing β-catenin and increasing GSK-3β expressions. Increased expression of E-cadherin, while decreased N-cadherin, indicating that JAT treatment suppressed the process of cell epithelial–mesenchymal transition (EMT). In HCT-116 nude mice xenograft model, JAT inhibited tumor growth and metastasis, and induced apoptosis of tumor cells.ConclusionThis study demonstrated that JAT efficiently inhibited colorectal cancer cells growth and metastasis, which provides a new point for clinical treatment of colorectal cancer.

Circular RNA hsa_circ_0007142 Is Upregulated and Targets miR-103a-2-5p in Colorectal Cancer.

Circular RNAs (circRNAs) are a large class of endogenous noncoding RNAs that regulate gene expression and mainly function as microRNA sponges. This study aimed to explore the aberrant expression of circRNAs in colorectal cancer (CRC). Using a circRNA microarray, we identified 892 differentially expressed circRNAs between six pairs of CRC and adjacent paracancerous tissues. Among them, hsa_circ_0007142 was significantly upregulated. Further analysis in 50 CRC clinical samples revealed that hsa_circ_0007142 upregulation was associated with poor differentiation and lymphatic metastasis of CRC. Bioinformatic analysis and luciferase reporter assay showed that hsa_circ_0007142 targeted miR-103a-2-5p in CRC cells. Moreover, the silencing of hsa_circ_0007142 by siRNAs decreased the proliferation, migration, and invasion of HT-29 and HCT-116 cells. Taken together, these findings suggest that hsa_circ_0007142 is upregulated in CRC and targets miR-103a-2-5p to promote CRC.