Metastasis Of Colorectal Cancer Cells Research Articles

MiR-20a/Foxj2 Axis Mediates Growth and Metastasis of Colorectal Cancer Cells as Identified by Integrated Analysis.

BackgroundMicroRNAs (miRNAs) have a significant regulatory effect on the proliferation, migration, and invasion of cells, and have been widely reported to have oncogenic or tumor-suppressive impacts on various tumors. In the present study we assessed the regulation and function of miR-20a on colorectal cancer (CRC) cell lines.Material/MethodsqPCR was used to quantify miR-20a expression. Luciferase reporter assay was conducted to confirm Foxj2 3′UTR associations. In addition, the function of miR-20a and Foxj2 in CRC was detected using MTT, colony formation, transwell assays, and cell cycle analysis.ResultsOur data revealed that miR-20a expression was elevated in the CRC cell lines, and cell migration, proliferation, and invasion abilities were promoted by the overexpression of miR-20a. Moreover, Foxj2 was authenticated as a direct target gene of miR-20a in CRC cells. Furthermore, we found that the ectopic Foxj2 dramatically suppressed miR-20a-promoted proliferation, migration, invasion, and xenografts in vitro and in vivo, and induced cell cycle arrest at G1 stage.ConclusionsOur results showing the roles of miR-20a/Foxj2 in carcinogenesis of CRC may help improve treatment of CRC.

Long Noncoding RNA DLX6-AS1 Regulates the Growth and Aggressiveness of Colorectal Cancer Cells Via Mediating the miR-26a/EZH2 Axis.

Objective: To understand the regulation of long noncoding RNA DLX6-AS1-mediated miR-26a/EZH2 axis in the growth of colorectal cancer (CRC) cells. Methods: The expression of DLX6-AS1, miR-26a, and EZH2 was detected in CRC tissues by quantitative reverse transcription-polymerase chain reaction. The CRC HT-29 cell line was selected for transfection and subjected to observe the growth by MTT and colony formation assays, cell cycle by flow cytometry, and migration and invasion by wound healing and Transwell assays, respectively. Finally, the expression of cycle- and metastasis-related proteins was detected by Western blotting. Results: DLX6-AS1 and EZH2 were increased, with a decreased miR-26a in CRC tissues, showing significant negative correlations between DLX6-AS1 and miR-26a, and between miR-26a and EZH2. CRC patients at advanced stage or with lymphatic metastasis had higher DLX6-AS1 expression. Dual-luciferase reporter gene assay uncovered the targeting correlations between DLX6-AS1 and miR-26a, or miR-26a and EZH2. After transfection of DLX6-AS1 siRNA or EZH2 siRNA, the growth and metastasis of CRC cells were suppressed, arresting the cells in G0/G1 phase, with a magnificent reduction in the ratio of cells in S phase or G2/M phase; meanwhile, Cyclin D1, Vimentin, and MMP9 expressions decreased evidently, whereas E-cadherin expression was upregulated. Changes above were fully reversed after transfection of miR-26a inhibitor, whereas si-EZH2 transfection abolished the positive role of miR-26a inhibitor on growth of CRC cells. Conclusion: Silencing DLX6-AS1 may block the malignant features of CRC cells by inhibiting the expression of EZH2 through upregulation of miR-26a. Thus, it is critical to the development and progression of CRC.

MIR600HG suppresses metastasis and enhances oxaliplatin chemosensitivity by targeting ALDH1A3 in colorectal cancer.

Background: Metastasis and chemoresistance indicate a poor prognosis in colorectal cancer (CRC) patients. However, the mechanisms that lead to the development of chemoresistance and metastasis in CRC remain unclear.Materials and methods: We combined clinical and experimental studies to determine the role of MIR600HG in CRC metastasis and chemoresistance. The statistical analysis was performed using GraphPad Prism software, version 8.0.Results: We detected down-regulated expression of long non-coding RNA (lncRNA) MIR600HG in CRC specimens and cell lines compared with normal controls, and the expression level of MIR600HG was inversely correlated with the overall survival of CRC patients. The inhibition of MIR600HG stimulated CRC cell metastasis and chemoresistance. In addition, our data showed that the inhibition of MIR600HG stimulated CRC stemness, while the overexpression of MIR600HG suppressed stemness. Importantly, our animal experiments showed that MIR600HG inhibited tumour formation and that the combination of MIR600HG inhibition and oxaliplatin (Oxa) treatment significantly inhibited tumour growth compared with that with either intervention alone. Furthermore, we demonstrated that MIR600HG exerts its anticancer role by targeting ALDH1A3 in CRC.Conclusions: Our data suggest that MIR600HG functions as a tumour suppressor and that the overexpression of MIR600HG inhibits tumour invasion and enhances chemosensitivity, providing a new strategy for CRC treatment.

DJ‑1 is a new prognostic marker and predicts chemotherapy efficacy in colorectal cancer.

Protein/nucleic acid deglycase DJ-1 (DJ-1) is a 20-kDa conserved protein, which belongs to the DJ-1/ThiJ/Pfp I protein superfamily. Immunohistochemistry was performed to investigate the expression of DJ-1 in a colorectal cancer (CRC) tissue microarray containing tumor and corresponding adjacent normal tissues. In the present study, DJ-1 expression was significantly upregulated in CRC cells and tissues, compared with that in normal colon cells and adjacent normal tissues, respectively. In addition, patients with high DJ-1 expression levels had a worse overall survival (OS) compared with patients with low expression levels. Multivariate Cox regression analysis revealed that high DJ-1 expression levels was an independent prognostic factor for patients with CRC. Moreover, DJ-1 was able to regulate the PI3K/Akt/p27/cyclin E and PI3K/Akt/mTOR signaling pathways to promote CRC cell growth and metastasis in vitro and in vivo. In addition, DJ-1 regulated the NF-κB/Snail signaling pathway to induce CRC cell epithelial-mesenchymal transition to promote migration and invasion. Notably, patients receiving LFP treatment (oxaliplatin, 5-FU and tetrahydrofolate) had an increased OS compared with patients who underwent only surgery and low DJ-1 expression levels. The findings from the present study suggest that DJ-1 may serve as a promising prognostic marker and predicts chemotherapy efficacy in patients with CRC.

HOXD9 promote epithelial-mesenchymal transition and metastasis in colorectal carcinoma.

BackgroundHOXD9, a Hox family member, is involved in cancer growth and metastasis. But, its regulation mechanism at the molecular level particularly in colorectal cancer (CRC), is mostly unknown.MethodsThe HOXD9 protein expression levels were analyzed using immunofluorescence, immunohistochemistry (IHC) assays, and western blot. The in vivo and in vitro roles of HOXD9 in CRC were determined using colony formation and EdU incorporation, CCK‐8, wound scratch and transwell invasion assay, and animal models.ResultsExpression of HOXD9 was higher in CRC than in matched healthy tissues. High expression of HOXD9 has significantly associated with the American Joint Committee on Cancer (AJCC) stages, tumor differentiation, lymph node metastasis, and other serious invasions, and it had a poor prognosis. In vitro, HOXD9 encouraged proliferation, movement and EMT processes in cells of CRC. Also, TGF‐β1 promoted the expression of HOXD9 and this effect was dependent on the dose and downregulation of HOXD9 repressed TGF‐β1 ‐induced EMT. In vivo, HOXD9 promoted the invasive and metastasis of CRC cells via orthotopic implantation.ConclusionsThe ectopic expression of HOXD9 promoted the invasion metastasis in cells of the colorectal tumor by induction of EMT in vitro and vivo.

Translation regulatory long non-coding RNA 1 represents a potential prognostic biomarker for colorectal cancer.

Long non-coding RNAs (lncRNAs) have attracted a lot of attention for their role in the development, progression and prognosis of colorectal cancer (CRC). However, little is known on the clinical significance of the translation regulatory lncRNA 1 (TRERNA1) in CRC. The present study aimed to explore the clinical value of TRERNA1 in patients with CRC. A total of 89 cancer-associated lncRNA genes were analyzed using the RT2 lncRNA PCR array Human Cancer PathwayFinder. Following the PCR array, reverse transcription-quantitative (RT-q)PCR was conducted to identify the differential expression of TRERNA1 between 130 CRC and corresponding non-tumorous adjacent tissues. Additionally, the association between TRERNA1 expression and clinical characteristics was analyzed. Furthermore, TRERNA1 expression was knocked down via small interfering RNAs. The results of the PCR array and RT-qPCR revealed that TRERNA1 expression was significantly upregulated in CRC tissues compared with in adjacent normal tissues. TRERNA1 upregulation was positively associated with distant metastasis, perineural invasion, TNM stage, node metastasis stage and tumor diameter. Multivariate analysis revealed that patients with higher TRERNA1 expression had a shorter overall survival (OS) time and a less favorable prognosis compared with those in the low TRERNA1 expression group. Knockdown of TRERNA1 inhibited invasion and metastasis of CRC cells via regulating Snail expression. In conclusion, TRERNA1 expression was upregulated in CRC tissues. High expression levels of TRERNA1 may be associated with poor OS times, a less favorable prognosis and lymph node metastasis in patients with CRC. TRERNA1 may therefore serve as a useful and novel biomarker for CRC lymph node metastasis and prognosis.

Interaction between linc01615 and miR-491-5p regulates the survival and metastasis of colorectal cancer cells.

BackgroundThe 5-year survival of colorectal cancer (CRC) has had no obvious improvement during the past decades, although a significant progress in treatments has been established. The molecular mechanisms that drive the progression of CRC are still unclear. This study aims to determine the biological activities of linc01615 and its regulatory microRNAs in CRC cells.MethodsThe expression of linc016150 and miR-491-5p was measured in six CRC cell lines and one normal colon mucosal epithelial cell line. Their effects on cell proliferation, apoptosis, invasion, and migration were tested in Caco-2 cells.ResultsLinc01615 mRNA expression was upregulated in six colorectal cancer cell lines compared to the normal colon mucosal epithelial cell line, which was negatively regulated by miR-491-5p in Caco-2 cells. Silencing of linc01615 gene expression significantly decreased cell proliferation, increased apoptosis, and inhibited invasion and migration in Caco-2 cells. Overexpression of linc01615 exhibited an opposite effect on silencing of linc01615 expression. Transfection with miR-491-5p mimics downregulated linc01615 expression and inhibited the biological activities of linc01615. In contrast, the inhibitor of miR-491-5p up-regulated linc01615 expression and subsequently enhanced the biological activities of linco1615. Also, overexpression of linc01615 can block the effects of miR-491-5p mimics in Caco-2 cells.ConclusionsLinc01615 functions as an oncogene while miR-491-5p functions as a tumor suppressor in colorectal cancer cells through negatively regulating each other; both are involved in cell proliferation, apoptosis, invasion, and migration in colorectal cancer cells.

Reprogramming of lipid metabolism in cancer-associated fibroblasts potentiates migration of colorectal cancer cells

Metabolic interaction between cancer-associated fibroblasts (CAFs) and colorectal cancer (CRC) cells plays a major role in CRC progression. However, little is known about lipid alternations in CAFs and how these metabolic reprogramming affect CRC cells metastasis. Here, we uncover CAFs conditioned medium (CM) promote the migration of CRC cells compared with normal fibroblasts CM. CAFs undergo a lipidomic reprogramming, and accumulate more fatty acids and phospholipids. CAFs CM after protein deprivation still increase the CRC cells migration, which suggests small molecular metabolites in CAFs CM are responsible for CRC cells migration. Then, we confirm that CRC cells take up the lipids metabolites that are secreted from CAFs. Fatty acids synthase (FASN), a crucial enzyme in fatty acids synthesis, is significantly increased in CAFs. CAF-induced CRC cell migration is abolished by knockdown of FASN by siRNA or reducing the uptake of fatty acids by CRC cells by sulfo-N-succinimidyloleate sodium in vitro and CD36 monoclonal antibody in vivo. To conclude, our results provide a new insight into the mechanism of CRC metastasis and suggest FASN of CAFs or CD36 of CRC cells may be potential targets for anti-metastasis treatment in the future.

Role of fibroblast growth factor 4 in the growth and metastasis of colorectal cancer.

The role of fibroblast growth factor receptor4 (FGFR4) in colorectal cancer (CRC) is poorly characterized. Therefore, the objective of the current study was to investigate the expression levels of FGFR4 in colorectal cancer and its prognostic value, and clarify the role of FGFR4 in the proliferation and metastasis of colorectal cancer cells. Immunohistochemistry was used to detect the association between FGFR4 expression and clinicopathological features in colorectal cancer tissues. The effect of FGFR4 silencing on tumor cell proliferation, cell cycle, apoptosis, migration and invasion was evaluated via lentiviral transfection of the colorectal cancer cell line SW620. Western blot analysis was used to detect the changes of epithelial‑mesenchymal transition (EMT) markers, following FGFR4 silencing. FGFR4 is upregulated in CRC tissues compared with normal tissues. Patients with high FGFR4 expression exhibited a lower 5‑year survival rate compared with patients with low FGFR4 expression (64 vs.74%). FGFR4 silencing reduced proliferation, inhibited cell invasion, arrested cells in S phase and promoted apoptosis in colorectal cancer cells. FGFR4 silencing partially reversed EMT progression and FGFR4 this effect was enhanced in the presence of XAV939 (a β‑catenin inhibitor). The current data suggest that FGFR4 may be associated with prognosis in patients with colorectal cancer. Invitro functional tests revealed that FGFR4 may represent an effective therapeutic target for colorectal cancer. FGFR4 may also regulate EMT via the Wnt/β‑catenin pathway.

Regulation of the small GTPase Ran by miR-802 modulates proliferation and metastasis in colorectal cancer cells

BackgroundThe small GTPase Ran is upregulated in multiple cancers and fundamental for cancer cell survival and progression, but its significance and molecular mechanisms in colorectal cancer (CRC) remain elusive.MethodsRan expression was detected in CRC cell lines and tumour tissues. In vitro and in vivo functional assays were performed to examine the effects of Ran on cell proliferation and metastasis. The pathways and effectors regulated by Ran were explored by an unbiased screening. Bioinformatics prediction and experimental validation were used to identify the miRNA regulator for Ran.ResultsRan expression was frequently increased in metastatic CRC cells and tissues, especially in metastatic tissues. The upregulation of Ran correlated with poor CRC patient prognosis. Ran silencing reduced proliferation and metastasis of CRC cells both in vitro and in vivo. Ran regulated the expression of EGFR and activation of ERK and AKT signalling pathways. miR-802 was identified as an upstream regulator of Ran and miR-802 overexpression resulted in antiproliferative and antimetastatic activities.ConclusionOur study demonstrates the oncogenic roles and underlying mechanisms of Ran in CRC and the novel miR-802/Ran/EGFR regulatory axis may provide potential biomarkers for the treatment of CRC.

MicroRNA-1224-5p Inhibits Metastasis and Epithelial-Mesenchymal Transition in Colorectal Cancer by Targeting SP1-Mediated NF-κB Signaling Pathways.

MicroRNAs (miRNAs) are small non-coding RNAs that play pivotal roles in cancer initiation and progression. However, the roles and molecular mechanisms of miRNAs in colorectal cancer (CRC) progression remain unclear. Here, we show that downregulation of miR-1224-5p in CRC is negatively correlated with SP1 expression and metastasis in patients and xenografted mouse models. Gain- and loss-of-function assays reveal that miR-1224-5p suppresses the migration, invasion, and epithelial–mesenchymal transition (EMT) of CRC cells in vitro and in vivo by directly targeting SP1. Moreover, SP1 promotes the phosphorylation of p65, which results in EMT progress in CRC cells. Clinical analysis reveals that miR-1224-5p and SP1 expression are remarkably associated with advanced clinical features and unfavorable prognosis of patients with CRC. Further study confirms that hypoxia accounts for the depletion of miR-1224-5p in CRC. The enhancement of hypoxia during epithelial–mesenchymal transition and metastasis of CRC cells is abolished by miR-1224-5p. Our findings provide the first evidence that miR-1224-5p is a potential therapeutic target and prognostic biomarker for patients with CRC.

Association of DCBLD2 upregulation with tumor progression and poor survival in colorectal cancer.

DCBLD2 expression dysregulation has been reported in several types of human cancer. As yet, however, the role of DCBLD2 in colorectal cancer (CRC) is not known. CRC tissues were obtained from patients undergoing surgery from February 2009 to May 2014 (n = 90). Tissue microarray construction and immunohistochemistry were carried out to determine DCBLD2 expression. In vivo studies were performed in 4-week-old BALB/c nude mice. In vitro studies were conducted using CRC-derived HT29 and HCT116 cell lines. DCBLD2 expression was found to be significantly increased in CRC tissues compared to adjacent normal tissues (p < 0.001). In addition, we found that DCBLD2 expression was positively correlated with the stage of the disease, the degree of differentiation and vascular invasion. High DCBLD2 expression was significantly associated with a poor overall survival. In vitro, DCBLD2 expression downregulation significantly reduced CRC cell proliferation and invasion. In a mouse xenograft model, DCBLD2 expression downregulation reduced lung metastasis and increased overall survival. Gene set enrichment analysis (GSEA) revealed that DCBLD2 overexpression induces epithelial-mesenchymal transition (EMT) and activates the JAK/STAT3 pathway. We found that high DCBLD2 expression correlated with a poor clinical outcome, as well as tumorigenesis, invasion and metastasis of CRC cells. DCBLD2 may serve as a prognostic biomarker and a novel therapeutic target for CRC.

SNP rs12982687 affects binding capacity of lncRNA UCA1 with miR-873-5p: involvement in smoking-triggered colorectal cancer progression

BackgroundThis investigation was arranged to elucidate whether single nucleotide polymorphisms (SNPs) of lncRNA UCA1 was implicated in elevating colorectal cancer (CRC) risk by interacting with environmental exposures.MethodsLncRNASNP database was firstly adopted to predict SNPs that possibly affected binding of UCA1 with miRNAs and then the interactive effect of SNPs and environmental exposure on CRC risk was evaluated by recurring to type 2 gene-environment interactions (GEI) model. Besides, MTT assay, colony formation assay, transwell assay and wound healing assay were performed to assess the activity of CRC cell lines which carried distinct genotypes of specific SNPs. The impact of nicotine on activity of CRC cells was also appraised.ResultsSNP rs12982687 of UCA1 intervened in the binding capacity of UCA1 with several miRNAs, especially miR-873-5p. MiRNAs regulated by UCA1, as predicted by mirPath software, shared genes that were enriched in HIF1 signaling pathway. Moreover, homozygote TT of rs12982687 reduced CRC risk among smokers, and CRC cells that carried rs12982687 (CC) displayed strong migration and invasion. By contrast, miR-873-5p mimic, which reduced UCA1 expression, delayed metastasis of CRC cells (all P < 0.05). Additionally, nicotine not merely elevated UCA1 and HIF-1α expressions in CRC cells, but also facilitated proliferation and metastasis of CRC cells (P < 0.05).ConclusionsSNP rs12982687 was involved in smoking-triggered CRC progression, given its influence on UCA1's binding with miR-873-5p and HIF-1 signaling.

Circular RNA 0060745, a Novel circRNA, Promotes Colorectal Cancer Cell Proliferation and Metastasis Through miR-4736 Sponging.

PurposeRecent studies have shown that noncoding RNAs (ncRNAs) play essential roles in the development of a number of cancers. Circular RNAs (circRNAs) have been shown to contribute to the progression of colorectal cancer (CRC).MethodsIn this study, the expression levels of circular RNA 0060745 (circ_0060745), and microRNA 4736 (miR-4736) were measured using qRT-PCR. Kaplan–Meier survival analysis and receiver operating characteristic (ROC) analysis were used to evaluate the diagnostic value of circ_0060745. Transwell assay and cell counting kit-8 (CCK8) assay were used to determine the metastatic and proliferative capacity of CRC cells. The expression of chromosome segregation one like (CSE1L) was measured using Western blotting and immunohistochemistry (IHC). In addition, RNA pull-down assay and luciferase assay were performed to verify the targeted binding between miR-473,6 and circ_0060745, and between as miR-4736 and CSE1L.ResultsWe showed that circ_0060745 was upregulated in CRC, and was associated with unfavorable clinicopathological characteristics. We also showed that circ_0060745 acted as an oncogene and promoted CRC cell proliferation and metastasis. Circ_0060745 was primarily located in the cytoplasm. Furthermore, miR-4736 was downregulated in CRC, was a downstream target of circ_0060745, and mediated proliferation and metastasis. We showed that circ_0060745 sequestered miR-4736, which resulted in CRC cell proliferation and metastasis. Finally, we showed that CSE1L, a downstream target of miR-4736, was upregulated in CRC and mediated suppression of proliferation and metastasis in CRC.ConclusionThe results of this study showed that circ_0060745 promoted CRC cell proliferation and metastasis via modulation of miR-4736/CSE1L signaling. The Circ_0060745/miR-4736/CSE1L axis might be a novel target for the treatment of CRC.

CircHIPK3 promotes colorectal cancer cells proliferation and metastasis via modulating of miR-1207-5p/FMNL2 signal

Increasing evidences demonstrate that circular RNAs (circRNAs) are extensively implicated in various cancers including colorectal cancer (CRC). In the present study, we found that circRNA HIPK3 (circPIK3) was upregulated in CRC. We identified that circHIPK3 was closely related with unfavorable clinicopathological features in patients with CRC. Functional transwell assay and proliferation assay indicated that circHIPK3 served as an oncogene and promoted CRC cells migration, invasion and proliferation. Meanwhile, we found that formin like 2 (FMNL2) was a key downstream molecule in circHIPK3-induced metastasis and proliferation in CRC cells. We further verified that circHIPK3 was mainly located at cytoplasm through an immunofluorescence assay. An online bioinformatics screening and a GEO datasets analysis showed that microRNA 1207-5p (miR-1207-5p) was downregulated in CRC. Also, we found that miR-1207-5p shared a similar miR-1207-5p response elements (MREs-1207-5p). Meanwhile, we showed that miR-1207-5p suppressed CRC cells migration, invasion and proliferation via directly targeting of FMNL2. Even further, via a constructed luciferase assay, we indicated that circHIPK3 was another target of miR-1207-5p. Functionally, we proved that circHIPK3 enhanced FMNL2 mediated promotion of migration, invasion and proliferation by sponging of miR-1207-5p in CRC cells.In summary, the outcomes of this study illustrated that circHIPK3 promoted CRC cells migration, invasion and proliferation modulating of FMNL2 by sponging of miR-1207-5p. Our findings indicated that circHIPK3/miR-1207-5p/FMNL2 axis might be a new strategy in molecular treatment of CRC.

MiR-645 promotes invasiveness, metastasis and tumor growth in colorectal cancer by targeting EFNA5

MicroRNA-645 (miR-645) has been implicated in numerous types of human cancers including colon cancer. However, the effects and mechanisms of action of miR-645 dysregulation on the growth and malignancy of colorectal cancer (CRC) remain unclear. In this study, we demonstrated that miR-645 knockdown significantly diminished CRC cell migration and invasion and repressed epithelial–mesenchymal transition (EMT). Conversely, miR-645 overexpression enhanced CRC cell migration, invasion, and EMT. In vivo assays confirmed that miR-645 knockdown substantially reduced CRC growth and metastasis. Regarding the mechanism, ephrin-A5 (EFNA5) was identified as a direct target gene of miR-645. MiR-645 specifically targeted the 3′-untranslated region of EFNA5 mRNA and hindered its expression. EFNA5 knockdown attenuated the effects of miR-645 knockdown on CRC cell migration and invasion. Additionally, we noted a statistically significant inverse correlation between EFNA5 mRNA and miR-645 levels in tumors from 28 patients with CRC. Hence, miR-645 acts as an oncogenic miRNA that may increase CRC cell migration, invasiveness, and metastasis by targeting EFNA5.

HBXIP: a potential prognosis biomarker of colorectal cancer which promotes invasion and migration via epithelial-mesenchymal transition

Hepatitis B X-interacting protein (HBXIP) is highly expressed in many cancers, but the correlation between the expression of HBXIP and the clinical significance and underlying molecular mechanisms in colorectal cancer (CRC) is still unclear. We selected 186 specimens from CRC patients for analyzing the relationship between the expression of HBXIP and the clinical-pathological features by immunohistochemistry. Migration and invasion experiments were performed to examine the effect of HBXIP on CRC cell metastasis. Besides, we also explored the possible molecular mechanism of HBXIP regulation of CRC cell metastasis by Western blot. Our data indicated that the HBXIP was overexpressed in CRC tissues. High HBXIP expression was correlated with metastasis and shorter survival times in patients with CRC and served as an independent factor for poor prognosis. Moreover, HBXIP promotes CRC metastasis by enhancing the epithelial-mesenchymal transition (EMT) process. Our findings provide the first evidence that HBXIP induces EMT to promote metastasis and predicts the poor prognosis of CRC. Therefore, HBXIP may become a new target for CRC treatment.

Linc00662 Promotes Tumorigenesis and Progression by Regulating miR-497-5p/AVL9 Axis in Colorectal Cancer.

BackgroundRecently, multiple lines of evidence have demonstrated that linc00662 serves as an oncogene in various cancers. However, the exact mechanism of oncogenesis mediated by linc00662 in colorectal cancer (CRC) remains unknown. In this study, we aimed to explore the biological role of linc00662 in the regulation of CRC progression.MethodsBoth gene expression omnibus (GEO) and the cancer genome atlas (TCGA) datasets were used to evaluate the expression of linc00662. RT-qPCR was used to analyze the expression of linc00662, miR-497-5p, and AVL9 in CRC clinical samples and cell lines. Cell Counting Kit-8 (CCK-8), flow cytometry, transwell assay, and xenograft model were used to investigate the effect of linc00662 on CRC cell proliferation, cell cycle, and metastasis. Western blot analysis was used to analyze the expression of the epithelial-mesenchymal transition (EMT)-associated markers. Furthermore, bioinformatics analysis and mechanism assays were used to elucidate the underlying mechanism. Dual-luciferase reporter assays were used to analyze the regulatory relationships among linc00662, miR-497-5p, and AVL9.ResultsIn this study, we found that the expression of linc00662 was significantly upregulated in CRC tissues compared to normal tissues and positively correlated with tissue differentiation, T stage, and lymphatic metastasis. Further, our data showed that the expression of linc00662 was positively associated with lymph node metastasis, TMN stage, and poor-moderate differentiation. Patients with higher linc00662 expression level were more likely to have poorer overall survival. Knockdown of linc00662 inhibited CRC cell growth, induced cell apoptosis, triggered cell cycle arrest at G2/M phase, and suppressed cell migration and invasion through regulating the EMT pathway. Further, mechanistic studies revealed that knockdown of linc00662 significantly reduced the expression of AVL9, a direct target of miR-497-5p.ConclusionsLinc00662 was significantly upregulated in CRC, and mediated CRC progression and metastasis by competing with miR-497-5p to modulate the expression of AVL9. Therefore, our result sheds light on the potential application of linc00662 in CRC diagnosis and therapy.

Activation of the pattern recognition receptor NOD1 augments colon cancer metastasis

While emerging data suggest nucleotide oligomerization domain receptor 1 (NOD1), a cytoplasmic pattern recognition receptor, may play an important and complementary role in the immune response to bacterial infection, its role in cancer metastasis is entirely unknown. Hence, we sought to determine the effects of NOD1 on metastasis. NOD1 expression in paired human primary colon cancer, human and murine colon cancer cells were determined using immunohistochemistry and immunoblotting (WB). Clinical significance of NOD1 was assessed using TCGA survival data. A series of in vitro and in vivo functional assays, including adhesion, migration, and metastasis, was conducted to assess the effect of NOD1. C12-iE-DAP, a highly selective NOD1 ligand derived from gram-negative bacteria, was used to activate NOD1. ML130, a specific NOD1 inhibitor, was used to block C12-iE-DAP stimulation. Stable knockdown (KD) of NOD1 in human colon cancer cells (HT29) was constructed with shRNA lentiviral transduction and the functional assays were thus repeated. Lastly, the predominant signaling pathway of NOD1-activation was identified using WB and functional assays in the presence of specific kinase inhibitors. Our data demonstrate that NOD1 is highly expressed in human colorectal cancer (CRC) and human and murine CRC cell lines. Clinically, we demonstrate that this increased NOD1 expression negatively impacts survival in patients with CRC. Subsequently, we identify NOD1 activation by C12-iE-DAP augments CRC cell adhesion, migration and metastasis. These effects are predominantly mediated via the p38 mitogen activated protein kinase (MAPK) pathway. This is the first study implicating NOD1 in cancer metastasis, and thus identifying this receptor as a putative therapeutic target.

JNK Pathway Mediates Low Oxygen Level Induced Epithelial–Mesenchymal Transition and Stemness Maintenance in Colorectal Cancer Cells

(1) Background: Epithelial–mesenchymal transition (EMT) and cancer cell stemness maintenance (SM) are important factors for cancer metastasis. Although hypoxia has been considered as a possible factor for EMT induction and promotion of SM, studies in this area, apart from hypoxia-inducible factor (HIF) pathways and severe hypoxia, are scant. This study aimed to evaluate the effects of different oxygen levels on EMT induction and SM and elucidate the signaling pathways involved in colorectal cancer cells. (2) Methods: Cell morphological analysis, migration assay, immunofluorescence staining of cytoskeleton and Western blotting were performed on human colorectal cancer cells HT-29, DLD-1, and SW-480 cultured at 1%, 10%, and normal (21%) O2 levels. The role played by c-Jun N-terminal kinase (JNK) was evaluated through the use of the specific JNK inhibitor SP600125. (3) Results: This study evaluated 1% and 10% O2 are possible conditions for EMT induction and SM. This study also demonstrated the partial relieve of EMT induction and SM by SP600125, showing the importance of the JNK pathway in these processes. Furthermore, this study proposed a novel pathway on the regulation of Akt by JNK-c-Jun. (4) Conclusions: This study suggests 10% O2 as another possible condition for EMT induction, and SM and JNK pathways play important roles in these processes through multiple factors. Inhibition of JNK could be explored as treatment for inhibiting metastasis in colorectal cancer cells.