Suppressed Colorectal Cancer Cell Migration Research Articles

CD44 Suppression Improved the Chemosensitivity of HT-29 Colorectal Cancer Cells to 5-Fluorouracil and Inhibited Cell Migration.

CD44 plays a pivotal role through tumorigenesis by regulating cancer cell metastasis, stemness, and chemosensitivity and is considered a promising therapeutic target for human cancers, including colorectal cancer (CRC). Therefore, the present research aimed to examine the simultaneous therapeutic effect of CD44 silencing and 5-fluorouracil (5-FU) on in vitro tumorigenesis of CRC cells. CD44 expression was initially evaluated in TCGA datasets and CRC tissues. Furthermore, functional analysis was performed on HT-29 CRC cells overexpressing CD44. The cells were transfected with CD44 siRNA and then treated with 5-FU. Consequently, to explore the combination therapy effect on cell viability, migration, apoptosis, and chromatin fragmentation, we performed MTT assay, scratch assay, Annexin V/PI staining and DAPI staining assays, respectively. The spheroid and colony formation assays were further employed to investigate stemness features. The gene expression at protein and mRNA levels were explored using western blotting and qPCR. Our findings illustrated that CD44 was significantly overexpressed in CRC tissues compared to normal samples. The suppression of CD44 considerably promoted the chemosensitivity of HT-29 cells to 5-FU by apoptosis induction. Also, the combination therapy led to overexpression of apoptotic genes, including P53, caspase-3, and caspase-9, as well as downregulation of AKT1 expression. Furthermore, CD44 suppression, separately or combined with 5-FU, hindered stemness properties in HT-29 cells via downregulation of Sox2 and Nanog expression. Besides, the combination therapy remarkably downregulated MMPs and suppressed CRC cell migration. Considering its involvement in chemosensitivity to 5-FU, CD44 could be suggested as a potential target for improving the efficiency of CRC chemotherapy.

PLHPP2 inhibits the stemness of colorectal cancer by inactivating the Nrf2 signaling pathway.

Pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase 2 (PHLPP2) is a critical regulator of cellular homeostasis and acts as a tumor suppressor in multiple human cancers. However, its exact biological function in colorectal cancer (CRC) and the underlying molecular mechanism remain poorly understood. The correlation between the transcription and protein abundance of PHLPP2 was analyzed using proteomic and corresponding transcriptional data. Immunohistochemistry was used to validate the protein expression and the role of PHLPP2 in patient prognosis. In addition, a series of experiments in vitro and in vivo were performed to investigate the underlying molecular mechanism. Immunohistochemical staining of a CRC tissue microarray revealed that PHLPP2 protein expression was significantly downregulated compared to that in adjacent normal tissues. Low expression of PHLPP2 was an independent prognostic risk factor for poor survival. A nomogram established by integrating PHLPP2 expression and traditional clinicopathological factors achieved more reliable prognostic assessment in CRC patients. Additionally, PHLPP2 overexpression suppressed CRC cell migration, invasion and stemness in vitro as well as tumorigenesis in vivo. Further experiments revealed that upregulation of PHLPP2 increased ROS levels by suppressing the Nrf2-ARE signaling pathway, which inhibited the stemness of CRC cells. Moreover, incubation with sulforaphane, a selective chemical agonist of Nrf2, reversed this inhibitory effect in CRC. PHLPP2 acts as a tumor suppressor gene in CRC by restraining the Nrf2-ARE signaling pathway and increasing ROS levels, affecting the stemness of CRC cells. These anticancer molecular mechanisms indicate PHLLPP2's significant clinical value in prognosis prediction and targeted therapy.

Inhibition of the cell migration, invasion and chemoresistance of colorectal cancer cells through targeting KLF3 by miR-365a-3p.

Background: Metastasis and chemoresistance limit treatment efficacy of colorectal cancer (CRC) patients. MicroRNAs (miRNAs) have been believed to be candidate biomarkers for tumor cell proliferation, metastasis and chemoresistance, but the related molecular mechanisms are not clear for prognosis prediction.Aims: We aimed to investigate the role of miR-365a-3p in metastasis and chemoresistance of CRC.Methods: The expression levels of miR-365a-3p in clinical CRC tissues were analyzed. The effects of miR-365a-3p expression levels on tumor chemoresistance, invasion and migration were also determined. A dual luciferase reporter gene assay was used to determine the effect of miR-365a-3p on its target gene, Kruppel-like factor 3 (KLF3), and the effect of the miR-365a-3p/KLF3 axis on CRC cell chemoresistance, migration and invasion was further investigated.Results: In patients with CRC with lymph node or distant organ metastasis or in CRC cell lines, the expression levels of miR-365a-3p were significantly downregulated. In addition, the findings of Transwell assays demonstrated that miR-365a-3p significantly suppressed CRC cell migration and invasion. The dual luciferase reporter gene assay results suggested that miR-365a-3p may play an important role in the regulation of migration, invasion and chemoresistance in CRC cells.Conclusions: The findings of present study provided evidence to suggest that miR-365a-3p may be a potential tumor suppressor gene in CRC and may inhibit the migration, invasion and chemoresistance of CRC cells. These results suggested that targeting miR-365a-3p/KLF3 axis may represent a potential therapeutic intervention for metastatic disease in patients with CRC.

MiR-144 inhibits colorectal cancer cell migration and invasion by regulating PBX3.

MicroRNAs (miRNA) are aberrantly expressed in various human cancers, including colorectal cancer (CRC). We aim to investigate the functional role and underlying mechanism of miR-144 in CRC. The expressional level of miR-144 and pre-leukemia transcription factor 3 (PBX3) in CRC tissues and cells was confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. The migration and invasion of CRC cells were detected by transwell assay. Luciferase reporter assay was performed to determine the specific target of miR-144 in CRC cells. The results displayed that miR-144 expression was significantly decreased in CRC tissues and cells compared to that in normal controls. Additionally, miR-144 mimic suppressed, while miR-144 inhibitor promoted the ability of CRC cell migration and invasion. More importantly, PBX3 was the direct target of miR-144 in regulating CRC development and PBX3 could reverse the inhibitory effect of miR-144 mimic on CRC cells. PBX3 expression was significantly increased in CRC and negatively correlated with miR-144 expression. In conclusion, miR-144 suppressed CRC cell migration and invasion by targeting PBX3, suggesting its potential value in the diagnosis and 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.

The lncRNA FEZF1-AS1 Promotes the Progression of Colorectal Cancer Through Regulating OTX1 and Targeting miR-30a-5p.

Long noncoding RNAs (lncRNAs) participate in and regulate the biological process of colorectal cancer (CRC) progression. Our previous research identified differentially expressed lncRNAs in 10 CRC tissues and 10 matched nontumor tissues by next-generation sequencing (NGS). In this study, we identified an lncRNA, FEZF1 antisense RNA 1 (FEZF1-AS1), and further explored its function and mechanism in CRC. We verified that FEZF1-AS1 is highly expressed in CRC tissues and cell lines. Through functional experiments, we found that reduced levels of FEZF1-AS1 significantly suppressed CRC cell migration, invasion, and proliferation and inhibited tumor growth in vivo. Mechanistically, we discovered that reduced levels of the lncRNA FEZF1-AS1 inhibited the activation of epithelial–mesenchymal transition (EMT); the overexpression of orthodenticle homeobox 1 (OTX1) partially rescued the FEZF1-AS1-induced inhibition of protein expression. It indicated that FEZF1-AS1 may play a role in the occurrence and development of CRC by regulating the FEZF1-AS1/OTX1/EMT pathway. Furthermore, it was reported that FEZF1-AS1 is located in both the nucleus and cytoplasm of HCT116 cells. Dual-luciferase reporter assays verified that FEZF1-AS1 directly binds miR-30a-5p and negatively regulated each other. Further, we showed that 5′-nucleotidase ecto (NT5E) is a direct target of miR-30a-5p, and the inhibition of miR-30a-5p expression partially rescued the inhibitory effect of FEZF1-AS1 on NT5E. Our results indicated that the mechanism by which FEZF1-AS1 positively regulates the expression of NT5E is through sponging miR-30a-5p. Our study demonstrated that lncRNA FEZF1-AS1 is involved in the development of CRC and may serve as a diagnostic and therapeutic target for CRC patients.

Long noncoding RNA SNHG6 functions as a competing endogenous RNA by sponging miR-181a-5p to regulate E2F5 expression in colorectal cancer.

BackgroundLong noncoding RNAs (lncRNAs) have been identified as a novel class of regulators implicated in diverse biological processes in human cancers. Currently, evidence have shown that SNHG6, a cancer-associated lncRNA, exerts critical functions in gastric cancer and hepatocellular carcinoma; however, its role in colorectal cancer (CRC) remains unclear.MethodsThe expression of SNHG6 was determined by quantitative real-time PCR in CRC tissues and cells. SNHG6 was downregulated by using RNAi technology. Cell proliferation was examined by MTT and clone formation assays. Cell migration and invasion were determined by wound healing and transwell assays. Fluorescence in situ hybridization assays were performed to examine subcellular localization of SNHG6 in CRC cells. Fluorescence reporter and Western blot assays were used to explore the potential mechanisms of SNHG6 in CRC progression.ResultsIn this study, we found that SNHG6 was significantly upregulated in CRC tissues and cell lines, compared with normal tissues and normal colorectal epithelial cell line NCM460, respectively. High expression of SNHG6 was positively correlated with tumor size, advanced TNM stage, and distant metastasis. Survival analyses revealed that SHNG6 was significantly associated with poor clinical outcomes and could serve as an independent prognostic factor. Loss-of-function studies demonstrated that SNHG6 knockdown inhibited CRC cell proliferation, induced G0/G1 arrest, promoted apoptosis, suppressed CRC cell migration and invasion, and restrained tumor growth. Mechanistic investigations showed that SNHG6 acted as a competing endogenous RNA for miR-181a-5p and attenuated the inhibitory effect of miR-181a-5p on E2F5.ConclusionTaken together, these results demonstrated that SNHG6 plays a crucial role in CRC progression via miR-181a-5p/E2F5 axis. Therefore, SNHG6 may serve as a prognostic and therapeutic biomarker in CRC.

NLRP3 inflammasomes in macrophages drive colorectal cancer metastasis to the liver

Inflammation and immunity are important determinants of cancer initiation, promotion, and progression to cancer equilibrium or suppression. The NOD-like receptor family pyrin domain containing 3 (NLRP3) is an oligomeric intracellular immune receptor, and the main component of inflammasome. As a widely distributed effector of innate immunity, NLRP3 inflammasome affects development of many cancer types, but its exact role in colorectal cancer (CRC) is controversial. We found that cells with the macrophage (MΦ) marker CD68 and strong NLRP3 expression densely surrounded CRC tissue. The NLRP3 inflammasome was activated in MΦs by MΦ–CRC cell crosstalk; it resulted in faster migration of CRC cells, whereas blocking NLRP3 signaling suppressed CRC cell migration in vitro, and metastatic ability in vivo. NLRP3 signaling activation in MΦs can contribute to CRC cell migration and invasion.

MiR-106b-5p inhibits the invasion and metastasis of colorectal cancer by targeting CTSA.

BackgroundAlthough miR-106b-5p has been reported to play a pivotal role in various human malignancies, its role in colorectal cancer (CRC) remains unknown. In this study, we comprehensively investigated miR-106b-5p expression and biologic functions in CRC and the molecular mechanism involved.Patients and methodsmiR-106b-5p expression was detected in CRC tissues and cell lines by quantitative reverse transcription-polymerase chain reaction. The effects of miR-106b-5p on metastasis were determined in vitro using transwell assays, and in vivo effects were evaluated using a mouse tail vein injection model. Downstream targets of miR-106b-5p were confirmed using bioinformatics programs, luciferase assays, and rescue experiments. Target gene expression and clinicopathologic parameters were also analyzed.ResultsmiR-106b-5p expression was lower in CRC tissues than in corresponding nontumorous tissues (P=0.009), and miR-106b-5p downregulation was negatively associated with lymph node metastasis (P=0.006). Functional assays demonstrated that miR-106b-5p overexpression suppressed CRC cell migration and invasion in vitro and lung metastasis formation in vivo. In addition, luciferase assays confirmed that miR-106b-5p directly bound to the 3′ untranslated region of cathepsin A (CTSA) and that miR-106b-5p suppressed CRC cell migration and invasion by targeting CTSA. Clinicopathologic analysis showed that CTSA was significantly upregulated in CRC, and increased CTSA was negatively associated with lymph node metastasis (P=0.012).ConclusionOur findings revealed that miR-106b-5p inhibits CRC metastasis by upregulating CTSA expression, which may lead to novel therapeutic strategies for CRC patients.

RETRACTED ARTICLE: LncRNA TUG1 promoted KIAA1199 expression via miR-600 to accelerate cell metastasis and epithelial-mesenchymal transition in colorectal cancer

BackgroundLncRNA TUG1 has been reported to be highly expressed in CRC samples and cells and promoted metastasis by affecting EMT, indicating a poor prognosis for colorectal cancer (CRC). In this study, we determined the underlying mechanism for tumor oncogenesis of lncRNA TUG1 in CRC metastasis.MethodsThe expressions of miR-600 and KIAA1199 in 76 CRC patients and CRC cells and CRC metastatic tissues were determined using qRT-PCR. Epithelial-mesenchymal transition (EMT)-related proteins were determined using western blot. CRC cell metastasis was assessed by colony formation, wound healing and transwell assay. Luciferase reporter gene assay was used to confirm miR-600 binding to KIAA1199 3’UTR.ResultsOur data showed that lncRNA TUG1 was upregulated in CRC cells, miR-600 was downregulated in CRC tissues, cell lines and CRC metastatic tissues, and low miR-600 expression predicted a poor clinical prognosis. Overexpression of miR-600 suppressed CRC cell migration/invasion and EMT-related proteins in vitro, inhibited tumor volume and weight, and decreased the number of CRC liver metastasis in vivo. KIAA1199 was upregulated in CRC tissues, and was negatively regulated by miR-600. KIAA1199 overexpression promoted CRC cell migration and invasion, which reversed the inhibition effect of miR-600 mimic on migration and invasion of CRC cells. Moreover, TUG1 negatively regulated miR-600, and inhibition of TUG1 suppressed CRC cell migration and invasion and EMT-related proteins via regulating miR-600.ConclusionOur study proved that TUG1 promoted KIAA1199 expression to accelerate EMT and metastasis of CRC cell through inhibition of miR-600 expression.

MiR-139-5p inhibits migration and invasion of colorectal cancer by downregulating AMFR and NOTCH1.

MicroRNAs (miRNAs) that exert function by posttranscriptional suppression have recently brought insight in our understanding of the role of non-protein-coding RNAs in carcinogenesis and metastasis. In this study, we described the function and molecular mechanism of miR-139-5p in colorectal cancer (CRC) and its potential clinical application in CRC. We found that miR-139-5p was significantly downregulated in 73.8% CRC samples compared with adjacent noncancerous tissues (NCTs), and decreased miR-139-5p was associated with poor prognosis. Functional analyses demonstrated that ectopic expression of miR-139-5p suppressed CRC cell migration and invasion in vitro and metastasis in vivo. Mechanistic investigations revealed that miR-139-5p suppress CRC cell invasion and metastasis by targeting AMFR and NOTCH1. Knockdown of the two genes phenocopied the inhibitory effect of miR-139-5p on CRC metastasis. Furthermore, the protein levels of the two genes were upregulated in CRC samples compared with NCTs, and inversely correlated with the miR-139-5p expression. Increased NOTCH1 protein expression was correlated with poor prognosis of CRC patients. Together, our data indicate that miR-139-5p is a potential tumor suppressor and prognostic factor for CRC, and targeting miR-139-5p may repress the metastasis of CRC and improve survival.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0093-5) contains supplementary material, which is available to authorized users.

MiR-150-5p suppresses colorectal cancer cell migration and invasion through targeting MUC4.

Growing evidence suggests that miR-150-5p has an important role in regulating genesis of various types of cancer. However, the roles and the underlying mechanisms of miR-150-5p in development of colorectal cancer (CRC) remain largely unknown. Transwell chambers were used to analyze effects on cell migration and invasion by miR-150-5p. Quantitative real-time PCR (qRT-PCR), Western blotting and dual-luciferase 3' UTR reporter assay were carried out to identify the target genes of miR-150-5p. In our research, miR-150-5p suppressed CRC cell migration and invasion, and MUC4 was identified as a direct target gene. Its effects were partly blocked by re-expression of MUC4. In conclusiomn, miR-150-5p may suppress CRC metastasis through directly targeting MUC4, highlighting its potential as a novel agent for the treatment of CRC metastasis.

The study on functions and mechanisms of miR-106b in the metastasis of colorectal cancer.

e14685 Background: Emerging evidence has shown that aberrantly expressed microRNAs (miRNAs) are highly associated with tumour initiation, development and progression. However, little is known about the potential role of miRNAs in colorectal cancer (CRC) metastasis. Methods: The miR-106b expression levels in CRC tissues were deteted by quantitative reverse transcription polymerase chain reaction (qRT-PCR) to investigate its relationship with clinical characteristics or survival of CRC patients. Using the CRC cell lines ectopically expressing miR-106b conducted by lentivirus transduction as experimental material, cell culture, cell growth analysis and transwell used to analyze the potential influence of miR-106b on CRC metastasis. In addition, luciferase assays and western blot were used for the target gene study. Results: In this study, we firstly investigate the role and related mechanism of miR-106b in CRC metastasis. We found that miR-106b expression was lower than in CRC compared with their corresponding non-tumorous tissues (P=0.009), and the downregulated miR-106b was negatively associated with lymph-node metastasis. Functional assays demonstrated that overexpression of miR-106b suppressed CRC cell migration and invasion in vitro. In addition, we confirmed that ATAD2 was a direct and functional target of miR-106b. Overexpression of miR-106b in CRC cells could reduce the mRNA and protein levels of ATAD2, whereas miR-106b silencing significantly increased ATAD2 expression. Luciferase assays confirmed that miR-106b could directly bind to 3′ untranslated region of ATAD2. Knockdown of ATAD2 significantly inhibited CRC cell migration and invasion. We further found that ATAD2 was involved in miR-106b-induced suppression of CRC cell migration and invasion. Conclusions: By understanding the functions and molecular mechanisms of miR-106b in CRC, we found mir-106b through targeting ATAD2 inhibited CRC cell migration and invasion. These results suggest that patients with downregulated miR-106b are prone to lymph node metastasis and tumour progression. miR-106b may have a therapeutic potential to suppress CRC metastasis.