Invasive Ability Of Colorectal Cancer Cells Research Articles

Silencing GDI2 inhibits proliferation, migration and invasion of colorectal cancer through activation of p53 signaling pathway

ObjectiveTo investigate the effect of silencing GDP dissociation inhibitor 2 (GDI2) on colorectal cancer development and possible mechanisms based on transcriptomic analysis. MethodsThe differences in the expression levels of GDI2 in normal colorectal tissues and tumor tissues of colorectal cancer (CRC) patients were detected. The correlation of GDI2 expression levels with survival and clinical characteristics of CRC patients was analyzed. The effects of GDI2 expression levels on the biological functions of CRC cells were examined by CCK-8 assay, plate clone formation assay, wound healing assay, and Transwell assay. The effect of GDI2 on the proliferation and growth of xenograft tumors was investigated by a xenograft tumor model of CRC in nude mice. Based on transcriptomics, we explored the possible mechanisms and associated pathways of the effect of silencing GDI2 on CRC cells. Cellular experiments and Western blot assays were performed to verify the potential mechanisms and related pathway of GDI2 action on CRC. ResultsThe expression levels of GDI2 in CRC tissues and cells were higher than those in normal tissues and cells. The expression level of GDI2 correlated with clinical characteristics such as lymphatic metastasis, tumor stage, tumor volume, and lymphocyte count. Silencing of GDI2 reduced the proliferative activity and migration and invasion ability of CRC cells, as well as inhibited the proliferation of CRC xenograft tumors. The differentially expressed genes were significantly enriched in biological processes such as cell cycle arrest and the p53 signaling pathway after GDI2 silencing. The percentage of G0/G1 phase cells in CRC cells was increased after silencing GDI2 as verified by flow cytometry. RAB5A was highly associated with the p53 pathway and could interact with TP53 via the ZFYVE20 protein. The mutual binding between GDI2 protein and RAB5A protein was verified by immunoprecipitation assay. Silencing GDI2 while overexpressing RAB5A reversed the reduced proliferation, migration, and invasion ability as well as cell cycle arrest of CRC cells. Meanwhile, the addition of p53 signaling pathway inhibitor Pifithrin-α (PFT-α) also reversed the biological effects of silencing GDI2 on CRC cells. The p-p21 and p-p53 protein expression levels were significantly greater in the sh-GDI2 group than in the sh-NC group. However, the p-p21 and p-p53 protein expression levels were reduced after silencing GDI2 while overexpressing RAB5A. ConclusionSilencing GDI2 activates the p53 signaling pathway by regulating RAB5A expression levels, which in turn induces cell cycle arrest and ultimately affects the proliferative activity, migration, and invasive ability of CRC cells.

Ubiquitin-specific peptidase 15 regulates the TFAP4/PCGF1 axis facilitating liver metastasis of colorectal cancer and cell stemness

The tumor recurrence and metastasis of colorectal cancer (CRC) are responsible for most of CRC-linked mortalities. It is an urgent need to deeply investigate the pathogenesis of CRC metastasis and look for novel targets for its treatment. The current study aimed to investigate the effects of ubiquitin-specific peptidase 15 (USP-15) on the CRC progression. In vivo, a mouse model of liver metastasis of CRC tumor was established to investigate the role of USP-15. In vitro, the migrated and invasive abilities of CRC cells were assessed by transwell assay. Cell stemness was evaluated by using sphere formation assay. The underlying mechanism was further explored by employing the co-immunoprecipitation, dual luciferase reporter assay, oligonucleotide pull-down assay, and chromatin immunoprecipitation assay. The results showed that USP-15 was upregulated in CRC patients with liver metastasis and high metastatic potential cell lines of CRC. Loss of USP-15 repressed the epithelial-to-mesenchymal transition (EMT), migration, invasion, and stemness properties of CRC cells in vitro. Downregulation of USP-15 reduced the liver metastasis of mice in vivo. USP-15 upregulation obtained the contrary effects. Subsequently, USP-15 deubiquitinated transcription factor AP-4 (TFAP4) and enhanced its protein stability. TFAP4 could transcriptionally activated polycomb group ring finger 1 (PCGF1). The pro-cancer effects of USP-15 were rescue by the knockdown of TFAP4 or PCGF1. In conclusions: USP-15 facilitated the liver metastasis by the enhancement of cell stemness and EMT in CRC, which was at least partly mediated by the deubiquitination of TFAP4 upon the upregulation of PCGF1.

LINC00339 accelerates invasion and migration of colorectal cancer via mediating miRNA-30a-5p.

To analyze the biological function of LINC00339 in the progression of colorectal cancer (CRC). We aim to provide directions in the early-stage treatment of CRC. LINC00339 level in 60 paired CRC tissues and paracancerous tissues was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The correlation between the LINC00339 level and clinical parameters was analyzed. Moreover, the LINC00339 level in CRC cell lines was determined as well. LINC00339 expression was changed in HCT-8 and HCT-116 cell lines by transfection of LINC00339 overexpression plasmid or anti-LINC00339. The regulatory effects of LINC00339 on the migratory and invasive abilities of CRC cells were evaluated through a series of functional experiments. Dual-luciferase reporter gene assay and rescue experiments were conducted to verify the interaction of LINC00339 and miRNA-30a-5p in mediating the progression of CRC. LINC00339 was upregulated in CRC tissues relative to paracancerous tissues. CRC patients with higher levels of LINC00339 had higher rates of lymph node metastasis and distant metastasis, and worse prognosis than those with lower levels. Knockdown of LINC00339 attenuated migratory and invasive abilities of HCT-116 cells. Overexpression of LINC00339 in HCT-8 obtained the opposite trends. In addition, we verified a negative correlation between LINC00339 and miRNA-30a-5p in CRC tissues. LINC00339 served as a ceRNA to absorb miRNA-30a-5p. Rescue experiments confirmed that miRNA-30a-5p knockdown revered the regulatory effects of LINC00339 on the migratory and invasive abilities of CRC cells. LINC00339 was closely correlated to metastasis and poor prognosis of CRC. It accelerates CRC cells to migrate and invade via mediating miRNA-30a-5p.

RNA demethylase ALKBH5 promotes colorectal cancer progression by posttranscriptional activation of RAB5A in an m6A‐YTHDF2‐dependent manner

N6-methyladenosine (m6A) modification is an emerging epigenetic regulatory mechanism in tumourigenesis. Considering that AlkB homolog 5 (ALKBH5) is a well-described m6A demethylase in previous enzyme assays, we aimed to investigate the role of m6A methylation alteration conferred by disturbed ALKBH5 in colorectal cancer (CRC) development. Expression of ALKBH5 and its correlation with clinicopathological characteristics of CRC were evaluated using the prospectively maintained institutional database. The molecular role and underlying mechanism of ALKBH5 in CRC were explored using in vitro and in vivo experiments with methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq, MeRIP-qPCR, RIP-qPCR and luciferase reporter assays. ALKBH5 expression was significantly upregulated in CRC tissues compared to the paired adjacent normal tissues, and higher expression of ALKBH5 was independently associated with worse overall survival in CRC patients. Functionally, ALKBH5 promoted the proliferative, migrative and invasive abilities of CRC cells in vitro and enhanced subcutaneous tumour growth in vivo. Mechanistically, RAB5A was identified as the downstream target of ALKBH5 in CRC development, and ALKBH5 posttranscriptionally activated RAB5A by m6A demethylation, which impeded the YTHDF2-mediated degradation of RAB5A mRNA. In addition, we demonstrated that dysregulation of the ALKBH5-RAB5A axis could affect the tumourigenicity of CRC. ALKBH5 facilitates the progression of CRC by augmenting the expression of RAB5A via an m6A-YTHDF2-dependent manner. Our findings suggested that ALKBH5-RAB5A axis might serve as valuable biomarkers and effective therapeutic targets for CRC.

NONHSAG028908.3 sponges miR‑34a‑5p to promote growth of colorectal cancer via targeting ALDOA.

Colorectal cancer (CRC) is an aggressive tumor, whose development is considered to be modulated by certain long non‑coding RNAs (lncRNAs). Therefore, the aim of the present study was to investigate the regulatory mechanism of lncRNA NONHSAG028908.3 on CRC. Data from The Cancer Genome Atlas (TCGA) database revealed that NONHSAG028908.3 was increased in CRC tissues compared with normal tissues (P<0.001). The results of reverse transcription‑quantitative PCR indicated that NONHSAG028908.3 was upregulated in four types of CRC cells compared with that in NCM460, a normal colorectal cell line. MTT, BrdU, and flow cytometric assays were applied to evaluate CRC cell growth. The migratory and invasive abilities of CRC cells were detected using wound healing and Transwell assays. Silencing of NONHSAG028908.3 inhibited proliferation, migration, and invasion of CRC cells. A dual‑luciferase reporter assay demonstrated that NONHSAG028908.3 served as a sponge to combine with microRNA (miR)‑34a‑5p. MiR‑34a‑5p suppressed the aggressiveness of CRC cells. The effects induced by NONHSAG028908.3 knockdown were partly reversed by inhibition of miR‑34a‑5p. Furthermore, miR‑34a‑5p, a target of NONHSAG028908.3, modulated aldolase, fructose‑bisphosphate A (ALDOA) expression in a negative feedback manner. Suppression of NONHSAG028908.3 notably decreased ALDOA expression, which was rescued via silencing of miR‑34a‑5p. Moreover, suppression of ALDOA revealed the inhibitory action on CRC cell growth and migration. In summary, the data of the present study indicate that NONHSAG028908.3 may positively regulate ALDOA via sponging miR‑34a‑5p, thereby promoting malignant activities in CRC.

USP20 is a predictor of poor prognosis in colorectal cancer and associated with lymph node metastasis, immune infiltration and chemotherapy resistance.

Colorectal cancer (CRC) is a highly prevalent malignancy with a poor prognosis. USP20 can support progression of variety of tumors. USP20 was shown to promote breast tumor metastasis, and proliferation of oral squamous carcinoma cells. However, the role of USP20 in CRC remains unclear. We used bioinformatics to analyze the expression and prognosis of USP20 in pan-cancer and explore the relationship between USP20 expression and immune infiltration, immune checkpoints, and chemotherapy resistance in CRC. The differential expression and prognostic role of USP20 in CRC was validated by qRT-PCR and immunohistochemistry. Cox univariate and multivariate analyses were performed to assess risk factors for poor prognosis of CRC, and new prognostic prediction models were constructed and evaluated by decision curve analysis (ROC) and receiver operating characteristic (DCA). USP20 was overexpressed in CRC cell lines to explore the effect of USP20 on the functionalities of CRC cells. Enrichment analyses were used to explore the possible mechanism of USP20 in CRC. The expression of USP20 was lower in CRC tissues than adjacent normal tissues. Compared with low USP20 expression patients, CRC patients with high USP20 expression level had shorter OS. Correlation analysis showed that USP20 expression was associated with lymph node metastasis. Cox regression analysis revealed USP20 as an independent risk factor for poor prognosis in CRC patients. ROC and DCA analyses showed that the performance of the newly constructed prediction model was better than the traditional TNM model. Immune infiltration analysis shown that USP20 expression is closely associated with T cell infiltration in CRC. A co-expression analysis showed that USP20 expression was positively correlated with several immune checkpoint genes including ADORA2A, CD160, CD27 and TNFRSF25 genes and positively associated with multiple multi-drug resistance genes such as MRP1, MRP3, and MRP5 genes. USP20 expression positively correlated with the sensitivity of cells to multiple anticancer drugs. Overexpression of USP20 enhanced the migration and invasive ability of CRC cells. Enrichment pathway analyses showed the USP20 may play a role via the Notch pathway, Hedgehog pathway and beta-catenin pathway. USP20 is downregulated in CRC and associated with prognosis in CRC. USP20 enhances CRC cells metastasis and is associated with immune infiltration, immune checkpoints, and chemotherapy resistance.

Nestin protein affects the maintenance of stem characteristics of colorectal cancer cells based on p53 dependent pathway.

Colorectal cancer (CRC) is a tumor with high incidence and poor prognosis. An increasing number of studies have shown that intermediate filament proteins, such as nestin, participate in the regulation of tumor progression. However, the mechanism related to CRC is complex, and the role and underlying mechanism of nestin have not been elucidated in CRC. We conducted quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blot analyses to examine the mRNA and protein levels in CRC and normal tissues. siRNAs targeting Nestin were transfected into CRC cells and then cell counting kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), sphere formation, and transwell analyses were used to assess the role of nestin in the proliferation, stem activity, migration, and invasive ability of CRC cells. Afterwards, nestin was overexpressed in CRC cells and P53 was overexpressed as a rescue group. CCK-8, EdU dyeing, sphere formation, and transwell assay was used to evaluated the role of Nestin/p53 axis in CRC cells. We found high nestin expression and low p53 expression in CRC tissues and cells. Functionally, silencing of nestin suppressed the multiplication, stemness, and metastatic ability of Caco-2 and RKO cells. Encouragingly, rescue experiments suggested that overexpression of p53 partly restored the impacts of nestin overexpression on the viability, proliferation, and metastatic ability of CRC cells. We confirmed that nestin and p53 play a functional role in the progression of CRC, and they may act as potential therapeutic targets for CRC treatment.

GLUT3 Promotes Epithelial-Mesenchymal Transition via TGF-β/JNK/ATF2 Signaling Pathway in Colorectal Cancer Cells.

Glucose transporter (GLUT) 3, a member of the GLUTs family, is involved in cellular glucose utilization and the first step in glycolysis. GLUT3 is highly expressed in colorectal cancer (CRC) and it leads to poor prognosis to CRC patient outcome. However, the molecular mechanisms of GLUT3 on the epithelial–mesenchymal transition (EMT) process in metastatic CRC is not yet clear. Here, we identified that activation of the c-Jun N-terminal kinase (JNK)/activating transcription factor-2 (ATF2) signaling pathway by transforming growth factor-β (TGF-β) promotes GLUT3-induced EMT in CRC cells. The regulation of GLUT3 expression was significantly associated with EMT-related markers such as E-cadherin, α- smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), vimentin and zinc finger E-box binding homeobox 1 (ZEB1). We also found that GLUT3 accelerated the invasive ability of CRC cells. Mechanistically, TGF-β induced the expression of GLUT3 through the phosphorylation of JNK/ATF2, one of the SMAD-independent pathways. TGF-β induced the expression of GLUT3 by increasing the phosphorylation of JNK, the nuclear translocation of the ATF2 transcription factor, and the binding of ATF2 to the promoter region of GLUT3, which increased EMT in CRC cells. Collectively, our results provide a new comprehensive mechanism that GLUT3 promotes EMT process through the TGF-β/JNK/ATF2 signaling pathway, which could be a potential target for the treatment of metastatic CRC.

LncRNA SNHG3 is responsible for the deterioration of colorectal carcinoma through regulating the miR-370-5p/EZH1 axis.

To illustrate the biological function of long non-coding RNA (lncRNA) SNHG3 in the deterioration of colorectal cancer (CRC) by regulating the miR-370-5p/EZH1 axis. SNHG3 levels in fifty pairs of CRC and non-tumor tissues were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Its correlation to tumor staging, lymph node metastasis and prognosis of CRC was analyzed. Cell counting kit-8 (CCK-8) and 5-Ethynyl-2'- deoxyuridine (EdU) assay were conducted to assess the influence of SNHG3 on CRC cell proliferation in vitro. In addition, invasive ability of CRC cells transfected with si-SNHG3 was explored by transwell assay. The binding and regulatory relations in the SNHG3/miR-370-5p/EZH1 axis were ascertained by Dual-Luciferase reporter assay. SNHG3 was upregulated in CRC tissues and cell lines. Its high level was correlated to advanced tumor staging, positive lymph node metastasis and poor prognosis of CRC. Knockdown of SNHG3 reduced proliferative and invasive rates of SW480 and HT29 cells. The SNHG3/miR-370-5p/EZH1 axis was ascertained. In addition, knockdown of miR-370-5p enhanced proliferative and invasive rates of SW480 and HT29 cells. LncRNA SNHG3 induces proliferative and invasive potentials of CRC by regulating the miR-370-5p/EZH1 axis.

M6A methylation of DEGS2, a key ceramide-synthesizing enzyme, is involved in colorectal cancer progression through ceramide synthesis

N6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulator in cancer. However, the understanding of m6A modification on lipid metabolism regulation in colorectal cancer (CRC) is very limited. Here, we observed that human CRCs exhibited increased m6A mRNA methylation mediated by dysregulation of m6A erasers and readers. By performing methylated RNA-immunoprecipitation sequencing (MeRIP-seq) and transcriptomic sequencing (RNA-seq), we identified DEGS2 as a downstream target of m6A dysregulation. Overexpression or knockdown of DEGS2 confirmed the role of DEGS2 in proliferation, invasion and metastasis of CRC both in vitro and in vivo. Mechanistic studies identified the specific m6A modification site within DEGS2 mRNA, and mutation of this target site was found to drastically enhance the proliferative and invasive ability of CRC cells in vitro and promote tumorigenicity in vivo. Lipidome analysis showed that lipid metabolism was dysregulated in CRC. Moreover, ceramide synthesis was suppressed due to DEGS2 upregulation mediated by m6A modification in CRC tissues. Our findings highlight that the function of DEGS2 m6A methylation in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.

Extracellular vesicles derived from cancer-associated fibroblast carries miR-224-5p targeting SLC4A4 to promote the proliferation, invasion and migration of colorectal cancer cells.

More and more studies indicated that extracellular vesicles (EVs) carrying miRNAs have been potential biomarkers of various cancers including colorectal cancer (CRC). This study aims to explore the function of miR-224-5p carried by EVs derived from cancer-associated fibroblasts (CAFs) in CRC. Here, we found that miR-224-5p was highly expressed while SLC4A4 was lowly expressed in CRC cells. Moreover, dual-luciferase reporter gene assay testified that miR-224-5p targeted SLC4A4. The expression of miR-224-5p in CAFs-derived EVs was found to be elevated. It was also testified that CAFs-derived EVs could transfer miR-224-5p into CRC cells. miR-224-5p in CAFs-derived EVs facilitated the proliferation, migration, invasion and anti-apoptosis of CRC cells. Overexpressing miR-224-5p increased the proliferative, migratory and invasive abilities of CRC cells and inhibit CRC cell apoptosis, while overexpressing SLC4A4 caused the opposite result. Research in vitro and in vivo further indicated that miR-224-5p promoted CRC cell progression via binding to its downstream target gene SLC4A4. Rescue assay also demonstrated that overexpressing miR-224-5p reversed the inhibitory effect of overexpressed SLC4A4 on cancer cell growth. In addition, in vivo assay identified that high level of miR-224-5p promoted the growth of cancer cells in mice in vivo. In conclusion, we explored the effect of miR-224-5p in CRC, which helps for further exploration of new methods for CRC targeted therapy.

Long noncoding RNA MCM3AP-AS1 enhances cell proliferation and metastasis in colorectal cancer by regulating miR-193a-5p/SENP1.

BackgroundAccumulating evidences have shown that long noncoding RNAs (lncRNAs) play key roles in many diseases, including cancer. Several studies reported that MCM3AP antisense RNA 1 (MCM3AP‐AS1) was associated with the tumorigenesis and progression. However, the specific function and mechanism of MCM3AP‐AS1 in colorectal cancer (CRC) have not been fully understood.MethodsThe expression of MCM3AP‐AS1 was detected by quantitative reverse transcription PCR (RT‐qPCR) in CRC tissues and matched noncancerous tissues (NCTs). CCK‐8 assay, colony formation assay, transwell assay, xenograft and lung metastasis mouse models were used to examine the tumor‐promoting function of MCM3AP‐AS1 in vitro and in vivo. The binding relationship between MCM3AP‐AS1, miR‐193a‐5p and sentrin‐specific peptidase 1 (SENP1) were screened and identified by databases, RT‐qPCR, dual luciferase reporter assay and western blot.ResultsIn the present study, we got that the expression of MCM3AP‐AS1 was higher in CRC tissues than in paired NCTs, and increased MCM3AP‐AS1 expression was associated with adverse outcomes in CRC patients. Functional experiments in vitro revealed that silencing of MCM3AP‐AS1 could inhibit the proliferation, colony formation, migratory, and invasive abilities of CRC cells. The mouse models of xenograft and lung metastasis further confirmed that in vivo silencing MCM3AP‐AS1 could significantly inhibit the growth and metastasis of CRC. Further mechanism studies indicated that MCM3AP‐AS1 could sponge miR‐193a‐5p and inhibit the activity of it. What is more, SENP1 was proved to be a novel target of miR‐193a‐5p and could be upregulated by MCM3AP‐AS1. At last, we observed that SENP1 overexpression in CRC tissues was closely related to unfavorable prognosis.ConclusionTaken together, we identified in CRC the MCM3AP‐AS1/miR‐193a‐5p/SENP1 regulatory axis, which affords a therapeutic possibility for CRC.

MiR-133b inhibits colorectal cancer metastasis via lncRNA-LUCAT1.

Aim: Colorectal cancer (CRC) is a common malignant tumor of the digestive system. Metastasis is the leading cause of poor prognosis of CRC patients, warranting further study of the molecular mechanism of metastasis in CRCand identification of new therapeutic targets. MiR-133b has been proven to play an important role in tumorigenesis by directly targeting coding genes. However, whether miR-133b can regulate tumorigenesis via long noncoding RNA (lncRNA) remains unclear. Methods: We systematically analyzed the expression level and correlation of miR-133b and LUCAT1 in cancer tissues and adjacent tissues from 30 patients with CRC. The effects of miR-133b and LUCAT1 on the invasive ability of CRC cells were detected by a transwell assay. The relationship between miR-133b and LUCAT1 was investigated by cells transfection experiments, rescue experiments and luciferase reporter assays. The binding of LUCAT1 and EZH2 was detected by RNA immunoprecipitation assay. Results: MiR-133b was expressed at low levels in CRC tissues, and LUCAT1 was highly expressed, with an inverse correlation between them. LUCAT1 promoted the migration and invasion of HCT116 and SW620 cells. Overexpression of LUCAT1 attenuated the inhibition of cell migration and invasion induced by miR-133b. However, the dual luciferase assay showed that miR-133b did not directly target LUCAT1. Conclusion: MiR-133b affects CRC metastasis via the LUCAT1/EZH2 complex. MiR-133b and LUCAT1 may be potential targets for antimetastasis therapy in CRC.

GJA1 is a Prognostic Biomarker and Correlated with Immune Infiltrates in Colorectal Cancer.

PurposePrevious studies found that gap junction alpha-1 protein (GJA1) was a potent tumor suppressor in colorectal cancer (CRC). We designed the present study to evaluate the clinical importance and molecular mechanisms of GJA1 in CRC.MethodsClinical and transcriptomic data from TCGA and GEO datasets were retrospectively collected. CRC patients were divided into two subgroups according to the expression level of GJA1 mRNA. Difference between survival time and response to neoadjuvant chemotherapy was then evaluated. Functional assays including wound-healing assay, transwell invasion assay and flow cytometry assay were performed to investigate the effects of GJA1 on invasive ability and response to chemotherapy drugs of CRC cells. Moreover, we explored the mechanisms of GJA1 by which it regulates CRC malignant phenotypes.ResultsThe expression level of GJA1 was significantly higher in normal tissue than cancer tissue, indicating a tumor suppressive role of GJA1 in CRC. Patients with higher expression of GJA1 showed better prognosis than those with low GJA1 expression level. Consistently, overexpression of GJA1 suppressed the invasive ability of CRC cells while enhancing the sensitivity of CRC cells to oxaliplatin-induced apoptosis. Mechanically, we found that GJA1 suppressed the epithelial mesenchymal transition process. Moreover, GJA1 could modulating infiltrating levels of several immune cells in the tumor microenvironment.ConclusionThese findings suggested that GJA1 was correlated with prognosis and immune infiltrating levels of CD8+ T cells, macrophages, neutrophils, and DCs in CRC. In addition, GJA1 expression contributes to regulation of tumor-associated macrophages (TAMs) and tumor infiltrating neutrophils (TINs) in CRC. These findings suggest that GJA1 is a promising biomarker for determining prognosis and immune infiltration in colorectal cancer.

LINC00346 accelerates the malignant progression of colorectal cancer via competitively binding to miRNA-101-5p/MMP9.

To clarify the promotive effect of LINC00346 on the malignant progression of colorectal cancer (CRC) by mediating miRNA-101-5p/MMP9 axis. Expression pattern of LINC00346 in 46 paired CRC tissues and adjacent normal tissues was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Correlation between LINC00346 level and prognosis of CRC patients was analyzed, and the LINC00346 level in CRC cell lines was examined as well. Subsequently, potential influences of LINC00346 on cellular behaviors of CRC cells were evaluated through cell counting kit-8 (CCK-8), colony formation, transwell, and wound healing assays. Finally, Dual-Luciferase reporter gene assay was conducted to verify the binding relationship between LINC00346 and miRNA-101-5p/MMP9. LINC00346 was upregulated in CRC tissues and cell lines. Compared with CRC patients with low level of LINC00346, those with high level suffered a poorer prognosis, and higher metastatic rates (lymph node metastasis and distant metastasis). Transfection of sh-LINC00346 attenuated proliferative, migratory, and invasive abilities of CRC cells. In addition, LINC00346 was confirmed to bind to miRNA-101-5p, and the latter was binding to MMP9. Moreover, the overexpression of miRNA-101-5p decreased colony number, viability, and numbers of migratory and invasive cells. LINC00346 is upregulated in CRC and correlated with metastasis and poor prognosis of CRC. LINC00346 accelerates the malignant progression of CRC via targeting miRNA-101-5p/MMP9.

Block of proliferation 1 promotes cell migration and invasion in human colorectal cancer cells via the JNK pathway.

Metastasis is one of the most common causes of death in patients with colorectal cancer (CRC). Block of proliferation 1 (BOP1) regulates tumorigenesis, epithelial‐to‐mesenchymal transition, migration, metastasis, and drug resistance in several tumor types. However, the role of BOP1 in the regulation of colorectal cancer cell migration and invasion is still largely unclear. In this study, the results of immunohistochemistry showed that BOP1 was upregulated in our cohort of CRC patients. BOP1 knockdown inhibited the migration and invasion of CRC cells, confirmed by the downregulation of the mRNA levels of MMP‐2 and MMP‐9. The overexpression of BOP1 in CRC cells exerted the opposite effect. SP600125, an inhibitor of JNK signaling, partially abolished the BOP1 overexpression‐mediated increase in the migratory and invasive ability of CRC cells. Our results indicated that BOP1 is an important regulator of CRC cell invasion and migration, predominantly through the JNK signaling pathway.

ADPGK-AS1 promotes the progression of colorectal cancer via sponging miR-525 to upregulate FUT1.

We sought to uncover the potential role of long non-coding RNA (lncRNA) ADPGK-AS1 in colorectal cancer (CRC). ADPGK-AS1 levels in 58 pairs of CRC tissues and paracancerous tissues and 30 normal colorectal tissues were determined. The in vitro level of ADPGK-AS1 in CRC cell lines was tested as well. The regulatory effects of ADPGK-AS1 on the proliferative, migratory, and invasive properties of HCT116 and SW480 cells were assessed. Using a Dual-Luciferase reporter gene assay, the interaction among ADPGK-AS1/miR-525/FUT1 was identified. Finally, potential influences of the regulatory loop ADPGK-AS1/miR-525/FUT1 on the phenotypes of CRC cells were explored. ADPGK-AS1 was upregulated in CRC tissues and cells. Knockdown of ADPGK-AS1 attenuated the proliferative, migratory, and invasive abilities of CRC cells. Meanwhile, miR-525 was confirmed to be the target of ADPGK-AS1 and FUT1 was the downstream gene binding miR-525. The regulatory loop ADPGK-AS1/miR-525/FUT1 was found to aggravate the malignant progression of CRC. ADPGK-AS1 is upregulated in CRC. The regulatory loop ADPGK-AS1/miR-525/FUT1 exacerbates the progression of CRC by promoting the proliferation, migration, and invasion of tumor cells.

METTL14 suppresses proliferation and metastasis of colorectal cancer by down-regulating oncogenic long non-coding RNA XIST

BackgroundN6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulation in eukaryotic cells. However, understanding of m6A in colorectal cancer (CRC) is very limited. We designed this study to investigate the role of m6A in CRC.MethodsExpression level of METTL14 was extracted from public database and tissue array to investigate the clinical relevance of METTL14 in CRC. Next, gain/loss of function experiment was used to define the role of METTL14 in the progression of CRC. Moreover, transcriptomic sequencing (RNA-seq) was applied to screen the potential targets of METTL14. The specific binding between METTL14 and presumed target was verified by RNA pull-down and RNA immunoprecipitation (RIP) assay. Furthermore, rescue experiment and methylated RNA immunoprecipitation (Me-RIP) were performed to uncover the mechanism.ResultsClinically, loss of METTL14 correlated with unfavorable prognosis of CRC patients. Functionally, knockdown of METTL14 drastically enhanced proliferative and invasive ability of CRC cells in vitro and promoted tumorigenicity and metastasis in vivo. Mechanically, RNA-seq and Me-RIP identified lncRNA XIST as the downstream target of METTL14. Knockdown of METTL14 substantially abolished m6A level of XIST and augmented XIST expression. Moreover, we found that m6A-methylated XIST was recognized by YTHDF2, a m6A reader protein, to mediate the degradation of XIST. Consistently, XIST expression negatively correlated with METTL14 and YTHDF2 in CRC tissues.ConclusionOur findings highlight the function and prognostic value of METTL14 in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.

Upregulated lncRNA CACNA1G-AS1 aggravates the progression of colorectal cancer by downregulating p53.

To investigate the role of long non-coding RNA (lncRNA) CACNA1G-AS1 in regulating proliferative and invasive abilities of colorectal cancer (CRC) cells by mediating p53, thus influencing the progression of CRC. CACNA1G-AS1 level in CRC tissues and adjacent normal tissues was first determined. Its level in CRC patients with different tumor stages was detected as well. Changes in proliferative and invasive abilities of HCT116 and SW480 cells influenced by CACNA1G-AS1 were evaluated. Subcellular distribution of CACNA1G-AS1 was analyzed. Through Western blot, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP) assay, the interaction between CACNA1G-AS1 and EZH2 was assessed. The biological function of the target gene of CACNA1G-AS1 was finally explored. CACNA1G-AS1 was upregulated in CRC tissues compared to adjacent normal ones. Its level remained higher in CRC patients with stage III-IV compared to those with stage I-II. Knockdown of CACNA1G-AS1 reduced proliferative and invasive abilities of HTC116 and SW480 cells. CACNA1G-AS1 was mainly distributed in the nucleus. Moreover, CACNA1G-AS1 was verified to interact with EZH2. Knockdown of CACNA1G-AS1 or EZH2 upregulated p53 level and decreased the recruitment ability of EZH2 on p53. Finally, p53 knockdown could partially reverse the regulatory effect of CACNA1G-AS1 on the proliferative ability of HCT116 cells. CACNA1G-AS1 downregulates p53 level by forming a carcinogenic complex with EZH2, thereby enhancing the proliferative and invasive abilities of CRC cells.

MicroRNA-362 Inhibits Cell Proliferation and Invasion by Directly Targeting SIX1 in Colorectal Cancer.

PurposeColorectal cancer (CRC) is the third most common cancer in China and poses high morbidity and mortality. In recent years, increasing evidence has indicated that microRNAs played important functions in the occurrence and development of tumors. The purpose of this study was to identify the biological mechanisms of miR-362 in CRC.Materials and MethodsQuantitative real-time PCR was carried out to assess the expression of miR-362 and SIX1. The Kaplan-Meier method was employed to evaluate the 5-year overall survival of CRC patients. The proliferative and invasive abilities of CRC cells were assessed by MTT and transwell assays.ResultsmiR-362 was significantly decreased in CRC tissues and cell lines, compared to the normal tissues and normal cells. A significant connection was confirmed between the overall survival of 53 CRC patients and low expression of miR-362. Downregulation of miR-362 inhibited the proliferation and invasion through binding to the 3′-UTR of SIX1 mRNA in CRC. Additionally, we discovered that SIX1 was a direct target gene of miR-362 and that the expression of miR-362 had a negative connection with SIX1 expression in CRC. SIX1 could reverse partial functions in the proliferation and invasion in CRC cells.ConclusionmiR-362 may be a prognostic marker in CRC and suppress CRC cell proliferation and invasion in part through targeting the 3′-UTR of SIX1 mRNA. The newly identified miR-362/SIX1 axis provides insight into the progression of CRC.