Molecular characteristics of early-onset compared with late-onset colorectal cancer: A case controlled study.

BACKGROUND: Early-Onset Colorectal Cancer (EOCRC) is associated with a poorer prognosis relative to Late-Onset Colorectal Cancer (LOCRC), and its incidence has witnessed a gradual escalation in recent years. This necessitates a comprehensive examination of the underlying pathogenesis and the identification of therapeutic targets specific to EOCRC patients. The present study aimed to delineate the distinct molecular landscape of EOCRC by juxtaposing it with that of LOCRC. METHODS: A total of 11,344 colorectal cancer patients, diagnosed between 2003 and 2022, were enrolled in this study, comprising 578 EOCRC cases and 10,766 LOCRC cases. Next-generation sequencing technology was employed to assess the tumor-related mutation and tumor mutation burden (TMB) in these patients. PD-L1 expression was quantified using immunohistochemistry. Microsatellite instability (MSI) was determined via capillary electrophoresis (2B3D NCI Panel). RESULTS: Upon comparing LOCRC with EOCRC patients, the latter group demonstrated a tendency towards advanced TNM stage, lower tumor differentiation, and less favorable histological types. Among LOCRC patients, those with MSI-H status were found to have an earlier TNM stage compared to those with MSI-L/MSS status. Significantly, the incidence of MSI-H was notably higher in EOCRC (10.2%) compared to LOCRC (2.2%). Mutations in the 7-gene panel (ARID1A, FANCI, CASP8, DGFRA, DPYD, TSHR, and PRKCI) were more prevalent in LOCRC. Within the EOCRC cohort, patients with the MSI-H subtype displayed an earlier TNM stage but concurrently exhibited poorer tissue differentiation and a higher frequency of mucinous adenocarcinoma. Among EOCRC patients, FBXW7, FAT1, ATM, ARID1A, and KMT2B mutations were significantly enriched in the MSI-H subgroup. A comparative analysis of MSI-H patients revealed heightened mutation frequencies of FGFBR2, PBRM1, RNF43, LRP1B, FBXW7, ATM, and ARID1A in the EOCRC group. Furthermore, EOCRC patients demonstrated a higher overall TMB, particularly in the MSI-H subtype. PD-L1 expression was elevated in EOCRC and positively associated with MSI status. CONCLUSIONS: This study revealed a significantly higher MSI-H distribution rate in early-onset colorectal cancer, and EOCRC exhibits a distinct mutational signature coupled with higher PD-L1 expression. These findings hold promise in guiding personalized therapeutic strategies for improved disease management in EOCRC patients.

The Uridylyl Transferase TUT7-Mediated Accumulation of Exosomal miR-1246 Reprograms TAMs to Support CRC Progression.

Tumor-associated macrophages (TAMs) play a crucial role in promoting tumor growth and dissemination, motivating a search for key targets to interfere with the activation of TAMs or reprogram TAMs into the tumor-suppressive type. To gain insight into the mechanisms of macrophage polarization, a designed co-culture system is established, allowing for the education of macrophages in a manner that closely mimics the intricacies of TAMs in the tumor immune microenvironment (TIME). Through database mining, exosomal miR-1246 is identified and is then validated. Exosomal miR-1246-driven polarization of TAMs disrupts the infiltration and function of CD8+ T cells. Mechanically, the amassment of exosomal miR-1246 stems from TUT7-mediated degradation of small noncoding RNA, a process stabilized by SNRPB, but not the precursor of miR-1246. Moreover, an Exo-motif is present in the exosomal miR-1246 sequence, enabling it to bind with the exosomal sorting protein hnRNPA2B1. RNA-seq analysis reveals that exogenous miR-1246 modulates the polarization of TAMs at a post-transcriptional level, emphasizing the pivotal role of the NLRP3 in macrophage polarization. In conclusion, the findings underscore the importance of exosomal miR-1246 as a trigger of macrophage reprogramming and uncover a novel mechanism for its enhanced presence in the TIME.

Upregulated circTMEM59 Inhibits Cell Growth and Metastasis by miR-668-3p/ID4 Axis in Colorectal Cancer.

The incidence and mortality of colorectal cancer (CRC) are ranked in the top three worldwide in 2020. Abundant studies have reported that circular RNAs (circRNAs) act critical roles in the genesis and development of tumors, including CRC. Nevertheless, the roles and detailed regulation mechanisms of circRNAs that are related to the initiation and development of CRC have not been fully found and clarified. This research primarily revealed that circTMEM59 was greatly downregulated in CRC tissues and cell lines via qRT-PCR. In addition, the decreased expression of circTMEM59 was closely related to adverse clinicopathological characteristics and the shorter survival time of CRC patients. Then, a further study found that the overexpression of circTMEM59 suppressed cell growth and accelerated the cell death of CRC via a series of experiments in vitro and in vivo. Furthermore, circTMEM59 also repressed the metastatic behaviors of CRC cells. Further study revealed that circTMEM59 played the role of competing endogenous RNAs (ceRNAs) by binding to miR-668-3p to increase the expression of inhibitor of DNA binding 4 (ID4) in CRC. In summary, the results of this study clarified the antitumor effects of circTMEM59/miR-668-3p/ID4 axis in CRC progression and provided potential therapeutic targets and clinical prognostic markers for CRC.

Total Mesorectal Excision vs. Transanal Endoscopic Microsurgery Followed by Radiotherapy for T2N0M0 Distal Rectal Cancer: A Multicenter Randomized Trial.

INTRODUCTION: Transanal endoscopic microsurgery (TEM) is an organ-preserving treatment alternative for patients with early rectal cancer. However, TEM alone is associated with greater risk of local recurrence and inferior survival in comparison with total meso-rectal excision (TME). As an important adjuvant therapy, radiotherapy can effectively reduce the local recurrence rate of rectal cancer. This study aimed to investigate whether TEM followed by radiotherapy can be a valid alternative to TME in T2N0M0 distal rectal cancer treatment. METHODS: We plan to recruit 168 participants meeting established inclusion criteria. Following informed consent, participants will randomly receive treatment protocols of TEM followed by radiotherapy (a total dose of 45-50.4 Gy given in 25-28 factions) or TME. Depending on post-operative pathology, the participants will receive either long-term follow-up or further treatment. The primary endpoint of this trial is 3-year local recurrence rate. The secondary end points include 3-year disease-free survival rate, 3-year overall survival rate, 3-year mortality rate, post-operative quality of life, post-operative safety index, intraoperative evaluation index and post-operative short-term evaluation index. DISCUSSION: This trial is the first prospective randomized trial to investigate the rectum preserving treatment by using transanal local excision followed by radiotherapy. CLINICAL TRIAL REGISTRATION: The trial was prospectively registered at ClinicalTrials.gov NCT04098471 on September 20, 2019.

Long non-coding RNA CCDC144NL-AS1 promotes cell proliferation by regulating the miR-363-3p/GALNT7 axis in colorectal cancer.

Colorectal cancer (CRC) is a burdensome health concern worldwide. Long non-coding RNA (lncRNA) have emerged as vital roles in multiple cancers, including CRC. Increasing evidence has demonstrated that lncRNA CCDC144NL-AS1 acts crucial roles in tumor developments. Nevertheless, its role in CRC remains largely unknown. The level of CCDC144NL-AS1 expression was detected in 100 CRC tissues and paired adjacent tissues. The gain- and loss-of-function experiments were conducted to investigate the biological functions of CCDC144NL-AS1 in vitro and in vivo. The potential mechanism of CCDC144NL-AS1 exerting as competing endogenous RNAs (ceRNAs) was demonstrated by bioinformatics, luciferase reporter assay and in vitro experiments. CCDC144NL-AS1 was up-regulated in CRC tissues and cells. High CCDC144NL-AS1 was connected with the adverse clinicopathological features and worse prognosis of CRC. Furthermore, knockdown of CCDC144NL-AS inhibited the cell proliferation and led to the cell cycle G0-1/S arrest, whereas upregulated CCDC144NL-AS1 obtained the inverse results. Further study found that CCDC144NL-AS1 functioned as ceRNAs in regulating CRC proliferation. MiR-363-3p was the target of CCDC144NL-AS1, which sponges GALNT7 in regulating cell growth of CRC. The study demonstrated that the CCDC144NL-AS1/miR-363-3p/GALNT7 axis exerts on key roles in cell proliferation and presents an emerging target for CRC therapy and prognostic biomarker.

Long non-coding RNA Lnc-LALC facilitates colorectal cancer liver metastasis via epigenetically silencing LZTS1.

Colorectal cancer (CRC) is one of the most common cancers around the world and endangers human health seriously. Liver metastasis is an important factor affecting the long-term prognosis of CRC and the specific mechanism of CRLM (colorectal cancer with liver metastasis) is not fully understood. LZTS1 has been found dysregulated in many cancers, especially in CRC. Theories suggested that hypermethylation of the promoter regions of LZTS1 was responsible for LZTS1 abnormal expression in multiple malignant tumors. Although the role of LZTS1 in CRC cell proliferation has been reported, its role in CRLM remains unclear. Numerous studies reported Long non-coding RNA (lncRNA) could regulate the gene expression level by regulating gene methylation status in many tumors. However, whether there were lncRNAs could change the methylation status of LZTS1 or not in CRLM was unknown. In this study, we aimed to investigate whether there are lncRNAs can regulate the expression of LZTS1 through affecting DNA methylation in CRLM. We found that upregulated Lnc-LALC in CRC was negatively correlated with LZTS1 expression, and Lnc-LALC could regulate LZTS1 expression in both mRNA and protein level in our study. Functionally, Lnc-LALC enhanced the CRC cells metastasis ability in vitro and vivo through inhibiting the expression of LZTS1. Furthermore, the precise mechanisms exploration showed that lnc-LALC could recruit DNA methyltransferases (DNMTs) to the LZTS1 promoter by combining with Enhancer of zeste homolog 2(EZH2) and then altered the expression of LZTS1 via DNMTs-mediated DNA methylation. Collectively, our data demonstrated the important role of Lnc-LALC/ LZTS1 axis in CRLM development.

Circulating non-coding RNA cluster predicted the tumorigenesis and development of colorectal carcinoma.

Carcinoembryonic antigen (CEA) is the most significant plasma biomarker in colorectal cancer (CRC), which is mainly used to diagnose and monitor the recurrence of CRC. However, due to the low sensitivity of CEA, it is more recommended for postoperative surveillance rather than early diagnosis. It is necessary to find efficient biomarkers for CRC. In this study, the expression of plasma non-coding RNAs was confirmed in three independent cohorts with total 1201 participants. First, 12 non-coding RNAs were screened from 9 plasma samples by using microarray. The expression of selected non-coding RNAs was further validated by multiphase detection and risk score analysis. We found that miR-20b-5p, miR-329-3p, miR-374b-5p, miR-503-5p, XLOC_001120 and ENSG00000243766.2 were significantly elevated in CRC plasma, and the AUC in training and validation set was 0.996 and 0.954, respectively. Moreover, miR-20b-5p, miR-329-3p and miR-503-5p were found elevated in plasma from larger tumors (5 cm as the cutoff) in CRC patients, and the merged AUC in training and validation set was 0.896 and 0.881. In conclusion, a panel of 6 non-coding RNAs showed their important clinical value for the early diagnosis of CRC. Among, miR-20b-5p, miR-329-3p and miR-503-5p might be the potential markers for evaluating larger tumor size of CRC.

Lnc-FAM84B-4 acts as an oncogenic lncRNA by interacting with protein hnRNPK to restrain MAPK phosphatases-DUSP1 expression.

The mitogen activated protein kinase (MAPK) pathway has been reported to be involved in many cancer developments. Normally, MAPK activity is self-limited between rapid phosphorylation and dephosphorylation. In abnormal conditions, however, this dynamic equilibrium is broken, trigging tumor-suppressing or -promoting roles. While dual-specificity MAPK phosphatases (MKP/DUSPs) are important for cascade control in MAPK pathway, their role in colorectal cancer (CRC) remains largely unknown. Here, we investigated lnc-FAM84B-4 and DUSP1 to systematically elucidate their underlying roles in MAPK singling pathway and functions in CRC. Upregulated lnc-FAM84B-4 was identified by re-mining CRC microarray. Functional assays were performed in vitro and in vivo. RNA-Seq, RNA pull-down, and RIP assays were used to investigate the mechanisms of Lnc-FAM84B-4 in regulating expression of DUSP1. The results indicated that Lnc-FAM84B-4 regulates MAPK pathway by restraining DUSP1 expression. Mechanistically, RNA pull-down followed by mass spectrum determined hnRNPK functions as a binding partner of lnc-FAM84B-4 in mediating DUSP1 expression. Our findings demonstrate the important role of lnc-FAM84B-4-hnRNPK-DUSP1 axis in CRC development, and suggest a therapeutic target for CRC treatment.

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.

TGM3 functions as a tumor suppressor by repressing epithelial­to­mesenchymal transition and the PI3K/AKT signaling pathway in colorectal cancer.

The dysregulation of transcription factors contributes to the unlimited proliferation of cancer cells. Transglutaminase 3 (TGM3) has been demonstrated to play a crucial role in physiology and pathology. However, the potential role of TGM3 in colorectal cancer (CRC) remains unknown. In the present study, reverse transcription­quantitative PCR and immunohistochemistry were utilized to analyze the expression of TGM3 in CRC and adjacent normal tissues. LoVo and HCT116 cell lines were then selected to further investigate the function of TGM3 in the proliferation, invasion and metastasis of CRC both in vitro and in vivo. Finally, western blotting was performed to investigate the molecular mechanisms underlying the effects of TGM3 in CRC. The expression level of TGM3 was significantly downregulated in CRC tissues, and was associated with tumor invasion, metastasis and patient prognosis. Following TGM3 inhibition and overexpression in CRC cells, it was revealed that TGM3 suppressed cell proliferation, potentially via the promotion of apoptosis and cell cycle regulation. Furthermore, TGM3 also inhibited invasion and metastasis. Finally, it was observed that TGM3 inhibited epithelial­to­mesenchymal transition and activated phosphorylated AKT serine/threonine kinase in CRC cells. The results from the present study revealed that TGM3 is a tumor suppressor in the progression of CRC, and may be used as a novel target for CRC treatment.

NMI promotes cell proliferation through TGFß/Smad pathway by upregulating STAT1 in colorectal cancer.

Colorectal cancer (CRC) is still a fatal health problem around the world. The underlying mechanisms of CRC have not been fully elucidated. N-myc interactor (NMI) acts as an oncogene or a tumor-suppressor gene in several kinds of cancers but CRC. Here, the expression of NMI was found higher in CRC tissues and cells. Higher expression of NMI indicated the poorer prognosis of CRC patients. Moreover, the proliferation of CRC cells was suppressed significantly after we silenced the expression of NMI, while overexpression of NMI promoted CRC cell proliferation. Flow cytometry demonstrated that NMI promoted cell proliferation through facilitating cell transition from the G1 phase to the S phase. Furthermore, it was found that NMI suppressed the phosphorylation of Smad3 by upregulating the expression of STAT1. The effect of NMI depletion on cell proliferation could be reversed by using Smad3 inhibitor SIS3. In summary, our findings demonstrated that NMI promoted cell proliferation via TGFß/Smad pathway and could indicate the prognosis of patients with CRC.

Upregulated METTL3 promotes metastasis of colorectal Cancer via miR-1246/SPRED2/MAPK signaling pathway.

BACKGROUND: m6A modification has been proved to play an important role in many biological processes. METTL3 as the main methyltransferase for methylation process has been found to be upregulated in many cancers, including CRC. Here, we investigate m6A modification and the underlying mechanism of METTL3 in the development of CRC. METHODS: The expression of METTL3 was detected in large clinical patient samples. To evaluate the function of METTL3 in vitro and in vivo, colony formation, CCK-8, cell migration and invasion assays were performed. To find out the downstream target of METTL3, GEO dataset was re-mined. We analyzed expression and metastasis-related miRNA by Pearson correlation, and miR-1246 was selected. Here, to identify the downstream target of miR-1246, Targetscan and miRWalk were used. RIP and luciferase reporter assay further confirmed SPRED2 as the direct target of miR-1246. RESULTS: We found that upregulated METTL3 is responsible for abnormal m6A modification in CRC and correlates positively with tumor metastasis. The gain- and loss-of-function indicates that METTL3 promotes cell migration and invasion in vitro and in vivo. Additionally, we confirmed that METTL3 can methylate pri-miR-1246, which further promotes the maturation of pri-miR-1246. By using bioinformatics tools, anti-oncogene SPRED2 was identified as the downstream target of miR-1246, wherein downregulated SPRED2 further reverses the inhibition of the MAPK pathway. CONCLUSIONS: The present study demonstrates that the METTL3/miR-1246/SPRED2 axis plays an important role in tumor metastasis and provides a new m6A modification pattern in CRC development.

CDCA2 promotes the proliferation of colorectal cancer cells by activating the AKT/CCND1 pathway in vitro and in vivo.

BACKGROUND: Cell division cycle associated 2 (CDCA2), upregulated in lung adenocarcinoma and oral squamous cell carcinoma, may be related to some malignant diseases. Nevertheless, its role in colorectal cancer (CRC) remains unknown. METHODS: CDCA2 expression was analyzed using The Cancer Genome Atlas (TCGA), quantitative real-time PCR (qRT-PCR), and immunohistochemistry. The impact of CDCA2 on cell proliferation was analyzed via loss- or gain-of-function assays. Furthermore, gene set enrichment analysis was conducted to explore the potential mechanism of CDCA2 in CRC. Lastly, the expression levels of CCND1 and AKT were measured in CRC cell lines. RESULTS: Our study revealed that CDCA2 expression was associated with tumor progression. Through loss- or gain-of-function assays, we found that upregulation of CDCA2 promoted the proliferation of DLD-1 cells, however, downregulation of CDCA2 in SW480 cells restrained proliferative capacity both in vitro and in vivo. The results of flow cytometry showed that CDCA2 promoted cell cycle progression via upregulation of CCND1 in CRC cell lines. In the following experiments, we found that CDCA2 regulated CCND1 expression through activating the PI3K/AKT pathway, and confirmed this using a specific PI3K inhibitor (LY294002). CONCLUSIONS: This study demonstrates that overexpression of CDCA2 might target CCND1 to promote CRC cell proliferation and tumorigenesis through activation of the PI3K/AKT pathway.

Long noncoding RNA Linc02023 regulates PTEN stability and suppresses tumorigenesis of colorectal cancer in a PTEN-dependent pathway.

Phosphatase and tensin homolog (PTEN), one of the most frequently mutated tumor suppressor genes in human cancer, is pivotal in the progression of colorectal cancer. Therefore, the regulation of PTEN has emerged as a theme of intense research in tumor biology. This study aims to show that long noncoding RNA (lncRNA) Linc02023 aberrant downregulation in colorectal cancer correlates positively with the expression of PTEN and CDKN2B but negatively with the tumor size in patients and xenografted mouse models. The gain- and loss-of-function investigation reveals that Linc02023 suppresses the proliferation of colorectal cancer cells in vitro and in vivo with apoptosis promotion and cell cycle rearrangement. Mechanistically, Linc02023 specifically binds to PTEN and blocks its interaction with and ubiquitination by WWP2, stabilizing it and suppressing its downstream expression. In conclusion, this study demonstrates that lncRNA Linc02023 may serve as a novel therapeutic target by restoring the PTEN tumor suppressor activity.

Construction of ceRNA networks reveals differences between distal and proximal colon cancers.

Although colon cancer is often referred to as a homogeneous entity, an increasing number of studies have revealed that colon cancer can be divided according to the anatomic site of the cancer. However, few studies have reported the difference between distal and proximal colon cancer with regard to molecular mechanism, and especially non­coding RNA molecules. In the present study, the data of 186 colon tumour tissues and 17 adjacent non­tumour colon tissues in the left colon and 229 colon tumour tissues and 21 adjacent non­tumour colon tissues in the right colon were obtained from The Cancer Genome Atlas (TCGA). A total of 879 lncRNAs, 165 miRNAs and 2,028 mRNAs were identified as left­specific RNAs [log2(fold change)>2, FDR<0.01]. There were 916 lncRNAs, 227 miRNAs and 2,069 mRNAs identified in right colon cancer. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways were analysed for 2,028 mRNAs from left colon cancer and 2,069 mRNAs from right colon cancer. After removing the elements of the intersection from side­specific lncRNAs of the left and right, we identified specific lncRNAs included exclusively in left or right colon cancer, including 277 lncRNAs in left colon cancer and 314 lncRNAs in right colon cancer. Among these lncRNAs, 20 lncRNAs from the left and 25 lncRNAs from the right were revealed to be associated with overall survival. Then, ceRNA networks were constructed. There were 18 lncRNAs, 22 miRNAs and 57 mRNAs included in the left colon cancer ceRNA network and 21 lncRNAs, 27 miRNAs and 55 mRNAs included in the right ceRNA network. In total, 15 lncRNAs were revealed to be significantly related to clinical features, two of which were ascertained by testing the mRNA expression of tissues. In conclusion, our research aimed to detect the difference between colon cancer in the left and the right colon and to assist in the identification of new potential biomarkers to be used for diagnostic and prognostic purposes.

CDCA3 mediates p21-dependent proliferation by regulating E2F1 expression in colorectal cancer.

Dysregulated cell cycle progression serves a crucial role in tumor development. Cell division cycle-associated 3 (CDCA3) is considered a trigger of mitotic entry; it is an important part of the S phase kinase-associated protein 1/Cullin/F-box ubiquitin ligase complex and mediates the destruction of mitosis-inhibitory kinase wee1. However, little is known about the role of CDCA3 in cancer, particularly colorectal cancer (CRC). The present study aimed to explore the biological and clinical significance of CDCA3 in CRC growth and progression. CDCA3 expression was significantly associated with tumor progression and poor survival. Overexpression of CDCA3 increased proliferation in LoVo CRC cells, whereas CDCA3 knockdown in SW480 CRC cells led to decreased proliferation, in vitro and in vivo. Further mechanistic investigations demonstrated that reduced CDCA3 expression resulted in G1/S phase transition arrest, which was attributed to a significant accumulation of p21 in SW480 cells; conversely, increased CDCA3 expression promoted G1/S phase transition through decreased p21 accumulation in LoVo cells. It was also demonstrated that CDCA3 was able to regulate the expression of transcription factor E2F1, thereby repressing p21 expression. Taken together, these results suggested that overexpression of CDCA3 may serve a crucial role in tumor malignant potential and that CDCA3 may be used as a prognostic factor and a potential therapeutic target in CRC.

Analysis of lncRNA-Associated ceRNA Network Reveals Potential lncRNA Biomarkers in Human Colon Adenocarcinoma.

BACKGROUND/AIMS: Long non-coding RNAs (lncRNAs) acting as competing endogenous RNAs (ceRNAs) play significant roles in the development of tumors, but the functions of specific lncRNAs and lncRNA-related ceRNA networks have not been fully elucidated for colon adenocarcinoma (COAD). In this study, we aimed to clarify the lncRNA-microRNA (miRNA)-mRNA ceRNA network and potential lncRNA biomarkers in COAD. METHODS: We extracted data from The Cancer Genome Atlas (TCGA) and identified COAD-specific mRNAs, miRNAs, and lncRNAs. The biological processes in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were analyzed for COAD-specific mRNAs. We then constructed a ceRNA network of COAD-specific mRNAs, miRNAs and lncRNAs and analyzed the correlation between expression patterns and clinical features of the lncRNAs involved. After identifying potential mRNA targets of 4 lncRNAs related to overall survival (OS), we conducted stepwise analysis of these targets through GO and KEGG. Using tissue samples from our own patients, we also verified certain analytical results using quantitative real-time PCR (qRT-PCR). RESULTS: Data from 521 samples (480 tumor tissue and 41 adjacent non-tumor tissue samples) were extracted from TCGA. A total of 258 specific lncRNAs, 206 specific miRNAs, and 1467 specific mRNAs were identified (absolute log2 [fold change] > 2, false discovery rate < 0.01). Analysis of KEGG revealed that specific mRNAs were enriched in cancer-related pathways. The ceRNA network was constructed with 64 lncRNAs, 18 miRNAs, and 42 mRNAs. Among these lncRNAs involved in the network, 3 lncRNAs (LINC00355, HULC, and IGF2-AS) were confirmed to be associated with certain clinical features and 4 lncRNAs (HOTAIR, LINC00355, KCNQ1OT1, and TSSC1-IT1) were found to be negatively linked to OS (log-rank p < 0.05). KEGG showed that the potential mRNA targets of these 4 lncRNAs may be concentrated in the MAPK pathway. Certain results were validated by qRT-PCR. CONCLUSION: This study providing novel insights into the lncRNA-miRNA-mRNA ceRNA network and reveals potential lncRNA biomarkers in COAD.

GPR56 promotes proliferation of colorectal cancer cells and enhances metastasis via epithelial­mesenchymal transition through PI3K/AKT signaling activation.

G protein­coupled receptor 56 (GPR56), a member of the orphan GPCR family, has been reported to be an oncogene in various malignancies. However, little is known regarding the detailed molecular mechanism of GPR56 in colorectal cancer (CRC). The present study aimed to detect the expression level and biological function of GPR56 in CRC. We examined the expression of GPR56 in CRC tissues and cell lines by quantitative real time (qRT)­PCR, immunohistochemistry, and western blot analysis. The prognostic significance of GPR56 in CRC patients was evaluated by Kaplan­Meier survival analysis. The influence of GPR56 on tumor cell proliferation (via Cell Counting Kit­8, and a tumor formation assay in mice), apoptosis (flow cytometry), cell cycle distribution (flow cytometry) and migration (Transwell assay) was explored. We also investigated the underlying mechanism of GPR56 by western blot analysis. We found GPR56 expression was significantly upregulated in CRC tissues and cell lines compared to corresponding normal controls. Higher GPR56 expression in patients predicted poorer prognosis. Depletion of GPR56 markedly suppressed cell proliferation, migration, and invasion. GPR56 overexpression promoted CRC cell metastasis by expediting epithelial­mesenchymal transition by activating PI3K/AKT signaling. In conclusion, GPR56 played an important role in CRC progression and may represent a new therapeutic target to reduce CRC metastasis.

Upregulated miR-1258 regulates cell cycle and inhibits cell proliferation by directly targeting E2F8 in CRC.

OBJECTIVES: MicroRNAs (miRNAs) as small noncoding RNA molecules function by regulating their target genes negatively. MiR-1258 was widely researched in multicancers, but its role remains unclear in colorectal cancer (CRC). METHODS: The expression of miR-1258 and its specific target gene were detected in human CRC specimens and cell lines by miRNA RT-PCR, qRT-PCR and Western blot. The effects of miR-1258 on CRC proliferation were evaluated using CCK-8 assays, EdU incorporation, colony formation assays and cell-cycle assays; in vitro and the in vivo effects were investigated using a mouse tumorigenicity model. Luciferase reporter and RIP assays were employed to identify interactions between miR-1258 and its specific target gene. RESULTS: MiR-1258 was downregulated in CRC tissues and CRC cell lines, and upregulated miR-1258 was proved to inhibit proliferation and arrest cell cycle at G0/G1 in vitro and vivo. Luciferase reporter, RIP and western blot assays revealed E2F8 to be a direct target of miR-1258. The effects of miR-1258 in proliferation and cell cycle regulation can be abolished by E2F8 through rescue experiments. By directly targeting E2F8, miR-1258 influenced the expression of several cell-cycle factors, including cyclin D1 (CCND1) and cyclin dependent kinase inhibitor 1A (p21). CONCLUSION: MiR-1258 may function as a suppressive factor by negatively controlling E2F8, thus, highlighting the potential role of miR-1258 as a therapeutic target for human CRC.

Angiogenesis and vasculogenic mimicry are inhibited by 8-Br-cAMP through activation of the cAMP/PKA pathway in colorectal cancer.

INTRODUCTION: Vasculogenic mimicry (VM) describes the formation of an epithelial-independent tumor microcirculation system that differs from traditional angiogenesis. Angiogenesis and the formation of VM are closely related through the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and the epithelial-mesenchymal transition (EMT) process. MATERIALS AND METHODS: In this study, 8-Br-cAMP, a cAMP analog and PKA activator, was used to activate the cAMP/PKA pathway to evaluate the effects of cAMP/PKA on angiogenesis and VM in colorectal cancer (CRC) cells. We used a syngeneic model of CRC in BALB/c mice. RESULTS: We discovered that treatment with 8-Br-cAMP significantly reduced tumor number compared to control mice after the 7th, 14th, and 28th days of treatment. VM was evaluated by periodic acid-schiff (PAS)-CD31 staining, and we found that VM was inhibited by 8-Br-cAMP treatment in vivo. Immunohistochemistry confirmed the inhibition of vascular endothelial growth factor (VEGF) and cAMP and the activation of PKA by 8-Br-cAMP; quantitative real-time-PCR (qRT-PCR) demonstrated that 8-Br-cAMP regulated the expression of vascular endothelial (VE)-cadherin, matrix metalloproteinase 2 (MMP2), ephrin type-A receptor 2 (EphA2), and VEGF in vivo. Experiments in vitro revealed that treatment with 8-Br-cAMP and U0126 decreased VEGF expression through PKA-ERK in CT26 cells by qRT-PCR. We further confirmed that tube formation of human umbilical vein endothelial cells was inhibited by 8-Br-cAMP in vitro. DISCUSSION: This study demonstrates that angiogenesis and VM are inhibited by 8-Br-cAMP treatment. Our data indicate that 8-Br-cAMP acts through the cAMP/PKA-ERK pathway and through EMT processes in CRC. These findings provide an insight into mechanisms of CRC and suggest that the cAMP/PKA-ERK pathway is a novel potential therapeutic target for the treatment of CRC.