BACKGROUND: Long non-coding RNA colon cancer-associated transcript 1 (CCAT1) is involved in transforming multiple cancers into malignant cancer types. Previous studies underlining the mechanisms of the functions of CCAT1 primarily focused on its decoy for miRNAs (micro RNAs). However, the regulatory mechanism of CCAT1-protein interaction associated with tumor metastasis is still largely unknown. The present study aimed to identify proteome-wide CCAT1 partners and explored the CCAT1-protein interaction mediated tumor metastasis. METHODS: CCAT1-proteins complexes were purified and identified using RNA antisense purification coupled with the mass spectrometry (RAP-MS) method. The database for annotation, visualization, and integrated discovery and database for eukaryotic RNA binding proteins (EuRBPDB) websites were used to bioinformatic analyzing CCAT1 binding proteins. RNA pull-down and RNA immunoprecipitation were used to validate CCAT1-Vimentin interaction. Transwell assay was used to evaluate the migration and invasion abilities of HeLa cells. RESULTS: RAP-MS method worked well by culturing cells with nucleoside analog 4-thiouridine, and cross-linking was performed using 365 nm wavelength ultraviolet. There were 631 proteins identified, out of which about 60% were RNA binding proteins recorded by the EuRBPDB database. Vimentin was one of the CCAT1 binding proteins and participated in the tumor metastasis pathway. Knocked down vimetin ( VIM ) and rescued the downregulation by overexpressing CCAT1 demonstrated that CCAT1 could enhance tumor migration and invasion abilities by stabilizing Vimentin protein. CONCLUSION CCAT1 may bind with and stabilize Vimentin protein, thus enhancing cancer cell migration and invasion abilities.
Humans ; HeLa Cells ; RNA, Long Noncoding/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Vimentin/metabolism* ; MicroRNAs/metabolism* ; Colonic Neoplasms/genetics* ; RNA-Binding Proteins/metabolism* ; Gene Expression Regulation, Neoplastic/genetics* ; Cell Movement/genetics*

This study aims to investigate the regulatory effects of Astragalus polysaccharide(APS) and APS combined with 5-fluorouracil(5-FU) on indoleamine-2,3-dioxygenase(IDO1) in the colon tumor microenvironment. Sixty Balb/c mice were randomized into a blank group, a model group, an APS group, an APS + 5-FU group, an APS + low-dose 5-FU group, and a 5-FU group. A tumor model was established by subcutaneous transplantation with CT-26 mouse colon cancer cells in other groups except the blank group. After successful modeling, each group was treated with corresponding drugs for 7 days. The general condition, body weight, and tumor volume of the mice were observed and measured daily during the treatment period. The mice were sacrificed at the end of treatment, and the tumor suppression rate and spleen index of the mice were calculated. Western blot and fluorescence quantitative PCR were employed to determine the protein and mRNA levels, respectively, of IDO1 in the tumor tissue of mice. High performance liquid chromatography was employed to measure the levels of tryptophan(Trp) and kynurenine(Kyn) in the tumor tissue of mice. Hematoxylin-eosin(HE) staining was performed to observe the histological changes of the tumor tissue, and immunohistochemistry to detect the changes of CD4 and CD8 expression in the tumor tissue. Compared with that in the model group, the tumor volume of mice in each treatment group significantly reduced. The body weights of mice in APS + 5-FU group and 5-FU group significantly reduced from day 4 to day 7 of treatment. In addition, the APS + 5-FU group and 5-FU group showed significantly decreased spleen index. The protein and mRNA levels of IDO1 were significantly down-regulated in the APS, APS + 5-FU, and APS + low-dose 5-FU groups. The drug interventions significantly increased the Trp content and decreased the Kyn content. The APS + 5-FU group showed significantly reduced infiltration of CD4~+ T lymphocytes and increased infiltration of CD8~+ T lymphocytes. APS inhibited the expression of IDO1 in the colon tumor microenvironment to increase CD8~+ T lymphocyte infiltration, and the combination of APS with 5-FU demonstrated better effect.
Mice ; Animals ; Tumor Microenvironment ; Colonic Neoplasms/genetics* ; Fluorouracil/pharmacology* ; Polysaccharides/pharmacology* ; CD8-Positive T-Lymphocytes/metabolism* ; RNA, Messenger/metabolism*

The epidermal cell differentiation regulator zinc finger protein 750 (ZNF750) is a transcription factor containing the Cys2His2 (C2H2) domain, the zinc finger structure of which is located at the N-terminal 25‍‍-‍46 amino acids of ZNF750. It can promote the expression of differentiation-related factors while inhibiting the expression of progenitor cell-related genes. ZNF750 is directly regulated by p63 (encoded by the TP63 gene, belonging to the TP53 superfamily). The Krüppel-like factor 4 (KLF4), repressor element-1 (RE-1)‍-silencing transcription factor (REST) corepressor 1 (RCOR1), lysine demethylase 1A (KDM1A), and C-terminal-binding protein 1/2 (CTBP1/2) chromatin regulators cooperate with ZNF750 to repress epidermal progenitor genes and activate the expression of epidermal terminal differentiation genes (Sen et al., 2012; Boxer et al., 2014). Besides, ZNF750 and the regulatory network composed of bone morphogenetic protein (BMP) signaling pathway, long non-coding RNAs (lncRNAs) (anti-differentiation non-coding RNA (ANCR) and tissue differentiation-inducing non-protein coding RNA (TINCR)), musculoaponeurotic fibrosarcoma oncogene (MAF)/MAF family B (MAFB), grainy head-like 3 (GRHL3), and positive regulatory domain zinc finger protein 1 (PRDM1) jointly promote epidermal cell differentiation (Sen et al., 2012).
Adenocarcinoma/metabolism* ; Carcinogenesis/genetics* ; Colonic Neoplasms/metabolism* ; Histone Demethylases/metabolism* ; Humans ; RNA, Long Noncoding/genetics* ; Transcription Factors/metabolism* ; Tumor Suppressor Proteins/metabolism*

Objective: To investigate the relationship between the expression of four mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) and NTRK genetic fusions in colorectal cancer. Methods: The paraffin-embedded tissue blocks of 830 cases of colorectal cancer were collected at the Affiliated Drum Tower Hospital, Nanjing University Medical School, China, from 2015 to 2019. Immunohistochemical and fluorescence in situ hybridization(FISH) method were used respectively to detect the expression of mismatch repair proteins and the break-apart of NTRKs; and the relationship between the expression of mismatch repair proteins and the NTRK genetic fusions was analyzed. Results: The overall mismatch repair protein deficiency (dMMR) rate was 9.88% (82/830), the mismatch repair proteins proficiency (pMMR) rate was 90.12%(748/830). The total deficiency rate of MLH1 protein was 9.04% (75/830), hPMS2 protein deficiency rate was 9.04% (75/830), MSH2 protein deficiency rate was 2.53% (21/830), MSH6 protein deficiency rate was 4.10% (34/830), the deficiency rate of synchronous MLH1 and PMS2 were 8.67% (72/830) and the deficiency rate of synchronous MSH2 and MSH6 were 2.17% (18/830). The dMMR group was associated with tumor location, different histological subgroups, tumor differentiation, AJCC stage and N stage (P<0.05). There were six cases (7.32%) carrying NTRK fusion by FISH among the 82 cases of dMMR, but only seven cases (0.94%) carrying NTRK fusion among the 748 cases of PMMR. The NTRKs translocation by FISH in all 13 cases were further confirmed by next generation sequencing. Among the clinicopathological characteristics, only differentiation showed significant difference between NTRK fusion positive and negative groups (P<0.05). More importantly, NTRK fusion was enriched in dMMR group (7.32% vs. 0.94%). Conclusion: In dMMR colorectal cancer group, the prevalence of NTRK fusion is higher than that in pMMR group.
Colonic Neoplasms ; Colorectal Neoplasms/genetics* ; DNA Mismatch Repair/genetics* ; Humans ; In Situ Hybridization, Fluorescence ; Mismatch Repair Endonuclease PMS2/metabolism* ; MutL Protein Homolog 1/metabolism* ; MutS Homolog 2 Protein/metabolism*

This study aims to investigate the potential mechanism of curcumin in mediating interleukin-6(IL-6)/signal transducer and activator of transcription 3(STAT3) signaling pathway to repair intestinal mucosal injury induced by 5-fluorouracil(5-FU) chemotherapy for colon cancer. SD rats were intraperitoneally injected with 60 mg·kg~(-1)·d~(-1) 5-FU for 4 days to establish a model of intestinal mucosal injury. Then the rats were randomly divided into model group(equal volume of normal saline), curcumin low, medium and high dose groups(50, 100, 200 mg·kg~(-1)), and normal SD rats were used as control group(equal volume of normal saline). Each group received gavage administration for 4 consecutive days, and the changes of body weight and feces were recorded every day. After administration, blood was collected from the heart, and jejunum tissues were collected. The levels of serum interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by ELISA, and at the same time, the concentration of Evans blue(EB) in jejunum was measured. Hematoxylin-eosin(HE) staining was used to observe the pathological state of jejunum, and the length of jejunum villi and the depth of crypt were measured. The positive expression levels of claudin, occludin and ZO-1 were detected by immunohistochemistry. Western blot was used to detect the protein expression of IL-6, p-STAT3, E-cadherin, vimentin and N-cadherin in jejunum tissues. The results showed that, curcumin significantly increased body weight and fecal weight(P<0.05 or P<0.01), decreased fecal score, EB concentration, IL-1β and TNF-α levels(P<0.05 or P<0.01) in rats. In addition, curcumin maintained the integrity of mucosal surface and villi structure of jejunum to a large extent, and reduced pathological changes in a dose-dependent manner. Meanwhile, curcumin could increase the positive expression of occludin, claudin and ZO-1(P<0.05 or P<0.01), repair intestinal barrier function, downregulate the protein expression of IL-6, p-STAT3, vimentin and N-cadherin in jejunum tissues(P<0.05 or P<0.01), and upregulate the protein expression of E-cadherin(P<0.05). Therefore, curcumin could repair the intestinal mucosal injury induced by 5-FU chemotherapy for colon cancer, and the mechanism may be related to the inhibition of IL-6/STAT3 signal and the inhibition of epithelial-mesenchymal transition(EMT) process.
Animals ; Colonic Neoplasms/drug therapy* ; Curcumin ; Fluorouracil/toxicity* ; Interleukin-6/genetics* ; Intestinal Mucosa/metabolism* ; Rats ; Rats, Sprague-Dawley ; STAT3 Transcription Factor/metabolism* ; Signal Transduction

OBJECTIVE: To explore the role of miR-593 in regulating the proliferation of colon cancer cells and the molecular mechanism. METHODS: Bioinformatics analysis identified PLK1 as the possible target gene of miR-593. Luciferase assay was employed to verify the binding between miR-593 and PLK1, and qRT-PCR and Western blotting were used to verify that PLK1 was the direct target gene of miR-593. CCK-8 assay was performed to test the hypothesis that miR-593 inhibited the proliferation of colon cancer cells by targeting PLK1. RESULTS: Luciferase assay identified the specific site of miR-593 binding with PLK1. Western blotting showed a significantly decreased expression of PLK1 in the colon cancer cells transfected with miR-593 mimics and an increased PLK1 expression in the cells transfected with the miR-593 inhibitor as compared with the control cells ( < 0.05). The results of qRT-PCR showed no significant differences in the expression levels of PLK1 among the cells with different treatments ( > 0.05). The cell proliferation assay showed opposite effects of miR-593 and PLK1 on the proliferation of colon cancer cells, and the effect of co-transfection with miR-593 mimic and a PLK1-overexpressing plasmid on the cell proliferation was between those in PLK1 over-expressing group and miR-593 mimic group. CONCLUSIONS miR-593 inhibits the proliferation of colon cancer cells by down-regulating PLK1 and plays the role as a tumor suppressor in colon cancer.
Binding Sites ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; metabolism ; pathology ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; In Vitro Techniques ; MicroRNAs ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sincalide ; metabolism ; Transfection

OBJECTIVE: To investigate the expression of cytokine signal suppressor 3 (SOCS3) in colon cancer tissue and the mechanism by which SOCS3 regulates the proliferation and invasion of colon cancer. METHODS: We collected the specimens of tumor tissues and paired adjacent tissues from 80 patients with colon cancer undergoing radical resection in our hospital between July, 2014 and May, 2017, and the expression of SOCS3 in the tissue samples was analyzed using Western blotting. We also transfected colon cancer cell line SW480 with a SOCS3-overexpressing plasmid or a small interference RNA (siRNA) for SOCS3 knockdown, and the changes in the cell proliferation and invasion capacity were evaluated using CCK-8 assay and Transwell assay, respectively. The effect of demethylation and IL-6 treatment on SOCS3 expression and the proliferation and invasion of SW480 cells were observed. RESULTS: Colon cancer tissues showed a lowered expression of SOCS3 compared with the adjacent tissues. Over-expression of SOCS3 significantly inhibited while SOCS3 knockdown obviously promoted the proliferation and invasion of SW480 cells . Demethylation treatment up-regulated SOCS3 expression and inhibited the proliferation and invasion capacity of SW480 cells; IL-6 treatment of the cells caused the reverse changes. CONCLUSIONS SOCS3 participates in the development and progression of colon cancer and serves as a potential target for colon cancer treatment. In patients with colon cancer, the low expression of SOCS3 possibly as a result of methylation may promote the proliferation and invasion of the cancer cells.
Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; etiology ; pathology ; Cytokines ; Demethylation ; Disease Progression ; Humans ; Interleukin-6 ; pharmacology ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; RNA, Small Interfering ; Signal Transduction ; Suppressor of Cytokine Signaling 3 Protein ; genetics ; metabolism ; Transfection

Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs) could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC) patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A) and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 "CRC genes." These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology.
Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; genetics ; immunology ; metabolism ; Case-Control Studies ; Colonic Neoplasms ; immunology ; metabolism ; Computational Biology ; methods ; Computer Simulation ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Immunoglobulin G ; immunology ; Male ; Middle Aged ; Protein Array Analysis ; methods

OBJECTIVE: To investigate the expressions of secreted frizzled-related protein 4 (SFRP4) in stage Ⅱ DNA mismatch repair-deficient (dMMR) and mismatch repair- proficient (pMMR) colorectal cancers and explore their clinical significance. METHODS: We collected fresh stage Ⅱ colon cancer tissues with different MMR status detected by immunohistochemistry (IHC). The differentially expressed mRNAs between dMMR and pMMR tumors were identified by Affymetrix Human oeLncRNA gene chip, and the expression of SFRP4 in these cancer tissues and in colorectal cancer cell lines were detected using Western blotting and real- time quantitative PCR. The apoptosis rates of HCT116 cells with and without siRNA- mediated transient SFRP4 knockdown were determined using flow cytometry. We further investigated the expression pattern of Ki-67 and its correlation with SFRP4 expression. RESULTS: Compared with pMMR colon cancer tissues or cells, both dMMR colon cancer tissues (=0.014) and cells (=0.0079) showed significantly increased expression of SFRP4, which was in negative correlation with Ki-67 (=0.041). In HCT116 cells, transient SFRP4 knockdown resulted in decreased cell apoptosis, including both early apoptosis (=0.003) and late apoptosis (=0.024). CONCLUSIONS Up-regulation of SFRP4 in dMMR stage Ⅱ colon cancer promotes apoptosis and inhibits proliferation of the cancer cells, and may improve the prognosis of dMMR colon cancer.
Apoptosis ; Cell Proliferation ; Colon ; metabolism ; pathology ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; DNA Mismatch Repair ; Gene Knockdown Techniques ; HCT116 Cells ; Humans ; Ki-67 Antigen ; metabolism ; Prognosis ; Proto-Oncogene Proteins ; genetics ; metabolism ; Up-Regulation

OBJECTIVETo explore the relationship between DNA mismatch repair (MMR) and clinicopathologic features and prognosis in patients with stages II and III colon cancers.

METHODSThe clinical and pathological data of 440 patients with stage II/III colon cancer after radical resection were retrospectively reviewed and analyzed. Immunohistochemical staining was used to assess the expression of MMR proteins (MLH1, MSH2, MSH6 and PMS2), and the correlation between DNA MMR and clinicopathological features and prognosis of colon cancers was analyzed.

RESULTSOf the 440 tumor samples tested for DNA mismatch repair status, 90 (20.5%) demonstrated defective DNA mismatch repair and 350 (79.5%) had proficient DNA mismatch repair. Defective DNA mismatch repair (dMMR) was associated with young patients (≤ 60), proximal colon cancer, stage II, poorly differentiated adenocarcinoma and mucinous adenocarcinoma (P<0.05 for all). Among the 440 patients, 126 (28.6%) cases had recurrence or metastasis and 93 (21.1%) died during the median follow-up of 61.0 months. The five-year disease-free survival (DFS) rate was 82.2% among the patients with tumor exhibiting dMMR, significantly higher than that in patients with tumors exhibiting pMMR (68.9%, P=0.02). The univariate and mutlivariate analyses showed that the MMR status is an independent factor affecting 5-year disease-free survival and overall survival (OS) in colon cancer patients (P<0.05 for both).

CONCLUSIONSDefective DNA mismatch repair (dMMR) is associated with patients with proximal colon cancer, stage II and poorly defferentiated adenocarcinoma and mucinous adenocarcinoma. The prognosis for patients with dMMR is better than those with pMMR. dMMR may be a useful biomarker for the prognosis of colon cancer.

Adaptor Proteins, Signal Transducing ; metabolism ; Adenocarcinoma ; genetics ; metabolism ; mortality ; pathology ; Adenocarcinoma, Mucinous ; genetics ; metabolism ; mortality ; pathology ; Adenosine Triphosphatases ; metabolism ; Age Factors ; Analysis of Variance ; Colonic Neoplasms ; genetics ; metabolism ; mortality ; pathology ; DNA Mismatch Repair ; DNA Repair Enzymes ; metabolism ; DNA-Binding Proteins ; metabolism ; Disease-Free Survival ; Humans ; Mismatch Repair Endonuclease PMS2 ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; metabolism ; Neoplasm Recurrence, Local ; Nuclear Proteins ; metabolism ; Prognosis ; Retrospective Studies ; Survival Rate