OBJECTIVE: To reveal the mechanisms by which miR-513b-5p inhibits metastasis of colon cancer stem cells (CCSCs) through IL-6/STAT3 in HCT116 cells.
METHODS: Sphere formation media and magnetic cell sorting were used to enrich and screen CCSCs. We used a colony formation assay, cell proliferation and viability assays, and a nude mouse transplantation tumor assay to identify CCSCs. ELISA was performed to identify IL-6 in the cell culture medium, and the growth, viability, wound healing, and transwell migration of distinct cell groups were compared to differentiate them. Dual-luciferase reporter assay, RT-PCR, and/or Western Blot analysis were conducted to determine the correlation between them.
RESULTS: CD133+CD44+ HCT116 cells were shown to have higher cloning efficiency, greater proliferation ability and viability, and stronger tumorigenicity. A dual-luciferase reporter assay revealed that miR-513b-5p negatively affected STAT3 expression. RT-PCR and/or Western Blot analysis suggested that miR-513b-5p negatively affected STAT3 and Vimentin, while positively affecting E-cadherin expression. The STAT3 overexpression vector + miR-513b-5p inhibitor cell group had the highest efficiency, greatest proliferation ability and viability, and the highest IL-6 level in the experiments.
CONCLUSIONS: Mir-513b-5p inhibited the epithelial-mesenchymal transition (EMT) of CCSCs through IL-6/STAT3. This potential mechanism may provide a new therapeutic target for colon cancer.

UNASSIGNED: To explore whether metformin (MET) can affect the biological behaviour and CD133 mRNA expression of CD133+ colon cancer stem cells (CCSCs) through miR-342-3p.
UNASSIGNED: The direct immunomagnetic bead method was used to select CD133+ CCSCs from the SW480 and HCT116 cell lines, and miRNA-tailing qRT-PCR was used to detect the expression changes of tumor suppressor-related miRNAs (miR-34a, miR-126, miR-143, miR-145, miR-342-3p, miR-342-5p) after MET intervention. Then, miR-342-3p with markedly significant differential expression was selected as the target miRNA. The lentiviruses LV16-hsa-miR-342-3p inhibitor and LV16-NC were used for the transfection inhibition test. CCK-8, flow cytometry, and qRT-PCR were used to detect the cell viability, apoptosis rate, and CD133 mRNA expression of CD133+ CCSCs.
UNASSIGNED: Under the high-glucose environment, the expression of tumor suppressor-related miRNAs in CCSCs changed differently (p <0.05), MET also had different effects on the expression of tumor suppressor-related miRNA under different glucose concentrations (p<0.05). Among them, MET upregulates the expression of miR-342-3p in CCSCs for the first time. The results of the lentiviruses transfection inhibition test showed that after miR-342-3p was inhibited, the cell viability and apoptosis rate of CD133+ CCSCs did not change significantly compared with before inhibition (p>0.05), but the expression of CD133 mRNA markedly increased (p<0.05). Meanwhile, after MET intervention, the apoptosis rate and the expression of CD133 mRNA of CD133+ CCSCs was significantly increased, and the proliferation of CD133+ CCSCs was obviously inhibited (p<0.05).
UNASSIGNED: MET upregulating the expression of miR-342-3p may not have a significant effect on the proliferation and apoptosis of CD133+ CCSCs, but it can reduce the expression of CD133 mRNA in CD133+ CCSCs.

Colon cancer stem cells (CCSCs) play an important role in facilitating colon cancer occurrence, metastasis and drug resistance. The results of our previous studies confirmed that the well-studied antioxidant gene peroxiredoxin-2 (PRDX2) promotes colon cancer progression. However, the underlying function and mechanisms associated with PRDX2 remodeling in the context of CCSCs have remained poorly studied. In our present study, we demonstrated that PRDX2 is highly expressed in CD133/CD44-positive colon cancer tissues and spheroid CD133+CD44+ CCSCs. PRDX2 overexpression was shown to be closely correlated with CD133+CD44+ CCSCs in colon cancer. Furthermore, PRDX2 depletion markedly suppressed CD133+CD44+ CCSC stemness maintenance, tumor initiation, migration and invasion and liver metastasis. Furthermore, the expression of various EMT markers and Wnt/β-catenin signaling proteins was altered after PRDX2 inhibition. In addition, PRDX2 knockdown led to increased ROS production in CD133+CD44+ CCSCs, sensitizing CCSCs to oxidative stress and chemotherapy. These results suggest that PRDX2 could be a possible therapeutic target in CCSCs.

Patients with advanced-stage colon cancer often exhibit resistance against treatment and distant metastasis, both key contributors to poor prognosis. Emerging evidence indicates that cancer stem cells (CSCs), characterized by the enhanced ability to self-renew, resist therapeutics, and promote metastasis, represents a clinical challenge to target. Alternative therapeutic approaches are urgently required. Here, we explored the feasibility of disrupting the intracellular communications between CSCs and the tumor microenvironment by way of exosomes. First, we demonstrated that exosomes secreted by colon tumorspheres (Exosp) promoted 5-FU resistance, migration, and tumorsphere formation. Exosp also increased the generation of cancer-associated fibroblasts and M2 polarized macrophages in vitro. Oncogenic molecules, including IL-6, p-STAT3, TGF-β1, and β-catenin, were identified as the cargoes of Exosp. Furthermore, the public database revealed the high abundance of miR-1246 in serum exosomes from colon cancer patients, and we verified in the Exosp from HCT116 and HT29 cells. Therapeutically, we demonstrated the ovatodiolide treatment reduced exosomal cargoes from tumorspheres (Exosp_OV). Exosp_OV were significantly less capable of promoting 5-FU resistance, migration, and tumorsphere formation when co-cultured with HCT116 and HT29 cells. Notably, Exosp_OV was less CAF- and M2 TAM-transformative. Computational docking analysis revealed that OV could bind and significantly reduced β-catenin activity. Finally, mouse xenograft data indicated that ovatodiolide suppressed tumor growth via down-regulating IL-6, STAT3, β-catenin expression, and serum exosomal miR-1246. In conclusion, our findings provided preclinical supports for ovatodiolide as a colon CSC inhibitor by reducing β-catenin/STAT3/miR-1246 signaling conveyed by CSC derived exosomes.

Colorectal cancer (CRC) is a fatal disease caused by the uncontrolled propagation and endurance of atypical colon cells. A person\'s lifestyle and eating pattern have significant impacts on the CRC in a positive and/or negative way. Diet-derived phytochemicals modulate the microbiome as well as targeting colon cancer stem cells (CSCs) that are found to offer significant protective effects against CRC, which were organized in an appropriate spot on the paper. All information on dietary phytochemicals, gut microbiome, CSCs, and their influence on CRC were accessed from the various databases and electronic search engines. The effectiveness of CRC can be reduced using various dietary phytochemicals or modulating microbiome that reduces or inverses the progression of a tumor as well as CSCs, which could be a promising and efficient way to reduce the burden of CRC. Phytochemicals with modulation of gut microbiome continue to be auspicious investigations in CRC through noticeable anti-tumorigenic effects and goals to CSCs, which provides new openings for cancer inhibition and treatment.

Cancer stem cells, which are a population of cancer cells sharing common properties with normal stem cells, have strong self-renewal ability and multi-lineage differentiation potential to trigger tumor proliferation, metastases, and recurrence. From this, targeted therapy for cancer stem cells may be one of the most promising strategies for comprehensive treatment of tumors in the future. We design a facile approach to establish the colon cancer stem cells-selective fluorescent probe based on the low-density lipoprotein (LDL) and the novel AgGa xIn(1- x)S2 quantum dots (AGIS QDs). The AGIS QDs with a high crystallinity are obtained for the first time via cation-exchange protocol of Ga3+ to In3+ starting from parent AgInS2 QDs. Photoluminescence peak of AGIS QDs can be turned from 502 to 719 nm by regulating the reaction conditions, with the highest quantum yield up to 37%. Subsequently, AGIS QDs-conjugated LDL nanocomposites (NCs) are fabricated, in which a cationic polyelectrolyte was used as a coupling reagent to guarantee the electrostatic self-assembly. The structural integrity and physicochemical properties of the LDL-QDs NCs are found to be maintained in vitro, and the NCs exhibit remarkable biocompatibility. The LDL-QDs can be selectively delivered into cancer stem cells that overexpress LDL receptor, and three-dimensional imaging of cancer stem cells is realized. The results of this study not only demonstrate the versatility of nature-derived lipoprotein nanoparticles, but also confirm the feasibility of electrostatic conjugation using cationic polyelectrolyte, allowing reseachers to design nanoarchitectures for targeted diagnosis and treatment of cancer.

BACKGROUND: Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality in both sexes globally. This is not unconnected with the heterogeneity and plasticity of CRC stem cells (CRC-SCs) which stealthily exploit the niche-related and (epi)genetic factors to facilitate metastasis, chemoresistance, tumor recurrence, and disease progression. Despite the accumulating evidence of the role of dysregulated microRNAs in malignancies, the therapeutic efficacy of pharmacological-targeting of CRC-SC-associated microRNAs is relatively under-explored.
METHODS: In this present study, we employed relatively new bioinformatics approaches, analyses of microarray data, Western blot, real-time polymerase chain reaction (RT-PCR), and functional assays to show that hsa-miR-324-5p expression is significantly suppressed in CRC cells, and inversely correlates with the aberrant expression of SOD2.
RESULTS: This converse hsa-miR-324-5p/SOD2 relationship is associated with enhanced oncogenicity, which is effectively inhibited by 4-acetylantroquinonol B (4-AAQB), as evidenced by inhibited cell viability and proliferation, as well as attenuated migration, invasion, and clonogenicity in 4-AAQB-treated DLD1 and HCT116 cells. Interestingly, 4-AAQB did not affect the viability and proliferation of normal colon cells. We also showed that 4-AAQB-induced re-expression of hsa-miR-324-5p, akin to short-interfering RNA, reduced SOD2 expression, correlates with the concurrent down-regulation of SOD2, N-cadherin, vimentin, c-Myc, and BcL-xL2, with concomitant up-regulation of E-cadherin and BAX2 proteins. Enhanced expression of hsa-miR-324-5p in the CRC cells suppressed their tumorigenicity in vitro and in vivo. Additionally, 4-AAQB synergistically potentiates the FOLFOX (folinate (leucovorin), fluorouracil (5FU), and oxaliplatin) anticancer effect by eliciting the re-expression of SOD2-suppressed hsa-miR-324, and inhibiting SOD2-mediated tumorigenicity.
CONCLUSIONS: Our findings highlight the pre-clinical anti-CSC efficacy of 4-AAQB, with or without FOLFOX in CRC, and suggest a potential novel therapeutic strategy for CRC patients.

BACKGROUND: Cancer stem cells (CSCs) are considered to be the major factor in tumor initiation, progression, metastasis, recurrence and chemoresistance. Maintaining the stemness and promoting differentiation of these cells involve various factors. Recently, long non-coding RNAs (lncRNAs) have been identified as new regulatory factors in human cancer cells. However, the function of lncRNAs in colon CSCs is still unknown.
METHODS: Primary colon cancer cells were maintained in serum-free medium to form spheres and CD133+/CD166+/CD44+ spheroid cells were selected using FACS technique. Then we detected growth curve, colony formation, invasion and migration ability, and tumorigenicity of CD133+/CD166+/CD44+ cells. LOCCS-siRNA and pcDNA-LOCCS plasmid vectors were constructed and transfected to evaluate impact of the lncRNA. We also performed dual luciferase reporter assay to verify the interaction of LOCCS and miR-93.
RESULTS: The research explored lncRNA expression and the regulatory role of novel lncRNAs in colon CSCs. Using the stem cell markers CD133, CD166 and CD44, we found a subpopulation of highly tumorigenic human colon cancer cells. They displayed some characteristics of stem cells, including the ability to proliferate and form colonies, to resist chemotherapeutic drugs, and to produce xenografts in nude mice. We also found an lncRNA, LOCCS, with obviously upregulated expression in colon CSCs. Knockdown of LOCCS reduced cell proliferation, invasion, migration, and generation of tumor xenografts. Furthermore, microRNA-93 (miR-93) and Musashi-1 mediated the tumor suppression of LOCCS knockdown.
CONCLUSIONS: There was reciprocal repression between LOCCS and miR-93. Research on mechanisms suggested direct binding, as a predicted miR-93 binding site was identified in LOCCS. This comprehensive analysis of LOCCS in colon CSCs provides insight for elucidating important roles of the lncRNA-microRNA functional network in human colon cancer.

To investigate whether Portulaca oleracea extract affects tumor formation in colon cancer stem cells and its chemotherapy sensitivity. In addition, to analyze associated genetic changes within the Notch signal transduction pathway. Serum-free cultures of colon cancer cells (HT-29) and HT-29 cancer stem cells were treated with the chemotherapeutic drug 5-fluorouracil to assess sensitivity. Injections of the stem cells were also given to BALB/c mice to confirm tumor growth and note its characteristics. In addition, the effect of different concentrations of P. oleracea extract was tested on the growth of HT-29 colon cancer cells and HT-29 cancer stem cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The effects of P. oleracea extract on the expression of β-catenin, Notch1, and Notch2 in the HT-29 cells were studied using reverse transcription polymerase chain reaction and Western blotting. The tumor volume of the HT29 cells was two times larger than that of HT29 cancer stem cells. Treatment with P. oleracea extract inhibited the proliferation of both HT-29 cancer cells and HT-29 cancer stem cells at doses from 0.07 to 2.25 µg/mL. Apoptosis of HT-29 cancer cells and HT-29 cancer stem cells was assessed by flow cytometry; it was enhanced by the addition of P. oleracea extract. Finally, treatment with P. oleracea extract significantly downregulated the expression of the Notch1 and β-catenin genes in both cell types. The results of this study show that P. oleracea extract inhibits the growth of colon cancer stem cells in a dose-dependent manner. Furthermore, it inhibits the expression of the Notch1 and β-catenin genes. Taken together, this suggests that it may elicit its effects through regulatory and target genes that mediate the Notch signal transduction pathway.

Recent evidences have unveiled critical roles of cancer stem cells (CSCs) in tumorigenicity, but how interactions between CSC and tumor environments help maintain CSC initiation remains obscure. The small GTPases Rab27A regulates autocrine and paracrine cytokines by monitoring exocytosis of extracellular vesicles, and is reported to promote certain tumor progression. We observe that overexpression of Rab27A increased sphere formation efficiency (SFE) by increasing the proportion of CD44+ and PKH26high cells in HT29 cell lines, and accelerating the growth of colosphere with higher percentage of cells at S phase. Mechanism study revealed that the supernatant derived from HT29 sphere after Rab27A overexpression was able to expand sphere numbers with elevated secretion of VEGF and TGF-β. In tumor implanting nude mice model, tumor initiation rates and tumor sizes were enhanced by Rab27A with obvious angiogenesis. As a contrast, knocking down Rab27A impaired the above effects. More importantly, the correlation between higher p65 level and Rab27A in colon sphere was detected, p65 was sufficient to induce up-regulation of Rab27A and a functional NF-κB binding site in the Rab27A promoter was demonstrated. Altogether, our findings reveal a unique mechanism that tumor environment related NF-κB signaling promotes various colon cancer stem cells (cCSCs) properties via an amplified paracrine mechanism regulated by higher Rab27A level.