scholarly journals Hiwi Promotes the Proliferation of Colorectal Cancer Cells via Upregulating Global DNA Methylation

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Lin Yang ◽  
Lei Bi ◽  
Qingwei Liu ◽  
Meng Zhao ◽  
Bin Cao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Cell Lines ◽  
Adenovirus Vector ◽  
Global Dna Methylation ◽  
Cell Renewal ◽  
Western Blot Assay ◽  
Real Time Quantitative Pcr ◽  
Protein Levels ◽  
Ht 29

Hiwi is well known for its role in stem cell renewal, maintaining the resting stage, and downregulating cell cycle of stem cells via RNA silencing. And Hiwi overexpression has been recognized in several types of cancers. In the present study, we examined the Hiwi expression in colorectal cancer (CRC) specimens in both mRNA and protein levels via real-time quantitative PCR, western blot assay, and immunohistochemical staining. Then we explored the role of Hiwi in the cancer cell proliferation and in the DNA methylation in human CRC Caro-2 and HT-29 cell lines. Results demonstrated that both mRNA and protein levels of Hiwi were significantly higher in 38 CRC tissues than in 38 peritumor tissues. Moreover, the Hiwi overexpression with an adenovirus vector significantly promoted the proliferation of Caro-2 and HT-29 cells, associated with significant increase in the global DNA methylation levels. And the chemical inhibition of DNA methylation significantly restrained such proliferation promotion. In summary, we confirmed that Hiwi was overexpressed in CRC tissues and that the forced Hiwi overexpression promoted the proliferation and global DNA methylation of CRC cell lines. Our results imply for the first time that Hiwi promotes the proliferation of CRC cells via promoting global DNA methylation.

microRNA-133b Regulates Cell Proliferation and Cell Cycle Progression via Targeting HuR in Colorectal Cancer

2022 ◽  
Vol 12 (2) ◽  
pp. 335-345
Author(s):  
Xiaoyan Zhang ◽  
Wei Zhu ◽  
Junjie Lu
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Viability ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter ◽  
Protein Levels ◽  
Ht 29

MicroRNAs (miRNAs/miRs) have been identified to serve a key role in the development of tumors. However, the role of miR-133b in colorectal cancer (CRC) remains largely unclear. This study will investigate the role and mechanism of miR-133b in CRC. Reverse transcription-quantitative polymerase chain reaction analysis was performed to detect the level of miR-133b in CRC cell lines. Bioinformatics software TargetScan predicted the potential target genes of miR-133b, and a dual luciferase reporter assay was used to confirm this. To investigate the role of miR-133b in CRC cells, miR-133b was upregulated or downregulated in CRC cell lines (SW620 and HT-29) by transfecting with a miR-133b mimic or inhibitor, respectively. Subsequently, cell viability was analyzed using MTT assay, whereas cell apoptosis and the cell cycle distribution were analyzed by flow cytometry. In addition, the associated protein levels were detected using western blot analysis. The results demonstrated that miR-133b was significantly downregulated in CRC cell lines when compared with the normal colonic epithelial NCM-460 cell line. Human antigen R (HuR; also termed ELAVL1) was demonstrated to be a direct target of miR-133b and was negatively regulated by miR-133b. HuR was also notably upregulated in the CRC cell lines when compared with the normal control. Transfection of SW620 and HT-29 cells with the miR-133b mimic significantly inhibited cell viability, and induced cell apoptosis and G1 phase arrest, while upregulation of HuR demonstrated the opposite effects. Furthermore, the present data demonstrated that the miR-133b mimic significantly enhanced the protein levels of p21 and p27, and downregulated cyclin D1 and cyclin A levels in SW620 and HT-29 cells; the opposite effects were observed following treatment with the miR-133b inhibitor. In conclusion, the data indicate that miR-133b suppressed CRC cell growth by targeting HuR.

scholarly journals Antiproliferative and Apoptotic Effects of Red Beetroot and Betanin on Human Colorectal Cancer Cell Lines

2021 ◽  
Author(s):  
Amir Saber ◽  
Nasim Abedimanesh ◽  
Mohammad-Hossein Somi ◽  
Ahmad Yari Khosroushahi
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Caspase 3 ◽  
Caspase 8 ◽  
Alcoholic Extract ◽  
Caspase 9 ◽  
Dapi Staining ◽  
Normal Cells ◽  
Colorectal Cancer Cell Lines ◽  
Ht 29

Abstract Background: Colorectal cancer (CRC) is the third most common type of cancer worldwide. Fruit and vegetables have some active compounds such as flavonoids and polyphenols that protect against malignancies through their antioxidative, anti-inflammatory, anti-proliferative, neuro, and hepatoprotective properties. Red beetroot (Beta vulgaris) contains red (betacyanins) and yellow (betaxanthins) pigments known as betalains. Betanin makes up 75-95% of the total betacyanins, possessed a wide range of favorable biological effects such as chemopreventive, anticarcinogenic, anti-tumorogenic, antiangiogenic, and proapoptotic effects. Methods: Red beetroot hydro-alcoholic extract and betanin were used to treat Caco-2 and HT-29 colorectal cancer cells, as well as KDR/293 normal epithelial cells. The half-maximal inhibitory concentration (IC50) was determined by prescreening MTT tests in the range of 20 to 140 µg/ml at 24 and 48 h. The cytotoxicity and apoptosis-inducing evaluations were performed via MTT assay, DAPI staining, and FACS-flow cytometry tests using determined times and doses. Moreover, the expression level of six important genes involving in the apoptosis pathway (Bcl-2, BAD, Caspase-3, Caspase-8, Caspase-9, and Fas-R) were determined using the real-time polymerase chain reaction (RT-PCR) method.Results: The IC50 doses for HT-29 and Caco-2 cell lines were determined to be about 92 μg/mL, 107 μg/mL for beetroot hydro-alcoholic extract, and 64 μg/mL, 90 μg/mL for betanin at 48 h, respectively. Our findings showed that beetroot extract and betanin significantly inhibit the growth of HT-29 and Caco-2 cell lines, time and dose-dependently, without considerable adverse effects on KDR/293 normal cells. Moreover, DAPI staining and flow cytometry results revealed significant apoptosis symptoms in treated cancerous cell lines. The expression level of pro-apoptotic genes involved in intrinsic and extrinsic apoptosis pathways (BAD, Caspase-3, Caspase-8, Caspase-9, and Fas-R) in treated HT-29 and Caco-2 cells was higher than untreated and normal cells, whereas the anti-apoptotic gene (Bcl-2) was downregulated. Conclusion: Beetroot hydro-alcoholic extract and betanin significantly inhibited cell proliferation and induced cell apoptosis (intrinsic and extrinsic pathways) via modification of effective genes in both colorectal cancer cell lines with no significant cytotoxic effects on KDR/293 normal cells. The mechanism of the anticancer effects of red beetroot extract and betanin needs to be further studied.

scholarly journals Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but not Decreased Promoter Methylation in Colorectal Cancer

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 539 ◽  
Author(s):  
Alexei J. Stuckel ◽  
Wei Zhang ◽  
Xu Zhang ◽  
Shuai Zeng ◽  
Urszula Dougherty ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Cell Lines ◽  
Promoter Methylation ◽  
Cpg Island ◽  
Methylation Status ◽  
Cxcr4 Expression ◽  
Normal Colonic Mucosa ◽  
Gene Body ◽  
Expression Levels

In colorectal cancer (CRC), upregulation of the C-X-C motif chemokine receptor 4 (CXCR4) is correlated with metastasis and poor prognosis, highlighting the need to further elucidate CXCR4’s regulation in CRC. For the first time, DNA methylation and 5-hydroxymethylcytosine aberrations were investigated to better understand the epigenetic regulation of CXCR4 in CRC. CXCR4 expression levels were measured using qPCR and immunoblotting in normal colon tissues, primary colon cancer tissues and CRC cell lines. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients. The DNA methylation status spanning CXCR4 gene was evaluated using combined bisulfite restriction analysis (COBRA). The methylation status in the CXCR4 gene body was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. CXCR4 expression levels were significantly increased in tumor stromal cells and in tumor colonocytes, compared to matched cell types from adjacent normal-appearing mucosa. CXCR4 promoter methylation was detected in a minority of colorectal tumors in the TCGA. The CpG island of the CXCR4 promoter showed increased methylation in three of four CRC cell lines. CXCR4 protein expression differences were also notable between microsatellite stable (MSS) and microsatellite instable (MSI) tumor cell lines. While differential methylation was not detected in CXCR4, enrichment of 5-hydroxymethylcytosine (5hmC) in CXCR4 gene bodies in CRC was observed compared to adjacent mucosa.

scholarly journals The Effect of Compound Sophora on Fluorouracil and Oxaliplatin Resistance in Colorectal Cancer Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
WeiHua Yin ◽  
GuPing Zhong ◽  
HuiZhen Fan ◽  
HongMei Xia
Keyword(s):  
Colorectal Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Sw480 Cell ◽  
Gastric Cancer Cells ◽  
Glycoprotein P ◽  
Chemotherapy Drugs ◽  
Protein Levels ◽  
Enhanced Expression

Fluorouracil (5-FU) and oxaliplatin (L-OHP) are the most commonly used chemotherapy drugs for colorectal cancer, though resistance is common. Compound Sophora injection is a traditional Chinese medicine that can protect the liver against oxidation, improve immunity, and enhance sensitivity to chemotherapy; it may have an effect of reversing resistance in 5-FU- and L-OHP-resistant gastric cancer cells (5-FU/SW480 and L-OHP/SW480, respectively). A concentration gradient experiment was performed to identify a nontoxic dose of compound Sophora injection. 5-FU/SW480 and L-OHP/SW480 cells were treated with the nontoxic dose of compound radix Sophorae injection for 48 h, and changes in drug resistance to 5-FU and L-OHP were detected. Alterations in apoptosis and the cell cycle were assessed, as were the mRNA and protein levels of permeability glycoprotein (P-gp), annexin A1 (ANXA1), and ATP-binding cassette superfamily G member 2 (ABCG2). Flow cytometry showed a reduction in the number of cells in the G1 phase and an increase of cells in the S phase (P<0.05). mRNA and protein expression of P-gp and ABCG2 was significantly higher in 5-FU/SW480 and L-OHP/SW480 cell lines, and ANXA1 expression decreased significantly (P<0.05). Compound Sophora injection can reverse the drug resistance of 5-FU/SW480 and L-OHP/SW480 cell lines to 5-FU and L-OHP, respectively, possibly through a mechanism involving reduced expression of P-gp and ABCG2 but enhanced expression of ANXA1, which is the basis for the identification of clinical drug resistance in colorectal cancer.

Analysis of RGS2 expression and prognostic significance in stage II and III colorectal cancer

2010 ◽  
Vol 30 (6) ◽  
pp. 383-390 ◽  
Author(s):  
Zheng Jiang ◽  
Zhimin Wang ◽  
Ye Xu ◽  
Beilan Wang ◽  
Wei Huang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Mrna Expression ◽  
Survival Time ◽  
Cell Lines ◽  
Prognostic Significance ◽  
Disease Free Survival ◽  
Stage Ii ◽  
Rank Test ◽  
Clinicopathological Factors ◽  
Real Time Quantitative Pcr

The role of RGS2 (regulator of G-protein signalling 2) has been studied in several tumours. The purpose of the present study is to investigate the correlations between clinicopathological factors and patients' survival time and RGS2 expression in stage II and III CRC (colorectal cancer) patients. Real-time quantitative PCR was performed in 36 CRC tissues with recurrence and 28 without recurrence, and in three CRC-metastasis-derived cell lines (SW620, LoVo and Colo205) and 3 primary-CRC-derived ones (SW480, Caco-2 and HCT116) to examine RGS2 mRNA expression. In addition, to provide visualized evidence for RGS2 mRNA expression, random CRC samples were also performed with RT–PCR (reverse transcription–PCR). RGS2 protein was detected by immunostaining in 118 paraffin-embedded specimens, and the correlations between clinicopathological factors and survival time and RGS2 expression were analysed. We found that RGS2 mRNA was down-regulated both in CRC tissues with recurrence and metastasis-derived cell lines, and the expression level of RGS2 was unrelated to gender, age, tumour grade, or lymphovascular or perineural invasion. However, it was positively related to disease-free survival time (P<0.05). Furthermore, low RGS2 expression indicated a poorer survival rate (P<0.05, log-rank test). Multivariate analysis also showed that weak RGS2 protein expression was an independent adverse prognosticator in CRC (P<0.05). Taken together, we suggested that down-regulation of RGS2 might play an important role in CRC metastasis and predict poor prognosis in stage II and III CRC patients.

Identification of epigenetic silencing of GCNT2 expression by comprehensive real-time PCR screening in colorectal cancer.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 506-506
Author(s):  
Kazunorii Nakamura ◽  
Horomichi Sawaki ◽  
Keishi Yamashita ◽  
Masahiko Watanabe ◽  
Hisashi Narimatsu
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Critical Role ◽  
Normal Mucosa ◽  
Primary Cancer ◽  
Normal Tissues ◽  
Cancer Tissues

506 Background: Glycoprotein expression profile has been proved to be dramatically altered in human cancers, however specific glycogenes which are aberrant in expression in cancer cells has not been fully identified. Recent accumulated evidence supported notion that the reduced expression of tumor suppressor genes is explained by DNA promoter methylation in human cancer. Methods: We used Comprehensive Real time PCR system (CRPS) for glycogenes (189 genes) to identify genes aberrantly expressed in colorectal cancer tissues (CRC) as compared to the corresponding normal mucosa tissues. GCNT2 was of particular interest among the identified genes in CRC. Results: (1) GCNT2 harbors 3 isoforms which have different promoter regions. (2) All of the 3 isoforms of GCNT2 genes were remarkably decreased in CRC as compared to the corresponding normal mucosa, and each isoform expression was strongly associated with other 2 isoforms in primary cancer tissues by TaqMan real time PCR (R = 0.99-995, p < 0.0001). (3) Among the 5 CRC cell lines (DLD1, HCT116, CACO2, LOVO), those which were silenced in expression were reactivated by demethylating agents such as 5-aza-2’ deoxycytidine and trichostatin A. (4) Promoter region of the variant 2 of GCNT2 was consistent with its silenced expression in CRC cell lines by cloned sequence, so we examined DNA methylation status of the promoter of the GCNT2 variant 2 in 50 primary cancer tissues and the corresponding normal tissues. Quantitative MSP revealed that almost half of normal tissues have methylation as high as tumor tissues, while, in the primary CRC with less methylation in the corresponding normal tissues, DNA methylation was higher in primary CRC tissues than in the corresponding normal tissues. Finally, GCNT2 variant 2 stable transfection induced expression of other 2 isoform variants. Conclusions: We identified novel methylation gene GCNT2 among the glycoenes. Glycoenes that were altered in genomic or epigenetic manner have been few, so GCNT2 may play a critical role in cancer progression through glycan change.

scholarly journals Hsa_circRNA_000166 facilitated cell growth and limited apoptosis through targeting miR-326 / LASP1 axis in colorectal cancer

2020 ◽  
Author(s):  
Qin Hao ◽  
Zhongtao Zhang
Keyword(s):  
Colorectal Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Direct Interaction ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Western Blot Assay ◽  
Apoptosis Assay ◽  
Geo Database

Abstract Background: Circular RNAs(circRNAs) belong to non-coding RNAs and widely expressed in a variety of cell species, including cancers. However, the function and mechanism of circRNAs in colorectal cancer (CRC) has not been well investigated. Methods: Microarray data of CRC from Gene Expression Omnibus (GEO) database was used to obtain DEGs. QRT-PCR and western blot assay were performed to determine the mRNA and protein levels of multiple genes, respectively. Cell growth and apoptosis assay were conducted to measure CRC cell proliferation and apoptosis, respectively. Luciferase assay was utilized to confirm the direct interaction between hsa_circRNA_000166 and miR-326. Results: We downloaded and analyzed the circRNA expression profile of CRC from the GEO database and identified 181 differentially expressed circRNAs between 10 pairs of CRC and adjacent normal tissues. Interestingly, we observed that the expression of hsa_circRNA_000166 was the top increased among these circRNAs. Then, we confirmed an upregulation of hsa_circRNA_000166 in CRC tissues and cell lines and observed that higher expression of hsa_circRNA_000166 was associated with poor 5-year survival rate of patients with CRC. Cell growth and apoptosis assay revealed that hsa_circRNA_000166 regulated the cell growth and apoptosis in CRC cell lines. Furthermore, we identified that hsa_circRNA_000166 targeted miR-326/LASP1 pathway using bioinformatic analysis and luciferase reporter assay. Finally, overexpression of miR-326 could sufficiently rescued the aberrant cell growth and apoptosis in CRC cell lines. Conclusion: Taken together, our results indicated that downregulation of hsa_circRNA_000166 inhibited the cell growth and facilitated apoptosis during CRC development by sponging miR-326 / LASP1 pathway.

scholarly journals Chrysin Modulates Aberrant Epigenetic Variations and Hampers Migratory Behavior of Human Cervical (HeLa) Cells

2022 ◽  
Vol 12 ◽  
Author(s):  
Ritu Raina ◽  
Abdulmajeed G. Almutary ◽  
Sali Abubaker Bagabir ◽  
Nazia Afroze ◽  
Sharmila Fagoonee ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Histone Modification ◽  
Hela Cells ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Global Dna Methylation ◽  
Dependent Manner ◽  
Suppressor Genes ◽  
Protein Levels

Purpose: Plant-derived phytochemicals have shown epigenetic modulatory effect in different types of cancer by reversing the pattern of DNA methylation and chromatin modulation, thereby restoring the function of silenced tumor-suppressor genes. In the present study, attempts have been made to explore chrysin-mediated epigenetic alterations in HeLa cells.Methods: Colony formation and migration assays followed by methylation-specific PCR for examining the methylation status of CpG promoters of various tumor-suppressor genes (TSGs) and the expression of these TSGs at the transcript and protein levels were performed. Furthermore, global DNA methylation; biochemical activities of DNA methyltransferases (DNMTs), histone methyl transferases (HMTs), histone deacetylases (HDACs), and histone acetyl transferases (HATs) along with the expression analysis of chromatin-modifying enzymes; and H3 and H4 histone modification marks analyses were performed after chrysin treatment.Results: The experimental analyses revealed that chrysin treatment encourages cytostatic behavior as well as inhibits the migration capacity of HeLa cells in a time- and dose-dependent manner. Chrysin reduces the methylation of various tumor-suppressor genes, leading to their reactivation at mRNA and protein levels. The expression levels of various chromatin-modifying enzymes viz DNMTs, HMTs, HDACs, and HATS were found to be decreased, and H3 and H4 histone modification marks were modulated too. Also, reduced global DNA methylation was observed following the treatment of chrysin.Conclusion: This study concludes that chrysin can be used as a potential epigenetic modifier for cancer treatment and warrants for further experimental validation.

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