ABSTRACT
As a top leading cause of cancer death in many countries, colorectal cancer (CRC) has drawn increasing attention to the study of the pathological mechanism. According to the "cancer stem cell hypothesis", malignancies originate from a small fraction of cancer cells that show self-renewal properties to initiate and sustain tumor growth and tumor metastasis. Therefore, these cancer stem cells (CSC) probably play important roles in tumor recurrence, metastasis, and drug resistance. Previous research reported that lysine-specific histone demethylase 1 (LSD1) maintains cancer stemness through up-regulating stemness markers SOX2 and OCT4. CD133 is believed to be the most robust surface marker for CRC stem cells, however the regulatory effect of LSD1 on stemness of CD133+ CRC has never been reported. In this study, our objectives included: 1) to isolate pure CD133+ and CD133− cells from SW620 cell line; 2) to investigate the effect of LSD1 on the characteristics of CD133+ stem cancer cells by knocking down the target gene. Results suggested that the SW620 cell line had both CD133+ and CD133− subsets. The CD133+ subset exhibited more CSC-like characteristics compared with the CD133− subset with higher viability, colony formation rate, migration and invasion rate, resistance to anti-cancer drugs, and apoptosis in vitro. The CD133+ also induced faster tumor formation and larger tumors in vivo. In the LSD1-knockdown CD133+ cells, the CSC-like characteristics had been all weakened. We conclude that LSD1 was important for CSCs to maintain their "stemness" features, which could be a potential therapeutic target of CRC.
Subject(s)
Humans , Animals , Rats , Neoplastic Stem Cells/drug effects , Colorectal Neoplasms/pathology , Cell Movement/drug effects , Apoptosis/drug effects , Cell Proliferation/drug effects , Histone Demethylases/pharmacology , Neoplastic Stem Cells/metabolism , Gene Expression Regulation, Neoplastic , Blotting, Western , Colony-Forming Units Assay , Cell Line, TumorABSTRACT
@#[Abstract] Objective: To investigate the effect of histone demethylase JMJD3 (jumonji domain-containing protein 3) on the stemness of diffuse large B-cell lymphoma (DLBCL) cells. Methods: The relationship between the expression of JMJD3 and the overall survival of DLBCL patients was analyzed using the clinical data of DLBCL patients in TCGA database. The control plasmid (pCMV) and JMJD3 expression plasmid(pCMV-JMJD3)weretransfectedintoDLBCLcellsofABCandGCBsubtype via lipofectamine transfection. Then, the mRNAlevels of JMJD3,ALDH1, OCT4 and SOX2 were detected by RT-PCR and qPCR; the activity ofALDH1 enzyme was detected by Flow cytometry; the protein expressions of OCT4 and SOX2 were detected by Western blotting. Gene enrichment in DLBCL patients with high JMJD3 expression was analyzed by gene set enrichment analysis (GSEA). Results: The result of prognosis analysis showed that high expression of JMJD3 was related with poor prognosis in DLBCL patients (P<0.05); however, multivariate analysis showed that the expression of JMJD3 was not the independent factor affecting the prognosis of DLBCL patients (all P>0.05). The expression of JMJD3 was remarkably increased in DLBCL cells transfected with pCMV-JMJD3, which led to significantly increased mRNA level and enzyme activity of ALDH1 as well as up-regulated mRNA and protein expressions of OCT4 and SOX2 (P<0.05 or P<0.01). GSEA analysis showed that enrichment of Wnt/β-catenin signaling pathway related gene set was observed in DLBCL patients with high JMJD3 expression (P<0.05). Conclusion: JMJD3 promotes the stemness of DLBCL cells, which may be a potential therapeutic target for DLBCL patients.
ABSTRACT
Lysine demethylase 6 (KDM6) is involved in the demethylation regulation of histone H3 as an important modification enzyme in epigenetic modification,and plays an important role in embryonic development,inflammation and disease development.Current researches indicate that KDM6 is involved in the occurrence and development of various tumors (pancreatic cancer,colon cancer,gastric cancer,breast cancer,bladder cancer,etc.),affects proliferation,metastasis,prognosis and chemotherapy resistance of tumors,and plays different roles due to different tumor backgrounds.
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Objective To construct rat lysine-specific histone demethylase 1 (LSD1) overexpression plasmids and LSD 1 demethylation fragment disfunction plasmids,and to evaluate their expression levels in HEK293T cells.Methods LSD1 fragments were amplified by PCR,and LSD1 demethylation disfunction fragments were amplified by overlap PCR.Rat-specific LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were constructed and verified by agarose gel electrophoresis and sequencing.HEK293T cells were infected using the validated recombinant plasmids and blank vectors,and the stably expressed cells were selected by puromycin.The expression of LSD1 in the stably expressed HEK293T was detected by Western Blot and real-time quantitative PCR.Results The results of agarose gel electrophoresis and sequencing showed that LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were successfully constructed.The Western Blot and real-time quantitative PCR results showed that compared with the blank group,the relative expression of LSD1 and mRNA in the LSD1 overexpression group and the LSD1 demethylation disfunction group were up-regulated,and the differences were statistically significant(all P＜0.01).Conclusions The constructed LSD1 overexpression plasmids and LSD1 demethylationi disfunction plasmids can achieve overexpression of LSD1 gene in HEK293T cells.This paper lays a foundation for further study of the relationship between LSD 1 gene and related diseases and demethylation of LSD 1.
ABSTRACT
Objective To investigate the expression of PHF8(PHD-finger protein 8) in human hepatocellular carcinoma (HCC) and its clinicopathologic significance.Methods The expression of PHF8 in 60 hepatocellular carcinoma samples as well as their natched paraneoplastic tissues,and 15 normal liver tissues were evaluated by immunohistochemistry.Statistical methods were used to analyse the relationship between the expression of PHF8 and the clinicopathological features of these patients.Results The PHF8-positive expression rate in the HCC samples was 55.0％ and it was significantly higher than that in the paraneoplastic tissues and the normal liver tissucs (16.7％,6.7％,respectively,P＜0.05).The expression of PHF8 was closely related to tumor size,tumor nodular numbers,pathological differentiation and TNM-staging (P＜0.05 for all).The 5-year disease-free survival and overall survival in the PHF8-positive group was significantly lower than that in the negative group (P＜0.05).Conclusions PHF8 was overexpressed in HCC samples,and its expression was closely associated with HCC clinicopathological features and prognosis of the patients.
ABSTRACT
Histone methylation is one of the epigenetic modifications. Histone methylation influences constitutive heterochromatin, genomic imprinting, inactivation of X-chromosome and gene transcription regulation. Abnormality of histone methylation is associated with several carcinomas. The discovery of enzymes that reverse histone methylation challenges the current understanding that histone methylation is a stable epigenetic marker and provides a novel way to study histone modifications.