Yuqin Liu

Expression of CD133 correlates with differentiation of human colon cancer cells.

CD133 has been identified as a cancer stem cell marker in colon and several other cancers, but its function is still unknown. We examined the CD133 expression in 44 human cancer cell lines, and found five of the 8 positive lines were from colon cancer. The CD133 positive subpopulation of colon cancer cells showed more vigorous growth and lower differentiation. Induction of differentiation reduced the CD133-positive population. Knockdown of CD133 expression in colon cancer cells could not induce cellular differentiation. Care must be taken if CD133 is used as the only marker of cancer stem cells in colon cancer, especially in established cell lines. CD133 negatively correlates with cell differentiation, but it is not a regulator of differentiation.

Ubiquitin-Specific Peptidase USP22 Negatively Regulates the STAT Signaling Pathway by Deubiquitinating SIRT1

Background/Aims: The ubiquitin-specific peptidase USP22 mediates various cellular and organismal processes, such as cell growth, apoptosis, and tumor malignancy. However, the molecular mechanisms that regulate USP22 activity remain poorly understood. Here we identify STAT3 as a new USP22 interactor. Methods:· We used western blotting and RT-PCR to measure key protein, acetylated STAT3, and mRNA levels in HEK293 and colorectal cancer cell lines transfected with expression plasmids or specific siRNAs. Co-immunoprecipitation was used to demonstrate protein-protein interaction and protein complex composition. Results: USP22 overexpression down-regulated STAT3 acetylation by deubiquitinating SIRT1. The three proteins were found to be present in a single protein complex. SiRNA-mediated depletion of endogenous USP22 resulted in SIRT1 destabilization and elevated STAT3 acetylation. Consistent with this finding, USP22 also down-regulated the expression of two known STAT3 target genes, MMP9 and TWIST. Conclusion: We show that USP22 is a new regulator of the SIRT1-STAT3 signaling pathway and report a new mechanistic explanation for cross talk between USP22 and the SIRT1-STAT pathways.

Ginsenoside Rb1 protects hippocampal neurons from high glucose-induced neurotoxicity by inhibiting GSK3β-mediated CHOP induction

Ginsenoside Rb1 is generally recognized as one of the principal bioactive ingredients in ginseng and shows neuroprotective effects in various neurons. Endoplasmic reticulum (ER) stress is considered to play an important role in numerous neurodegenerative disorders. Recently, glucogen synthase kinase 3β (GSK3β) was reported to regulate ER stress-induced C/EBP homologous protein (CHOP) in neuronal cells. Therefore, in this study, we investigated the effects of ginsenoside Rb1 on GSK3β-mediated ER stress in high glucose-treated hippocampal neurons. Results from the MTT assay showed that treatment with 1 µM Rb1 for 72 h protected neurons from high glucose-induced cell injury. Using western blot analysis, we found that treatment with Rb1 effectively inhibited the phosphorylation of the high glucose-induced protein kinase RNA-like ER kinase (PERK) and of GSK3β, and reduced the level of the CHOP protein. The levels of these proteins were also decreased by treatment with the GSK3β inhibitor Licl. Rb1 also significantly decreased the mRNA expression of the gene CHOP, as shown by quantitative RT-PCR analysis. Taken together, the present results suggested that Rb1 may protect neurons from high glucose-induced cell damage by inhibiting GSK3β‑mediated CHOP induction, providing a potentially new strategy for preventing and treating cognitive impairment caused by diabetes.

miR-935 suppresses gastric signet ring cell carcinoma tumorigenesis by targeting Notch1 expression

Gastric signet ring cell carcinoma (GSRCC) is a unique pathological type of gastric carcinoma that is extremely invasive and has a poor prognosis. Expression of microRNAs (miRNAs) has been closely linked to the carcinogenesis of gastric cancer and has been considered as a powerful prognostic marker. The function of miR-935 has never been reported in cancer before. We found, using microRNA array, that expression of miR-935 in GSRCC cell lines is lower than in non-GSRCC cell lines, and enhanced expression of miR-935 in GSRCC cell-lines inhibit cell proliferation, migration and invasion. We also identified Notch1 as a direct target of miR-935. Knockdown of Notch1 reduced proliferation, migration/invasion of GSRCC cells, and overexpression Notch1s activated form (Notch intracellular domain) could rescue miR-935s tumor suppressive effect on GSRCC. Expression of miR-935 was lower in gastric carcinoma tissue than in paired normal tissue samples, and lower in GSRCC than in non-GSRCC. Our results demonstrate the inverse correlation between the expression of miR-935 and Notch1 in gastric tissues. We conclude that miR-935 inhibits gastric carcinoma cell proliferation, migration and invasion by targeting Notch1, suggesting potential applications of the miR-935-Notch1 pathway in gastric cancer clinical diagnosis and therapeutics, especially in gastric signet ring cell carcinoma.

Ubiquitin-specific peptidase 22 inhibits colon cancer cell invasion by suppressing the signal transducer and activator of transcription 3/matrix metalloproteinase 9 pathway.

Colon cancer is associated with increased cell migration and invasion. In the present study, the role of ubiquitin-specific peptidase 22 (USP22) in signal transducer and activator of transcription 3 (STAT3)-mediated colon cancer cell invasion was investigated. The messenger RNA levels of STAT3 target genes were measured by reverse transcription-quantitative polymerase chain reaction, following USP22 knockdown by RNA interference in SW480 colon cancer cells. The matrix metalloproteinase 9 (MMP9) proteolytic activity and invasion potential of SW480 cells were measured by zymography and Transwell assay, respectively, following combined USP22 and STAT3 short interfering (si)RNA treatment or STAT3 siRNA treatment alone. Similarly, a cell counting kit-8 assay was used to detect the proliferation potential of SW480 cells. The protein expression levels of USP22, STAT3 and MMP9 were detected by immunohistochemistry in colon cancer tissue microarrays (TMAs) and the correlation between USP22, STAT3 and MMP9 was analyzed. USP22/STAT3 co-depletion partly rescued the MMP9 proteolytic activity and invasion of SW480 cells, compared with that of STAT3 depletion alone. However, the proliferation of USP22/STAT3si-SW480 cells was decreased compared with that of STAT3si-SW480 cells. USP22 expression was positively correlated with STAT3 and MMP9 expression in colon cancer TMAs. In conclusion, USP22 attenuated the invasion capacity of colon cancer cells by inhibiting the STAT3/MMP9 signaling pathway.

A Combination of Species Identification and STR Profiling Identifies Cross-contaminated Cells from 482 Human Tumor Cell Lines

Human tumor cell lines are extremely important tools for cancer research, but a significant percentage is cross-contaminated with other cells. Short tandem repeat (STR) profiling is the prevailing standard for authenticating cell lines that originate from human tissues. Based on the analysis of 482 different human tumor cell lines used in China by STR, up to 96 cell lines were misidentified. More importantly, the study has found that STR profiling alone is insufficient to exclude inter-species cross-contamination of human cell lines. Among the 386 cell lines which had a correct STR profile, 3 of them were inter-species cross-contaminated. Careful microscopic examination may be helpful in some cases to detect changes in morphology but additional testing is needed. Additionally, species verification by PCR could easily identify the contaminants, even with a low percentage of contaminating cells. Combining STR profiling with species identification by PCR, more than 20.5% (99/482) of tumor cell lines were revealed as having been incorrectly identified, including intra-species (14.5%), inter-species (4.4%) cross-contamination and contaminating cell lines (1.7%). Therefore, quality control of cell lines is a systemic issue. Each cell line should undergo a full QA (Quality Assurance) assessment before it is used for research.

Intrinsic gene changes determine the successful establishment of stable renal cancer cell lines from tumour tissue.

Human tumor cell lines, especially those with complete data and follow‐up, are important tools in tumor biological studies. Clear cell renal cell cancer (ccRCC) is not sensitive to radiotherapy or chemotherapy, and treatment of patients with distant metastasis relies on targeted therapy. Here, we report the establishment of seven new ccRCC stable cell lines that were continuously cultured for more than 20 generations among 81 cases of renal cell cancer. Moreover, gene expression and methylation in the established cell lines, in those that had a finite in vitro life span of less than 10 generations, and in cells that originated from the same culture at different generations were profiled using microarrays. Genes including SLC34A2, SEPP1, SULT1C4 and others were differentially expressed in established cell lines and finite cell lines, and changes in their expression might be caused by methylation or demethylation. The expression level of SLC34A2 was related not only to the life span in vitro culture but also to tumor size. Additionally, six of the seven new ccRCC cell lines had VHL deletions or termination mutations. So in addition to the establishment of seven new ccRCC cell lines with complete clinical data, we conclude that genes such as SLC34A2 and VHL play key roles in the continuous in vitro growth and development of ccRCC.

ALDH1A3 affects colon cancer in vitro proliferation and invasion depending on CXCR4 status

Background:Aldehyde dehydrogenase (ALDH) has been widely used as a marker of cancer stem cells (CSCs). However, the ALDH family includes 19 members, and the most relevant isoforms and their biological functions in cancer biology are still controversial.Methods:We examined ALDH enzyme activity and the mRNA expression of 19 ALDH members in 58 human cell lines. The biological effect and mechanism of knocking down ALDH1A3 with siRNA and shRNA in cell lines were explored. Finally, the relationship between ALDH1A3 and CXCR4 was analysed in a large panel of cell lines.Results:ALDH1A3 is the key isoform that contributed to Aldefluor positivity in cell lines. Knocking down ALDH1A3 in different cancer cells conferred opposite phenotypes due to differential effects on CXCR4 expression. There was a significant negative correlation between ALDH1A3 and CXCR4 in 58 human cell lines.Conclusions:ALDH1A3 was the main contributor to Aldefluor positivity in human cell lines, and its contrasting effects might arise from differences in CXCR4 expression.

Effect of Chinese herbal compound Naofucong (脑复聪) on the inflammatory process induced by high glucose in BV-2 cells

ObjectiveTo determine the effect of medicated serum of Chinese herbal compound Naofucong (脑复聪, NFC) on the microglia BV-2 cells viability and the transcription and expression of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in microglia BV-2 cells to further explore the mechanisms underlying the protective effect of NFC on inflammatory process induced by high glucose.MethodsThe microglia BV-2 cells incubated in vitro were divided into different groups: the control group (25 mmol/L glucose), the model group (75 mmol/L glucose), high glucose media containing different dose medicated serum of NFC. After being cultured for 24 h, changes in IL-6 and TNF-α were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The expression of surface marker CD11b of activated microglia was measured by confocal laser scanning microscope and Western blot. Nuclear factor-κB (NF-κB) p-p65 expression was analyzed by Western blot.ResultsThe model group obviously increased the expression of microglial surface marker CD11b and NF-κB p-p65 (all P<0.01), induced a signifificant up-regulation of release and the mRNA expression of IL-6 and TNF-α (P<0.01 or P<0.05). The medicated serum of NFC could obviously down-regulate the transcription and expression of surface marker CD11 b and NF-κB p-p65 (all P<0.01), and inhibit the mRNA and protein expression (P<0.01 or P<0.05) of inflflammatory cytokines, such as IL-6 and TNF-α, in microglia BV-2 cells cultured with high glucose for 24 h.ConclusionsThe inhibition of microglial activation and IL-6 and TNF-α expression induced by high glucose may at least partly explain NFC therapeutic effects on diabetes-associated cognitive decline diseases. Its underlying mechanism could probably be related to the inhibition of NFC on NF-κB phosphorylation.

Cloning and Expression of a Novel Target Fusion Protein and its Application in Anti- Tumor Therapy

Backgrounds: Epidermal growth factor (EGF) is a 53 amino acid polypeptide and its receptor EGFR is an established therapeutic target for anti-tumor therapy. Two major categories of EGFR-targeted drugs include monoclonal antibodies (mAbs) and small molecular tyrosine kinase inhibitors (TKIs). However, drug resistance occurs in a significant proportion of patients due to EGFR mutations. Since EGFR can maintain activation while abrogating the activity of mAbs or TKIs, or bypass signaling functions while successfully circumventing the EGF-EGFR switch, developing new mechanism-based inhibitors is necessary. Methods: In this study, based on the principle of tumor immunotherapy, a recombinant protein pLLO-hEGF was constructed. The N-terminal portion contains three immunodominant epitopes from listeriolysin O (LLO) and the C-terminal includes EGF. To use EGF as a target vector to recognize EGFR-expressing cancer cells, immunodominant epitopes could enhance immunogenicity of tumor cells for immune cell activation and attack. Results: Recombinant protein pLLO-hEGF was successfully expressed and showed strong affinity to cancer cells. Also, pLLO-hEGF could significantly stimulate human lymphocyte proliferation and the lymphocytes demonstrated enhanced killing potency in EGFR-expressing cancer cells in vitro and in vivo. Conclusion: This study can provide novel strategies and directions in tumor biotherapy.