Integrative analysis of long non-coding RNA and messenger RNA expression in toll-like receptor 4-primed mesenchymal stem cells of ankylosing spondylitis.

BACKGROUND: The precise pathogenesis of ankylosing spondylitis (AS) is still largely unknown at present. Our previous study found that toll-like receptor 4 (TLR4) downregulated and performed immunoregulatory dysfunction in mesenchymal stem cells from AS patients (AS-MSCs). The aim of this study was to explore the expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in TLR4-primed AS-MSCs, and to clarify the potential mechanisms. METHODS: The immunoregulatory effects of MSCs were determined after TLR4 activation. Next, the differentially-expressed (DE) lncRNAs and mRNAs between AS-MSCs and TLR4-primed AS-MSCs [stimulated by lipopolysaccharide (LPS)] were identified via high-throughput sequencing followed by quantitative real-time PCR (qRT-PCR) confirmation. Finally, bioinformatics analyses were performed to identify the critical biological functions, signaling pathways, and associated functional networks involved in the TLR4-primed immunoregulatory function of AS-MSCs. RESULTS: A total of 147 DE lncRNAs and 698 DE mRNAs were identified between TLR4-primed AS-MSCs and unstimulated AS-MSCs. Of these, 107 lncRNAs were upregulated and 40 were downregulated (fold change ≥2, P<0.05), while 504 mRNAs were upregulated and 194 were downregulated (fold change ≥2, P<0.05). Five lncRNAs and five mRNAs with the largest fold changes were respectively verified by qRT-PCR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that the DE mRNAs and lncRNAs were highly associated with the inflammatory response, such as NOD-like receptor (NLR) signaling pathway, the TNF signaling pathway and the NF-κB signaling pathway. Cis-regulation prediction revealed eight novel lncRNAs, while trans-regulation prediction revealed 15 lncRNAs, respectively. Eight core pairs of lncRNA and target mRNA in the lncRNA-transcription factor (TF)-mRNA network were as follows: PACERR-PTGS2, LOC105378085-SOD2, LOC107986655-HIVEP2, MICB-DT-MICB, LOC105373925-SP140L, LOC107984251-IFIT5, LOC112268267-GBP2, and LOC101926887-IFIT3, respectively. CONCLUSIONS: TLR4 activation in AS can enhance the immunoregulatory ability of MSCs. Eight core pairs of lncRNA and target mRNA were observed in TLR4-primed AS-MSCs, which could contribute to understanding the potential mechanism of AS-MSC immunoregulatory dysfunction.

[Isolation and characterization of side population cells in human gastric cancer cell line BGC-823].

OBJECTIVE: Isolate and characterize the side population (SP) cells with potency of stem cells from human gastric carcinoma cell line BGC-823. METHODS: SP and non-SP cells were sorted from BGC-823 cells by fluorescence-activated cell sorting (FACS) using Hoechst33342 staining. The tumorigenic ability of the SP cells was assessed by in vivo transplantation into non-obese diabetic/severe combined immunodeficiency mice. RESULTS: SP cells were isolated from BGC-823 cells in a proportion of 0.9% to 2.1% with respect to the whole cell population. The colony formation assay showed that the colony formation rate of the SP cells was significantly higher than that of the non-SP cells (72.56% vs. 49.00%, P < 0.01). The drug sensitivity test showed that the SP cells showed stronger drug resistance to 5-Fu than the non-SP cells. The in vivo transplantation of SP cells in mice showed that the tumor weight was (0.176 ± 0.034) g, significantly higher than that of non-SP cells (0.045 ± 0.046) g (P < 0.01). CONCLUSIONS: The results of this study indicate the existence of cancer stem-like SP cells in the human gastric cancer line BGC-823 cells. Further characterization of this SP cell population may provide new insights for diagnosis and treatment of gastric cancer.

Inhibition effects of all trans-retinoic acid on the growth and angiogenesis of esophageal squamous cell carcinoma in nude mice.

BACKGROUND: The potential application of retinoic acid receptor activators, such as all trans-retinoic acid (ATRA), for treating various cancers have been studied both pre-clinically and clinically. Whether ATRA has an anticancer effect on human esophageal squamous cancer cell (ESCC) is still unknown. We have explored the anticancer effect of ATRA in ESCC, and in this study, the effects of ATRA on levels and patterns of expression of the vascular endothelial growth factor (VEGF) signal transduction pathway in transplantable tumor growth of the human ESCC cell line, EC9706, in nude mice. METHODS: The animal model of the ESCC xenograft was made by subcutaneous implantation of tumor cells into nude mice. Reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical assays were used to detect the expression of the VEGF signal transduction pathway in ESCC xenograft tissues. RESULTS: Compared to the control group, the tumor inhibition rates in the low dose ATRA, high dose ATRA, and 5-FU groups were 83.21%, 88.32%, 91.02%, respectively. The protein and mRNA levels of VEGF were down-regulated after being treated with ATRA and 5-FU compared to the control group (P < 0.05). The study also revealed that ATRA specifically down-regulated VEGF and the component of the VEGF signal transduction pathway of CD31, CD34, and CD105 (component of the TGF-ß receptor) in ESCC xenograft tissues (P < 0.05). CONCLUSIONS: ATRA can significantly inhibit tumor growth and has anticancer effects on transplantable tumor growth of human ESCC cell line EC9706 in nude mice. These findings indicate that ATRA specifically down regulated VEGF and the components of VEGF signal transduction, which may be an important mechanism responsible for the neoangiogenesis inhibition of ESCC cells.

[Research advances in induced pluripotent stem cells].

Differentiated somatic cells can be directly reprogrammed into induced pluripotent stem (iPS) cells in vitro. Similarly to embryonic stem (ES) cells, iPS cells have pluripotency to differentiate into all cell types and capability to self-renew themselves indefinitely. Without immune rejection and ethical issues, patient-specific iPS cells promise to be an ideal tool for regenerative medicine, drug screening, and toxicity testing.

[Mechanism of loss of human esophageal cancer-related gene 4 (ECRG4) gene expression in esophageal squamous cell carcinoma cell line EC9706].

OBJECTIVE: To investigate the mechanism of loss of human esophageal cancer-related gene 4 (ECRG4) expression in esophageal squamous cell carcinoma (ESCC.) METHODS: PCR-SSCP and DNA sequencing analysis were used to detect the mutation of ECRG4 exons in esophageal cancer and matched adjacent normal tissues of 80 patients. DNA bisulfite-modifying ssPCR sequencing assay was used to examine the methylation status of ECRG4 promoter in human esophageal squamous cell carcinoma EC9706 cells. The re-expression of ECRG4 mRNA was examined by RT-PCR in EC9706 cells, after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. RESULTS: No mutation in the four ECRG4 exons was found in all the ESCC and matched normal adjacent tissues. RT-PCR showed that 11 of 16 CpG islands of ECRG4 promoter were hypermethylated, while ECRG4 mRNA expression level was undetectable in the EC9706 cells. The ECRG4 mRNA was re-expressed after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. CONCLUSION: The epigenetic mechanism of methylation is a reason of loss of ECRG4 gene expression in the ESCC cell line EC9706.

[Expression of IGF-1R in esophageal squamous cell carcinoma and the effect of its silencing by siRNA on the proliferation of esophageal cancer EC9706 cells in vitro].

OBJECTIVE: To explore the correlation of IGF-1R expression with clinical features of esophageal squamous cell carcinoma (ESCC) and to investigate the effect of silencing IGF-1R by siRNA on the proliferation of esophageal cancer cell line EC9706 cells. METHODS: Immunohistochemistry was used to detect the expresion of IGF-1R in 80 specimens of ESCC and 18 specimens of normal esophageal mucosa. IGF-1R siRNA was transfected into esophageal squamous cell carcinoma EC9706 cells, and the effect of RNAi was assessed by Western blot. The proliferation of EC9706 cells was determined by drawing growth curve, MTT assay and plate colony-forming assay. RESULTS: The total and strong positive rates of IGF-1R expression were 86.3% and 51.3% in ESCC, and 61.1% and 11.1% in normal esophageal epithelium, respectively. The total and strong positive rates of IGF-1R expression in patients with lymph node metastasis were 94.4% and 74.1%, significantly higher than 69.2% and 3.9%, respectively, in those without lymph node metastasis (P<0.01). A significantly higher IGF-1R expression was associated with lower histological grade (P<0.05). The total and strong rates of IGF-1R expression in 39 patients of stages III and IV were 97.4% and 71.8% , significantly higher than the 75.6% and 31.7%, respectively, in 41 cases of stages I and II (P<0.01). IGF-1R RNAi significantly inhibited IGF-1R expression and the growth of EC9706 cells. The clone formation rate of RNAi-IGF-1R transfected cells was 19.1%, significantly lower than that of 52.3% in non-transfected cells and 49.0% in empty vector-transfected EC9706 cells (P<0.05). CONCLUSIONS: The overexpression of IGF-1R is colerated with lymph node metastasis, differentiation and clinical stage. Down-regulation of IGF-1R can inhibit the proliferation of esophageal cancer EC9706 cells in vitro.

[Promoting effect of all-trans retinoic acid on the chemosensitivity of esophageal cancer EC9706 cells].

OBJECTIVE: To investigate the impact of all-trans retinoic acid (ATRA) on chemosensitivity to esophageal squamous cell carcinoma EC9706 cells in vitro and its mechanism. METHODS: EC9706 cells were routinely cultured as the control group. The experimental group was divided into three groups. The ATRA group with ATRA in final concentration of 1 µmol/L; the 5-Fu group with 5-Fu in final concentration of 50 mg/L; the combined treatment group with ATRA in final concentration of 1 µmol/L and 5-Fu 50 mg/L. The cell apoptosis was detected by terminal deoxynucleotidy transferase mediated dUTP nick end labelling (TUNEL). The cell cycle and apoptosis were detected by flow cytometry. RESULTS: The results of TUNEL showed that in the combined treatment group appeared a large number of apoptotic cells, and their nuclei were stained brown, with a positive rate of 89.7%. There was a significant difference in the comparison with the ATRA group (38.3%) and 5-Fu group (40.3%) (P < 0.05). The flow cytometry showed that the ATRA + 5-Fu group had a significantly higher apoptosis rate (76.9% ± 2.7%) than that in the ATRA group (38.2% ± 2.6%) and 5-Fu group (45.2% ± 2.3%) (P < 0.05). The ratio of cells in G(1) phase increased in the ATRA + 5-Fu group (83.4% ± 3.0%), significantly higher than (48.2% ± 2.5%) in the ATRA group and (53.2% ± 2.6%) in the 5-Fu group (P < 0.05). The ratio of cells in S + G(2)/M phase was decreased in the ATRA + 5-Fu group, with a significant difference (P < 0.05) when compared with other groups. There was no significant difference between the ATRA group and 5-Fu group (P > 0.05) in the apoptosis rate and the proportion of cells at different phases. CONCLUSION: ATRA can induce apoptosis of esophageal carcinoma EC9706 cells in vitro. The combination of ATRA and 5-Fu may enhance the chemotherapeutic efficacy.

[The relationship between retinoic acid receptor-ß expression and chemotherapy response in esophageal squamous cell carcinoma].

OBJECTIVE: To observe the relationship between expression of retinoic acid receptor-ß(RAR-ß) in esophageal squamous cell carcinoma (ESCC) and chemotherapy response. METHODS: Fifty-two cases advanced ESCC patients treated by DDP and 5-FU, DDP 80 mg/m(2), divided into 5 days; 5-FU 375 mg/m(2), d1-5. Immunohistochemistry was used to examine the expression of RAR-ß in ESCC. Fifty cases normal esophageal tissue were used as controls. RESULTS: RAR-ß immunoreactivity was recognized in both cytoplasm and nucleus, RAR-ß positive rate was lower in ESCC compared with normal tissue (61.5% vs 92%, P < 0.05). The 52 cases ESCC patients were treated 228 chemotherapy cycles, the overall response rate (OR) was 71.2%. The OR in RAR-ß positive patients was 84.4% (27/32), significant higher than RAR-ß negative patients 50.0% (10/20) (P < 0.05). The time-to-progression (TTP) for RAR-ß positive patients was 5.9 months, the median survival period was 12.1 months, 2 years survival rate was 56.7%; whereas TTP for RAR-ß negative patients was 2.1 months, the median survival period was 5.8 months, 2 years survival rate was 32.9%. There was significant difference between the 2 groups (P < 0.05). CONCLUSION: RAR-ß protein expression by immunohistochemistry may be a useful indicator to predict the chemotherapy response and clinical outcome for ESCC, meanwhile it may be an avenue for target therapy.

[Tumor-suppressing function of human esophageal cancer related gene 4 in esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the tumor-suppressing function of human esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). METHODS: The recombinant plasmid pcDNA3.1-ECRG4 with ECRG4 open reading frame was constructed. The EC9706 cell was transfected with either pcDNA3.1 or pcDNA3.1-ECRG4. And the effects of ECRG4 on tumor cell were examined by in vivo assays of cell proliferation, adhesion, migration, invasion and tumor growth. The expression levels of P53 and P21 proteins were detected in EC9706 cell with transfection of ECRG4 gene by Western blotting. RESULTS: The final tumor volume and weight in ECRG4 transfection group were (264±43) mm3 and (0.39±0.09) g, versus (464±128) mm3 and (0.76±0.13) g in control group (both P<0.05). And the capacities of tumor cells adhesion, migration and invasion decreased in ECRG4 transfection group versus that in control group (all P>0.05). Furthermore, the expression levels of P53 and P21 proteins were higher in ECRG4 transfection group than those in control group (100.00±3.87, 35.71±2.36 vs 16.6±1.92, 1.09±0.11, both P<0.05). CONCLUSION: As a novel candidate tumor suppressor in ESCC, ECRG4 may induce the up-regulation of p21 protein through p53 pathway to inhibit tumor growth in ESCC.

[Insulin induces anticancer cytotoxicity of 5-Fu to two human colon cancer cell lines].

OBJECTIVE: To explore the possibility of use of insulin as a potentiator of 5-Fu to human colon cancer cell lines HCT-8 and HT-29 and study its mechanism. METHODS: MTT assay was used to examine the inhibition rate of cell growth after treatment with 5-Fu and insulin. Cell cycle was determined by flow cytometry. RESULTS: Insulin showed an enhancing effect on the chemotherapeutic response of 5-Fu when insulin was applied at a dose of exceeding 0.8 mU/ml 0 approximately 8 h before 5-Fu. Within the range of from 0.8 mU/ml to 8 mU/ml, a higher concentration of insulin gave a higher proportion of inhibited cells. But when the insulin concentration exceeds 8 mU/ml, the proportion became stable as that of 8 mU/ml. Insulin increased the percentage of S phase cells and decreased the percentage of G(1) phase cells (P < 0.01). The percentage of S phase cells reached a peak when the cells were treated with insulin for 6 hours. CONCLUSION: Insulin can enhance the anticancer toxicity of 5-Fu to human colon cancer cell lines HCT-8 and HT-29 cells. Insulin increases the percentage of S phase cells, which may be one of the main mechanisms of insulin-induced enhancement of anticancer response of cancer cells to 5-Fu chemotherapy.

[Mechanism of apoptosis of esophageal cancer EC9706 in nude mice induced by all-trans retinoic acid].

OBJECTIVE: To investigate the mechanism of apoptosis of EC9706 tumor-bearing nude mice induced by all-trans retinoic acid (ATRA). METHODS: Human esophageal carcinoma cell line EC9706 cells were inoculated into nude mice to establish the solid tumor model. The tumor models were divided into the following groups: ATRA group, fluorouracil group, the two-drugs combination group, and with an equal volume fraction of solvent as the control group. The nude mice were sacrificed after 10 days of medication. TUNEL staining was used to detect cell apoptosis. RT-PCR was used to detect the expression level of mRNA and immunohistochemistry was used to detect the expression level of protein of caspase-3 and survivin, the apoptosis-related genes in the tumor tissue. RESULTS: The apoptosis rates of the ATRA group, 5-Fu group and ATRA + 5-Fu group were 44.3%, 39.7% and 91.0%, respectively. There was a significant difference in comparison with the control group (0.7%), and the ATRA group had no significant difference compared with that of the fluorouracil group (P > 0.05), but the apoptosis rate of the two-drugs combination group was significantly higher than that in the two single-drug groups (P < 0.05). The average gray value of caspase-3 protein expressed in the control group was 46.12 ± 0.33 and the relative expression of caspase-3 mRNA was 0.14 ± 0.03, both were significantly lower than that in the ATRA group, 5-Fu group and the two-drugs combination group (P < 0.05). The average gray value of survivin protein expressed in the control group was 96.07 ± 0.13 and the relative expression of survivin mRNA was 0.84 ± 0.04, both were significantly higher than those of other groups (P < 0.05). The ATRA group had no significant difference compared with the fluorouracil group (P > 0.05), but the two-drugs combination group was significantly different compared with the single-drug groups (P < 0.05). CONCLUSION: Apoptosis in the EC9706 tumor cells in nude mice can be induced by ATRA. The mechanism may be related with down-regulation of the level of survivin gene expression and up-regulation of the level of caspase-3 gene expression.

[Expression of ezrin and CD44-v6 in human esophageal squamous cell carcinoma and its clinical significance].

OBJECTIVE: To evaluate the expression of ezrin and CD44-v6 in esophageal squamous cell carcinoma, and to evaluate its relationship with lymph node metastasis (LNM) and histological grading. METHODS: The expression of ezrin and CD44-v6 in 71 patients with esophageal squamous cell carcinoma was studied using immunohistochemical (SP) method. The correlation of their expression with relevant clinical data was statistically analyzed. RESULTS: In normal esophageal squamous epithelia, the expression of ezrin was found in 33 cases among 71 cases and the expression of CD44-v6 in 18 cases among 71 cases. In esophageal squamous cell carcinoma, the expression of ezrin was found in 64 cases among 71 cases and CD44-v6 in 58 cases among 71 cases. The expression of ezrin was closely related to LNM. The positive rate of ezrin expression in LNM cases was significantly higher than that in cases without LNM. The expression of CD44-v6 had a close relation to tumor differentiation and LNM. The positive rate of CD44-v6 expression in LNM cases was significantly higher than that in patients without LNM. The expression of ezrin in CD44-v6 positive cases was significantly higher than that of CD44-v6 negative cases. The LNM rate was 60.0% in 48 patients with positive expression of both ezrin and CD44-v6, while none of lymph node metastasis was found in the 6 patients with both negative. CONCLUSION: The test of CD44-v6 and ezrin expression may have significant prognostic value for assessing the degree of malignancy and potential LNM probability of ESCC. Ezrin may become a new target in evaluation of tumor prognosis.

[Apoptosis in esophageal cancer cells induced by all-trans retinoic acid].

OBJECTIVE: To study the anti-tumor effects of all-trans retinoic acid (ATRA) and mechanisms of its action. METHODS: Human esophageal carcinoma cell line EC9706 cells were treated with ATRA at different concentration. The proliferation inhibition was examined by MTT assay. Morphological examination, TUNEL method and flow cytometry were used to detect the apoptosis and changes of cell cycle. Immunohistochemical method was used to detect the expression of apoptosis-related genes caspase-3 and bcl-2. The semi-quantification of protein expression was analyzed by pathological image analysis. RESULTS: ATRA inhibited the proliferation of EC9706 cells moderately. Apoptosis in EC9706 cells was induced by ATRA treatment. The morphology of EC9706 cells showed changes such as nuclear chromatin condensation and fragmentation. Sub-G1 peak was found by flow cytometry. The maximal apoptosis rate was 32.6%. The expression of caspase-3 gene was enhanced. The expression of bcl-2 gene was decreased. All these effects were presented in a dose-dependent and time-depend manner. CONCLUSION: Apoptosis is one of the key mechanisms of ATRA action on EC9706 cells.

[Cellar senescence and tumor].

Cells withdrawing from the cell cycle and entering the terminally non-dividing state are referred senescence. With few exceptions,normal cells necessarily enter this process. Molecular analyses have identified some changes in gene expressions as cells become senescent, including repression of positive-acting transcriptional regulators, over-expression of CDK inhibitor, and interference with downstream pathways, and changes in telomere and telomerase. Current findings show that senescence is well connected with tumorigenesis and tumor therapy. Studies with cell types other than fibroblast will better define the roles of cellar senescence in tumorigenesis, moreover, it may provide a novel therapeutic approach to tumor repression.

[The effect of insulin on chemotherapeutic drug sensitivity in human esophageal and lung cancer cells].

OBJECTIVE: To discuss the effect of insulin, as a metabolic promoter, on chemotherapeutic drug sensitivity in human esophageal and lung cancer cells. METHODS: Human esophageal cancer cells NEC and human lung adenocarcinoma cells GLC were cultured and then inoculated into the wells. MTT was added. Chemotherapeutic drugs, etopside, cisplatin, or 5-fluoto-uracilum was added to examine their cytotoxity activity. Then insulin of the final concentration of 5 mU/ml was added 8 hours before etopside (30 - 40 micro g/ml), cisplatin (2.5 micro g/ml), or 5-Fu (50 micro g/ml) was added. MTT A value was tested by colorimetry to evaluate the number of cancer cells, cell activity, and metabolism status so as to reflect the cytotoxity of the anti-tumor agent. Insulin was added into the suspension of cancer cells. Flow cytometry was used to detect the cell-cycle progresses. RESULTS: Insulin alone did not inhibit the cell growth and mildly promoted the cell metabolism with the concentration > 5 mU/ml. Insulin (2.0 - 15.0 mU/ml) enhanced the chemocytotoxity of etopside (30 micro g/ml) on human esophageal and lung cancer cells as indicated by MTT colorimetry. GLC cell cycle assay showed that the S phase block induced by etopside, cisplatin and 5-FU and the G(2)/M block induced by 5-FU were enhanced by insulin with the increased block rates of 80% and 90% respectively. The increased block rate induced by insulin in NEC cells was lower than in GLC cells. CONCLUSION: A reversible metabolic promoter, insulin enhances the cytotoxity of the chemotherapeutic agents. It is possible to increase the growth and metabolism of cancer cells first so as to enhance the chemosensibility, and then administer chemotherapeutic agents, thus improving their therapeutic effects.

Using Lab On-line to Clone and Identify the Esophageal Cancer Related Gene 4.

Using Internet as platform, databases as materials and software as tools to assemble a lab on-line is revolutionizing in bioscience research. The major works of lab on-line are cloning, identification, localization of genes, and the structural and functional analysis of proteins. In this report, the esophageal cancer related gene 4(ECRG-4)(accession number AF325503) was successfully isolated. The 97 bp ECRG-4 EST was initiallyusedto fish the human EST databases. Five pieces of ESTs showed strong homology to it, and they were assembled to one 772 bp cDNA sequence by DNASTAR software. Then the 447 bp full open reading frame of ECRG-4 was determined by ORF FINDER to encode 148 amino acids. Sequence of ECRG-4 did not reveal remarkable similarity to the known sequences in a homology analysis with the public database of GenBank, showing that it is a new gene. Homology analysis of protein coding by ECRG-4 showed a 31% homology with mouse IgG V region. ECRG-4 gene is expressed in normal esophagus, bladder and brain tissues, but its expression was significantly down-regulated in prostate tumors and tumor cell lines. ECRG-4 gene was located in 2q14.1--4.3 by HTGS and STS, and was conformed by radiation hybrid (RH) method. We propose that this purely lab on-line cloning approach can be used by modestly sized laboratories to rapidly and accurately characterize human genes without wasting too much money and time.