The interaction of lncRNA EZR-AS1 with SMYD3 maintains overexpression of EZR in ESCC cells.

EZR, a member of the ezrin-radixin-moesin (ERM) family, is involved in multiple aspects of cell migration and cancer. SMYD3, a histone H3-lysine 4 (H3-K4)-specific methyltransferase, regulates EZR gene transcription, but the molecular mechanisms of epigenetic regulation remain ill-defined. Here, we show that antisense lncRNA EZR-AS1 was positively correlated with EZR expression in both human esophageal squamous cell carcinoma (ESCC) tissues and cell lines. Both in vivo and in vitro studies revealed that EZR-AS1 promoted cell migration through up-regulation of EZR expression. Mechanistically, antisense lncRNA EZR-AS1 formed a complex with RNA polymerase II to activate the transcription of EZR. Moreover, EZR-AS1 could recruit SMYD3 to a binding site, present in a GC-rich region downstream of the EZR promoter, causing the binding of SMYD3 and local enrichment of H3K4me3. Finally, the interaction of EZR-AS1 with SMYD3 further enhanced EZR transcription and expression. Our findings suggest that antisense lncRNA EZR-AS1, as a member of an RNA polymerase complex and through enhanced SMYD3-dependent H3K4 methylation, plays an important role in enhancing transcription of the EZR gene to promote the mobility and invasiveness of human cancer cells.

Mig-2 attenuates cisplatin-induced apoptosis of human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways.

AIM: Mig-2 (also known as Kindlin-2 and FERMT2) is an important regulator of integrin activation and cell-extracellular matrix adhesion, and involved in carcinogenesis and tumor progression. The aim of this study was to investigate the role of mig-2 in cisplatin-induced apoptosis of human glioma cells in vitro. METHODS: The expression of mig-2 was modulated in human glioma H4, HS 683 and U-87 MG cells by transfection with a plasmid carrying mig-2 or mig-2 siRNA. Cisplatin-induced apoptosis was detected using Annexin V/PI staining and flow cytometry, as well as MTS analyses. The expression of apoptosis-related or signaling proteins was examined using Western blotting analysis. H4 cells were transfected with plasmids carrying mig-2 mutants to determine the functional domain of mig-2. RESULTS: In the 3 glioma cell lines tested, overexpression of mig-2 significantly attenuated cisplatin-induced apoptosis, whereas knock-down of mig-2 potentiated the apoptosis. The mechanisms of action of mig-2 were further addressed in H4 cells: overexpression of mig-2 markedly reduced cleaved caspase-9, caspase-8, caspase-3 and PARP, as well as p-JNK and p-p38, and increased p-AKT in cisplatin-treated H4 cells, whereas mig-2 siRNA reversely changed these apoptosis-related and signaling proteins. Furthermore, pretreatment with JNK inhibitor SP600125 and p38 inhibitor SB203580, or with AKT inhibitor LY294002 abolished the effects of mig-2 on cisplaxtin-induced apoptosis. In H4 cells, GFP-mig-2 F3 plasmid that contained only the F3 subdomain showed the same efficiency in attenuating cisplatin-induced apoptosis, as the mig-2 wild-type vector did, whereas GFP-mig-2 (1-541) plasmid that lacked the F3 subdomain was inactive. CONCLUSION: Mig-2 significantly attenuates the antitumor action of cisplatin against human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways. The F3 subdomain of mig-2 is necessary and sufficient for this effect.

[Molecular mechanism and effect of microRNA185 on proliferation, migration and invasion of esophageal squamous cell carcinoma].

OBJECTIVE: To explore the role and mechanism of microRNA185 (miR-185) on proliferation, migration and invasion of esophageal squamous cell carcinoma (ESCC). METHODS: Samples were obtained from 23 ESCC patients undergoing surgery whose were confirmed by pathological diagnosis of esophageal carcinoma from 2002 to 2012,at Department of Thoracic Surgery,Cancer Institute and Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. Real-time PCR was used to measure the expression of miR-185. The xCELLigence RTCA MP system and Transwell assay were performed to detect the effect of miR-185 on proliferation, migration and invasion of ESCC respectively. After transfecting of miR-185 mimic into KYSE150, the expression of Six1's downstream gene cyclin A1 was evaluated by real-time PCR. After transfection of miR-185 inhibitor into KYSE30, the expression of E-cadherin, a downstream protein of Six1, was observed under confocal microscope. RESULTS: The expression level of miR-185 was down-regulated in ESCC compared with adjacent normal tissue (0.006 vs 0.039,P = 0.016). After transfection of miR-185 mimic, miR-185 significantly inhibited proliferation, migration and invasion of ESCC.Transwell assay showed, in comparison with the control group, the number of KYSE150 metastatic and invasive cells was respectively decreased(146 ± 15 vs 64 ± 11, 110 ± 12 vs 67 ± 5, both P < 0.05). And the expression level of cyclin A1 decreased. After transfection of miR-185 inhibitor,the expression level of E-cadherin decreased. CONCLUSION: miR-185 may inhibit proliferation, migration and invasion of ESCC through its target gene Six1.

Novel HER2 aptamer selectively delivers cytotoxic drug to HER2-positive breast cancer cells in vitro.

BACKGROUND: Aptamer-based tumor targeted drug delivery system is a promising approach that may increase the efficacy of chemotherapy and reduce the related toxicity. HER2 protein is an attractive target for tumor-specific drug delivery because of its overexpression in multiple malignancies, including breast, gastric, ovarian, and lung cancers. METHODS: In this paper, we developed a new HER2 aptamer (HB5) by using systematic evolution of ligands by exponential enrichment technology (SELEX) and exploited its role as a targeting ligand for delivering doxorubicin (Dox) to breast cancer cells in vitro. RESULTS: The selected aptamer was an 86-nucleotide DNA molecule that bound to an epitope peptide of HER2 with a Kd of 18.9 nM. The aptamer also bound to the extracellular domain (ECD) of HER2 protein with a Kdof 316 nM, and had minimal cross reactivity to albumin or trypsin. In addition, the aptamer was found to preferentially bind to HER2-positive but not HER2-negative breast cancer cells. An aptamer-doxorubicin complex (Apt-Dox) was formulated by intercalating Dox into the DNA structure of HB5. The Apt-Dox complex could selectively deliver Dox to HER2-positive breast cancer cells while reducing the drug intake by HER2-negative cells in vitro. Moreover, Apt-Dox retained the cytotoxicity of Dox against HER2-positive breast cancer cells, but reduced the cytotoxicity to HER2-negative cells. CONCLUSIONS: The results suggest that the selected HER2 aptamer may have application potentials in targeted therapy against HER2-positive breast cancer cells.

Migfilin sensitizes cisplatin-induced apoptosis in human glioma cells in vitro.

AIM: Filamin binding LIM protein 1, also known as migfilin, is a skeleton organization protein that binds to mitogen-inducible gene 2 at cell-extracellular matrix adhesions. The aim of this study was to investigate the role of migfilin in cisplatin-induced apoptosis in human glioma cells, to determine the functional domains of migfilin, and to elucidate the molecular mechanisms underlying the regulation of cisplatin-related chemosensitivity. METHODS: The human glioma cell lines Hs683, H4, and U-87 MG were transfected with pEGFP-C2-migfilin to elevate the expression level of migfilin. RNA interference was used to reduce the expression of migfilin. To determine the functional domains of migfilin, U-87 MG cells were transfected with plasmids of migfilin deletion mutants. After treatment with cisplatin (40 µmol/L) for 24 h, the cell viability was assessed using the MTS assay, and the cell apoptotic was examined using the DAPI staining assay and TUNEL analysis. Expression levels of apoptosis-related proteins were detected by Western blot analysis. RESULTS: Overexpression of migfilin significantly enhanced cisplatin-induced apoptosis in Hs683, H4, and U-87 MG cells, whereas downregulation of migfilin expression inhibited the chemosensitivity of these cell lines. The N-terminal region of migfilin alone was able to enhance the cisplatin-induced apoptosis. However, despite the existence of the N-terminal region, mutants of migfilin with any one of three LIM domains deleted led to a function loss. Furthermore, apoptotic proteins (PARP and caspase-3) and the anti-apoptotic protein Bcl-xL were modulated by the expression level of migfilin in combination with cisplatin. CONCLUSION: The LIM1-3 domains of migfilin play a key role in sensitizing glioma cells to cisplatin-induced apoptosis through regulation of apoptosis-related proteins.

[Effect of peritoneal fibrosis induced by transforming growth factor-beta 1 on the adhesion of gastric cancer cell].

OBJECTIVE: To elucidate the effect of transforming growth factor-beta 1 (TGF-ß1) on peritoneal fibrosis and the regulation of gastric cancer adhering to mesothelial cells. METHODS: The peritoneal mesothelial cell line of HMrSV5 was used to determine the role of TGF-ß1 in the regulation of gastric cancer cell adhering to mesothelial cells. And the mRNA and protein expressions of collagen III and fibronectin were detected by adhesion assay, Western blot, immunofluorescent staining and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: (1) The treatment of 5 ng/ml TGF-ß1 could induce the expressions of collagen III and fibronectin in mesothelial cells at 24, 48 and 72 h (P < 0.01). (2) As compared with the controls, the percentages of adhered HGC-27 and HSC-39 gastric cancer cells significantly increased under the treatment of TGF-ß1 for 24 and 72 h. The increased adhesion percentages of HGC-27 were 65% ± 5% and 124% ± 11% (P < 0.05) while those of HSC-39 85% ± 9% and 146% ± 17% respectively (P < 0.05). (3) Arginyl-glycyl-aspartic acid (RGD) (knockdown of minimal sites for cell-binding domain of extracellular matrix) decreased the number of cancer cells adhering to mesothelial cells under the stimulation of TGF-ß1. And the decreased adhesion percentage of HGC-27 was 65% ± 8% (P < 0.05). CONCLUSIONS: TGF-ß1 significantly stimulates the expressions of collagen III and fibronectin in mesothelial cells. And it is associated with the increased adhesion of gastric cancer cell and offers a favorable environment for the dissemination of gastric cancer.

Application of network pharmacology and molecular docking approach to explore active compounds and potential pharmacological mechanisms of Aconiti Lateralis Radix Praeparata and Lepidii Semen Descurainiae Semen for treatment of heart failure.

BACKGROUND: Heart failure (HF) is the end stage of the development of heart disease, whose prognosis is poor. The previous research of our team indicated that the formulae containing Aconiti Lateralis Radix Praeparata and Lepidii Semen Descurainiae Semen (ALRP-LSDS) could inhibit myocardial hypertrophy, inhibit cardiomyocyte apoptosis, delay myocardial remodeling (REM), and improve the prognosis of patients with HF effectively. In order to explore the mechanism of ALRP-LSDS for the treatment of HF, a combined approach of network pharmacology and molecular docking was conducted. METHODS: Public database TCMSP was used to screen the active compounds of ALRP-LSDS. The targets of screened active compounds were obtained from the TCMSP database and predicted using the online analysis tool PharmMapper. The targets of HF were obtained from 6 databases including GeneCards, OMIM, DrugBank, TTD, PharmGKB, and DisGeNET. Protein-protein interaction and enrichment analysis were performed, respectively, by STRING and Metascape online tools after merging the targets of active compounds and HF. Cytoscape software was used to conduct networks. Finally, molecular docking was performed by Vina to verify the correlation between key targets and active compounds. RESULTS: Final results indicated that the active compounds including ß-sitosterol, isorhamnetin, quercetin, kaempferol, and (R)-norcoclaurine, the targets including AKT1, CASP3, and MAPK1 might be the main active compounds and key targets of ALRP-LSDS for the treatment of HF separately. The binding ability of AKT1 to the main active compounds was better compared with the other 2 key targets, which means it might be more critical. The pathways including AGE-RAGE signaling pathway in diabetic complications, Pathways in cancer, and Fluid shear stress and atherosclerosis might play important roles in the treatment of HF with ALRP-LSDS. In general, ALRP-LSDS could inhibit cardiomyocyte apoptosis, delay REM, and improve cardiac function through multicompound, multitarget, and multipathway, which contributes to the treatment of HF. CONCLUSIONS: Based on the combined approach of network pharmacology and molecular docking, this study screened out the main active compounds, key targets, and main pathways of ALRP-LSDS for the treatment of HF, and revealed its potential mechanisms, providing a theoretical basis for further research.

Efficacy and Safety of Banxia Formulae for Insomnia: A Systematic Review and Meta-Analysis of High-Quality Randomized Controlled Trials.

OBJECTIVE: To systematically evaluate the efficacy and safety of Banxia (Pinellia Tuber) formulae in the treatment of insomnia compared with those of conventional western medicines. METHODS: Randomized controlled trials (RCTs) evaluating the efficacy and safety of Banxia formulae in the treatment of insomnia were searched from the following databases: PubMed, Cochrane Library, EMBASE, the China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Database (VIP), and Wanfang database. The literature collected was from the time when the databases were established to April 2020. Quality assessment and meta-analysis were conducted by using Cochrane bias risk assessment tool and RevMan 5.2, respectively. Publication bias was assessed by Egger's test. RESULTS: Fourteen RCTs with 910 participants were identified. A total of 46 traditional Chinese medicines involving 2 different dosage forms were used in the included studies. Meta-analysis indicated that Banxia formulae had more significant effects on improving the total effective rate (RR = 1.23, 95% CI 1.16 to 1.31), Pittsburgh Sleep Quality Index (PSQI, MD = -1.05, 95% CI -1.63 to -0.47), and the TCM syndrome score (SMD = -0.78, 95% CI -1.18 to -0.39). Meanwhile, on reducing adverse events, Banxia formulae also showed an advantage (RR = 0.48, 95% CI 0.24 to 0.93). CONCLUSION: According to the current studies, the efficacy of Banxia formulae in the treatment of insomnia is better than that of the conventional western medicines, and its safety is relatively stable. However, due to the limitations of this study, further research and evaluation are needed.

[Specifically up-regulated non-coding RNA gene HULC in tumor cell lines and its effects on the expression of neighboring gene SLC35B3].

OBJECTIVE: To study the expression of non-coding RNA HULC in different tumor cell lines and its effects on the expression of neighboring genes. METHODS: The expression levels of HULC were detected in different tumor cell lines. And the neighboring genes of HULC were searched by bioinformatics. Then its effects on the neighboring genes were examined by real-time polymerase chain reaction (PCR). RESULTS: The expression levels of HULC RNA were detected in the 293T, EC9706, KYSE150, HCT116, SMMC-7721, HepG2, H446 and H1299 cell lines. Non-coding RNA HULC was specifically highly up-regulated in hepatocarcinoma cell line HepG2. Little coding genes existed around HULC gene (< 1000 bp) and the nearest coding gene was SLC35B3 at 200 000 bp away from the location of HULC gene. The SMMC-7721 cell line had a low endogenous expression of HULC gene. In comparison with SMMC-7721 cell line, the expression of SLC35B3 gene in HepG2 cell line was higher [(0.477 ± 0.040)% vs (0.129 ± 0.004)%, P < 0.01]. The exogenous expression of HULC had little effects on SLC35B3 gene. But knocking down the expression of HULC gene by siRNA attenuated the expression of SLC35B3 [siA (0.283 ± 0.007)%, siB (0.387 ± 0.015)%, control (0.477 ± 0.042)%]. CONCLUSION: The specifically up-regulated non-coding gene HULC in HepG2 cell line has some effects on the expression of its neighboring gene SLC35B3.

Identification of key genes and pathways in gastric signet ring cell carcinoma based on transcriptome analysis.

BACKGROUND: Gastric signet ring cell carcinoma (GSRCC) is one of the most malignant tumors. It has the features of high invasiveness, rapid progression, and resistance to chemotherapy. However, systematic analyses of mRNAs have not yet been performed for GSRCC. AIM: To identify key mRNAs and signaling pathways in GSRCC. METHODS: A transcriptome analysis of two GSRCC and two non-GSRCC samples was performed in this study. Differentially expressed mRNAs and pathways were identified based on the KEGG and PANTHER pathway annotations. The interactive relationships among the differential genes were mapped with the STRING database. Quantitative real-time polymerase chain reaction was used to validate the key gene expression in GSRCC. RESULTS: About 1162 differential genes (using a 2-fold cutoff, P < 0.05) were identified in GSRCC compared with non-GSRCC. The enriched KEGG and PANTHER pathways for the differential genes included immune response pathways, metabolic pathways, and metastasis-associated pathways. Ten genes (MAGEA2, MAGEA2B, MAGEA3, MAGEA4, MAGEA6, MUC13, GUCA2A, FFAR4, REG1A, and REG1B) were identified as hub genes in the protein-protein interaction network. The expression levels of five genes (MAGEA2, MAGEA3, MAGEA4, MAGEA6, and REG1B) showed potential clinical value. CONCLUSION: We have identified the potential key genes and pathways in GSRCC, and these hub genes and pathways could be diagnostic markers and therapeutic targets for GSRCC.

Efficacy and Safety of Fuzi Formulae on the Treatment of Heart Failure as Complementary Therapy: A Systematic Review and Meta-Analysis of High-Quality Randomized Controlled Trials.

OBJECTIVE: Heart failure is a major public health problem worldwide nowadays. However, the morbidity, mortality, and awareness of heart failure are not satisfied as well as the status of current treatments. According to the standard treatment for chronic heart failure (CHFST), Fuzi (the seminal root of Aconitum carmichaelii Debx.) formulae are widely used as a complementary treatment for heart failure in clinical practice for a long time. We are aiming to assess the efficacy and safety of Fuzi formulae (FZF) on the treatment of heart failure according to high-quality randomized controlled trials (RCTs). METHODS: RCTs in PubMed, Cochrane Library, China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Database (VIP), and Wanfang Database were searched from their inception until June 2019. In addition, the U.S. National Library of Medicine (clinicaltrials.gov) and the Chinese Clinical Trial Registry (http://www.chictr.org.cn) were also searched. We included RCTs that test the efficacy and safety of FZF for the treatment of heart failure, compared with placebo, CHFST, or placebo plus CHFST. The methodological quality of included studies were evaluated by the Cochrane Collaboration's tool for assessing risk of bias. RCTs with Cochrane risk of bias (RoB) score ≥4 were included in the analysis. The meta-analysis was conducted through RevMan 5.2 software. The GRADE approach was used to assess the quality of the evidence. RESULTS: Twelve RCTs with 1490 participants were identified. The studies investigated the efficacy and safety of FZF, such as FZF plus the CHFST vs placebo plus CHFST (n = 4), FZF plus CHFST vs CHFST (n = 6), FZF plus digoxin tablets (DT) plus CHFST vs placebo plus DT plus CHFST (n = 1), and FZF plus placebo plus CHFST vs placebo plus DT plus CHFST (n = 1). Meta-analysis indicated that FZF have additional benefits based on the CHFST in reducing plasma NT-proBNP level, MLHFQ scores, Lee's heart failure scores (LHFs), and composite cardiac events (CCEs). Meanwhile, it also improved the efficacy on TCM symptoms (TCMs), NYHA functional classification (NYHAfc), 6MWD, and LVEF. Adverse events were reported in 6 out of 12 studies without significant statistical difference. However, after assessing the strength of evidence, it was found that only the quality of evidence for CCEs was high, and the others were either moderate or low or very low. So we could not draw confirmative conclusions on its additional benefits except CCEs. Further clinical trials should be well designed to avoid the issues that were identified in this study. CONCLUSION: The efficacy and additional benefits of FZF for CCEs were certain according to the high-quality evidence assessed through GRADE. However, the efficacy and additional benefits for the other outcomes were uncertain judging from current studies. In addition, the safety assessment has a great room for improvement. Thus, further research studies are needed to find more convincing proofs.

MicroRNA-886-3P functions as a tumor suppressor in small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive disease and miRNAs may play an important role in modulating SCLC progression. We have previously screened 924 miRNAs and found that miR-886-3P was negatively associated with SCLC survival. In the current study, we further investigated the role of miR-886-3P mimic in regulating SCLC cell phenotypic alteration in vitro and xenograft tumor formation in vivo. We found that transfection of miR-886-3P mimic significantly inhibited SCLC cell proliferation, migration, and colony formation, and induced mesenchymal-epithelial transition (MET) by suppressing TGF-ß1 synthesis in vitro. Furthermore, intra-tumor injection of miR-886-3P mimic lead to necrosis and suppression of tumor invasion to the surrounding tissue in the subcutaneous xenograft tumor, and intra-vein injection of miR-886-3P mimic suppressed xenograft lung cancer growth in vivo. These findings suggested that miR-886-3P functions as a tumor suppressor in SCLC and thus, it might be a potential therapeutic molecule in the treatment of lung cancer.

Clinicopathological parameters predicting recurrence of pT1N0 esophageal squamous cell carcinoma.

AIM: To identify the clinicopathological characteristics of pT1N0 esophageal squamous cell carcinoma (ESCC) that are associated with tumor recurrence. METHODS: We reviewed 216 pT1N0 thoracic ESCC cases who underwent esophagectomy and thoracoabdominal two-field lymphadenectomy without preoperative chemoradiotherapy. After excluding those cases with clinical follow-up recorded fewer than 3 mo and those who died within 3 mo of surgery, we included 199 cases in the current analysis. Overall survival and recurrence-free survival were assessed by the Kaplan-Meier method, and clinicopathological characteristics associated with any recurrence or distant recurrence were evaluated using univariate and multivariate Cox proportional hazards models. Early recurrence (≤ 24 mo) and correlated parameters were assessed using univariate and multivariate logistic regression models. RESULTS: Forty-seven (24%) patients had a recurrence at 3 to 178 (median, 33) mo. The 5-year recurrence-free survival rate was 80.7%. None of 13 asymptomatic cases had a recurrence. Preoperative clinical symptoms, upper thoracic location, ulcerative or intraluminal mass macroscopic tumor type, tumor invasion depth level, basaloid histology, angiolymphatic invasion, tumor thickness, submucosal invasion thickness, diameter of the largest single tongue of invasion, and complete negative aberrant p53 expression were significantly related to tumor recurrence and/or recurrence-free survival. Upper thoracic tumor location, angiolymphatic invasion, and submucosal invasion thickness were independent predictors of tumor recurrence (Hazard ratios = 3.26, 3.42, and 2.06, P < 0.001, P < 0.001, and P = 0.002, respectively), and a nomogram for predicting recurrence-free survival with these three predictors was constructed. Upper thoracic tumor location and angiolymphatic invasion were independent predictors of distant recurrence. Upper thoracic tumor location, angiolymphatic invasion, submucosal invasion thickness, and diameter of the largest single tongue of invasion were independent predictors of early recurrence. CONCLUSION: These results should be useful for designing optimal individual follow-up and therapy for patients with T1N0 ESCC.

[Expression of fascin and cytokeratin 14 in esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the expression of fascin and cytokeratin 14 (CK14) in human esophageal squamous cell carcinoma (ESCC) and the association of these two proteins with ESCC malignant progression and the possibility of application of these 2 proteins in the diagnosis of ESCC. METHODS: A tissue microarray composed of the representative regions of ESCC and corresponding normal epithelium was constructed. Immunohistochemistry was conducted to examine the expression of fascin and CD14 in 116 specimens of ESCC and the normal tissues near the cancerous tissues. The relation of these two proteins with the invasive depth, node involvement, differentiated grade, pTNM stages was analyzed. Disease-free survival analysis was carried out using Kaplan-Meier method. The correlation of the two proteins was analyzed using Spearman's correlation test. ESCC cells of the lines EC9706, TE12, COLO-680N, KYSE510, KYSE450, KYSE410, KYSE180, KYSE150, KYSE140, KYSE70, KYSE30, and YES2 were cultured and underwent SDS-PAGE and Western blotting to examine the expression of fascin and CD14. And the correlation of the two proteins with the characteristics of the cell lines was analyzed too. RESULTS: Fascin and CK14 were negative in the normal esophageal epithelia except in the basal cells. The positive rates of fascin and CK14 in the ESCC cells were 79.3% and 67.0% respectively. The positive rate of either fascin or CK14 was 86.2%. The expression rates of fascin and CK14 in well- and moderately-differentiated ESCCs were significantly higher than that in the poorly-differentiated ones (P = 0.054 and P < 0.01). The patients with positive expression of fascin and those with negative expression of CK14 had a poorer survival in comparison with those with negative fascin expression and those with positive CK14 expression respectively, however, without statistical significances (P = 0.8980 and P = 0.2610). The positive rates of fascin in the well-, moderately-, and poorly- differentiated ESCCs were 87.1%, 83.9%, and 62.1% respectively (P = 0.054). The positive rates of CK14 in the well-, moderately-, and poorly-differentiated ESCCs were; 87.1%, 76.4%, and 27.6% respectively (P < 0.01). The prognosis was not significantly correlated with the expression of both proteins (P = 0.8980 and P = 0.2610). There was an significantly positive correlation between the expression levels of these 2 proteins (r = 0.487, P < 0.01). Fascin was highly expressed in most of the ESCC lines, except in the slowly growing and weakly migrating TE12 line. High expression of CK14 was only seen in the line KYSE180. CONCLUSION: Fascin and CK14 may play important roles in the carcinogenesis and progression of ESCC. Combination of fascin and CK14 would be valuable markers in diagnosis of ESCC.

Tissue microarray analysis reveals a tight correlation between protein expression pattern and progression of esophageal squamous cell carcinoma.

BACKGROUND: The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. METHODS: Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5gamma2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5gamma2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. RESULTS: In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5gamma2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5gamma2 and SPARC. CONCLUSION: Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5gamma2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC.

[Suppression of HCT116 growth of cells by Gadd45].

OBJECTIVE: To study the mechanism of suppression of growth of HCT116 human colon carcinoma cells by Gadd45 gene. METHODS: HCT116 human colon carcinoma cells were transfected with pTRE-Gadd45 vector so as to establish Gadd45-inducible cell line that was cultured in medium with tetracycline. Then tetracycline was withdrawn. The number of cell clones was counted. Flow cytometry was used to detect the percentages of cells at the G1, S, and G2-M phases. TUNEL technique was used to detect the apoptosis of cells. Western blotting was used to analyze the expression of Gadd45 protein, and apoptosis-related proteins: poly-ADP-ribose polymerase (PARP), and caspase 3 protein. RESULTS: Gadd45 protein was not expressed in the HCT116 cells cultured in the medium with tetracycline, however, it was expressed with a gradually increased level in the cells cultured in the medium from which tetracycline was withdrawn, The clone formation rate of HCT116 cells was 100% in the medium with tetracycline, however, was only 14.2% in the medium with tetracycline withdrawal, with a suppression rate of more than 85%. The percentage of cells in G(2)-M phase was significantly increased in the cells cultured in the medium with tetracycline withdrawal. 24-36 hours after the withdrawal of tetracycline, PARP and caspase 3 protein were activated remarkably. CONCLUSION: High expression of Gadd45 inhibits the growth of HCT116 cells, through inducing G2-M arrest and activating apoptotic pathway.

A preliminary study of genes related to concomitant chemoradiotherapy resistance in advanced uterine cervical squamous cell carcinoma.

BACKGROUND: Tumor intrinsic chemoradiotherapy resistance is the primary factor in concomitant chemoradiotherapy failure in advanced uterine cervical squamous cell carcinoma. This study aims to identify a set of genes and molecular pathways related to this condition. METHODS: Forty patients with uterine cervical squamous cell carcinoma in International Federation of Gynecology and Obstetrics stage IIb or IIIb, treated with platinum-based concomitant chemoradiotherapy between May 2007 and December 2012, were enrolled in this trial. Patients included chemoradiotherapy resistant (n = 20) and sensitive (n = 20) groups. Total RNA was extracted from fresh tumor tissues obtained by biopsy before treatment and microarray analysis was performed to identify genes differentially expressed between the two groups. RESULTS: Microarray analysis identified 108 genes differentially expressed between concomitant chemoradiotherapy resistant and sensitive patients. Functional pathway cluster analysis of these genes revealed that DNA damage repair, apoptosis, cell cycle, Map kinase signal transduction, anaerobic glycolysis and glutathione metabolism were the most relevant pathways. Platelet-derived growth factor receptor alpha (PDGFRA) and protein kinase A type 1A (PRKAR1A) were significantly upregulated in the chemoradiosensitive group, while lactate dehydrogenase A (LDHA), bcl2 antagonist/killer 1 (BAK1), bcl2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and cyclin-dependent kinase 7 (CDK7) were upregulated in the chemoradiotherapy resistant group. CONCLUSION: We have identified seven genes that are differentially expressed in concomitant chemoradiotherapy resistant and sensitive uterine cervical squamous cell carcinomas, which may represent primary predictors for this condition.

Optimization of transfection efficiency of small interfering RNA in purified human prolactinoma cells.

BACKGROUND: Control of hypersecretion of certain hormones is one of the key targets in the treatment of pituitary adenomas. RNA interference has been shown to inhibit protein expression, and thus it may represent a promising method for the treatment of pituitary adenomas. In the present study, transfection efficiency of small interfering RNA (siRNA) was optimized in human prolactinoma cells. METHODS: First, a method was optimized to extract highly purified human prolactinoma cells in vitro. The extracted cells were verified to retain the physiological features of prolactin (PRL) secretion. Second, three conditions for siRNA transfection were tested by the evaluation of transfection efficiency and cell viability. The proper transfection condition was verified for human prolactinoma cells. Third, the siRNA for prolactin was transfected into the human prolactinoma cells, and the suppression of PRL mRNA was evaluated by quantitative real-time reverse transcription-PCR. RESULTS: The siRNA of 100 pmol with Lipofectamine 2000 of 5 µl for 1 × 10(6) cells was proved preferable, with transfection efficiency being 53.3% and cell viability being 69.7%. In the preliminary experiment the siRNA against PRL decreased the mRNA of PRL by 34.0%. CONCLUSION: It is possible to inhibit hormone hypersecretion by RNA interference, that may eventually enable therapeutic siRNA drugs developed.

[Gadd45 mediated G2/M cell cycle arrest induced by BRCA1].

BACKGROUND & OBJECTIVE: It is considered that tumor suppressor gene BRCA1 is an important factor in the regulation of cell cycle checkpoint, but the molecular mechanism by which BRCA1 regulates cell cycle G(2)/M arrest is less known. The objective of this study was to investigate the effects of Gadd45 on the BRCA1-induced cell growth suppression. METHODS: BRCA1 induction of Gadd45 protein was analyzed using Western-blot assay following cells transfection with BRCA1 expression vector and cell sorting. Activation of the Gadd45 promoter by BRCA1 was determined by CAT assay. Effect of antisense Gadd45 on the BRCA1-induced cell cycle G(2)-M arrest was examined by flow cytometry analysis. And the effects of antisense Gadd45 on BRCA1-mediated growth suppression in HeLa and HCT116 cell lines was determined by colony formation assay. RESULTS: Gadd45 protein was highly induced after expression of BRCA1. BRCA1 strongly activated the Gadd45 promoter. Antisense Gadd45 substantially abrogated BRCA1-activated cell cycle G(2)-M arrest and BRCA1-induced cell growth suppression on HeLa and HCT116 cell lines. CONCLUSION: Gadd45 is a BRCA1-regulated downstream gene and mediates the role of BRCA1 in the control of cell cycle G(2)/M arrest and growth suppression.