ncRNAs-mediated overexpression of STIL predict unfavorable prognosis and correlated with the efficacy of immunotherapy of hepatocellular carcinoma.

BACKGROUND: STIL centriolar assembly protein (STIL) is a cytoplasmic protein implicated in cellular growth and proliferation as well as chromosomal stability, which abnormal condition affected tumor immunity and tumor progression. However, the role of STIL in the biological mechanism of hepatocellular carcinoma (HCC) remains unclear. METHODS: Comprehensive bioinformatic approaches, in vitro functional assays, and validation were conducted to elucidate the oncogenic value of STIL in HCC. RESULTS: In the present study, we found that STIL may serve as an independent prognostic indicator and a potential oncogene in HCC. Gene set enrichment analysis (GSEA), and Gene set variation analysis (GSVA) showed that upregulated expression of STIL was positively associated with pathways enriched in the cell cycle and DNA damage response. Subsequently, we identified several non-coding RNAs (ncRNAs) accounting for the upregulation of STIL expression using a combination of in silico bioinformatics approaches (including expression analysis, correlation analysis, and survival analysis). Finally, CCNT2-AS1/SNHG1-has-miR-204-5p-STIL axis was screened out as the most potential upstream ncRNA-related pathway of STIL in HCC. Moreover, STIL expression is highly associated with the infiltration of immune cells, the expression of immune checkpoints, as well as the survival benefit of immunotherapy/chemotherapy. CONCLUSIONS: Our study discloses that ncRNAs-mediated overexpression of STIL independently predicted poor prognosis and correlated with the efficacy of PD-1-targeted immunotherapy in HCC.

LncRNA SNHG12 Decreases Non-Small Cell Lung Cancer Cell Sensitivity to Cisplatin by Repressing miR-525-5p and Promoting XIAP.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is recognized as one of the primary causes of global cancer-related mortality. Long noncoding RNAs (lncRNAs) participate in NSCLC cell progression. This study probed the potential mechanism of lncRNA small nucleolar RNA host gene 12 (SNHG12) in cisplatin (DDP)-resistance in NSCLC cells. METHODS: The intracellular expressions of SNHG12, miR-525-5p, and XIAP were examined via reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Afterwards, small interfering RNAs (siRNAs) of SNHG12, microRNA (miR)-525-5p inhibitor, and X-linked inhibitor of apoptosis (XIAP) pcDNA3.1 were transfected into NSCLC cells. Subsequently, changes in half-maximal (50%) inhibitory concentration (IC50) of NSCLC cells to DDP were detected through the cell counting kit-8 (CCK-8) method. NSCLC proliferative ability and apoptosis rate were determined with the help of colony formation and flow cytometry assays. The subcellular localization of SNHG12 was analyzed by nuclear/cytosol fractionation assay and binding relationships between miR-525-5p and SNHG12 or XIAP were analyzed via dual-luciferase reporter gene assay. Furthermore, rescue experiments were designed to detect the effects of miR-525-5p and XIAP on NSCLC sensitivity to DDP. RESULTS: SNHG12 and XIAP were up-regulated in NSCLC cells while miR-525-5p was down-regulated. After DDP treatment and SNHG12 repression, NSCLC proliferative ability was decreased whereas apoptosis rate was increased, and NSCLC sensitivity to DDP was enhanced. Mechanically, SNHG12 repressed miR-525-5p expression, and miR-525-5p could targeted inhibit XIAP transcription level. miR-525-5p repression or XIAP overexpression reduced NSCLC sensitivity to DDP. CONCLUSION: SNHG12 was overexpressed in NSCLC cells and promoted XIAP transcription by repressing miR-525-5p expression, enhancing DDP-resistance in NSCLC cells.

UHRF1 plays an oncogenic role in small cell lung cancer.

Small cell lung cancer (SCLC) is a malignant tumor characterized by aggressiveness and dismal prognosis. The specific role of ubiquitin-like PHD and RING finger domain (UHRF1), a frequently overexpressed cancer-promoting gene in various tumors, is poorly understood in SCLC. Herein, we explored the potential carcinogenic role of UHRF1 in SCLC. First, public databases were used to analyze the expression of UHRF1 in SCLC, and tissue specimens in our center were examined to confirm the results while clinical outcomes were collected to analyze its relationship with UHRF1. Then, UHRF1 knockdown and overexpression cell lines were established to evaluate the carcinogenic function of UHRF1 in vitro and in vivo. The mechanism of the biological consequences was determined by co-inmunoprecipitation. Moreover, we also analyzed the influence of UHRF1 on cisplatin (DDP) sensitivity of SCLC. The expression of UHRF1 was significantly higher in SCLC tissues than in normal tissues, and high levels of UHRF1 suggested a poor prognosis for SCLC. Mechanistically, UHRF1 promoted SCLC growth through yes-associated protein 1 (YAP1). Specifically, UHRF1 bound to YAP1 and inhibited YAP1 ubiquitin degradation, thus stabilizing the YAP1 protein in SCLC cells. UHRF1 downregulation enhanced DDP sensitivity in SCLC cells and was correlated with a favorable prognosis in patients with SCLC treated with platinum-based chemotherapy. UHRF1 plays an oncogenic role in SCLC by modulating YAP1. Therefore, UHRF1 could be used as a biomarker to predict the prognosis of SCLC patients and serve as a potential therapeutic target for SCLC patients.

ASF1B is a Promising Prognostic Biomarker and Correlates With Immunotherapy Efficacy in Hepatocellular Carcinoma.

Background: Anti-silencing function 1B (ASF1B), a histone H3-H4 chaperone, is crucial for S-phase progression and cell proliferation. Recent studies have shown that ASF1B may be used as a new proliferation marker for cancer prognosis. However, the prognostic value and effect of ASF1B on tumor cells and the immune microenvironment in hepatocellular carcinoma (HCC) remain unclear. Methods: We analyzed the expression of ASF1B and its prognostic value using The Cancer Genome Atlas (TCGA) database (as a training set) and other databases, and we validated the findings by immunohistochemistry in our clinical database, containing 141 HCC patients (as a validation set). Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were performed to probe the tumor-associated biological processes of ASF1B in HCC. The interrelationships between ASF1B expression and tumor immunological characteristics were analyzed by multiple databases. The Imvigor210 cohort was retrieved to assess the ability of ASF1B to predict immunotherapy efficacy. Results: ASF1B was highly expressed in tumor tissue compared to paracancerous tissue. High ASF1B expression was associated with worse overall survival (OS) and progression-free survival (PFS) in the training set (p = 0.005, p < 0.001) and validation set (p < 0.001, p < 0.001). Multivariate analysis revealed that ASF1B was an independent prognostic factor associated with OS and PFS. GSEA and GSVA suggested that ASF1B was involved in tumor-associated biological processes, including the cell cycle, DNA replication, base excision repair, mismatch repair, RNA degradation, ubiquitin-mediated proteolysis, and nucleotide excision repair. Further analysis revealed that the levels of ASF1B were positively correlated with the immune cells infiltration of B cells, CD8+ T cells, CD4+ T cells, neutrophils, and dendritic cells. However, ASF1B was positively correlated with Treg cell infiltration and inhibitory immune checkpoints in exhausted T cells. Patients who received anti-PD-L1 immunotherapy with high ASF1B expression had a higher objective response. Conclusion: The ASF1B level is an independent prognostic factor and may serve as a potential immunotherapeutic target.

LncRNA SNHG12 in extracellular vesicles derived from carcinoma-associated fibroblasts promotes cisplatin resistance in non-small cell lung cancer cells.

Non-small-cell lung cancer (NSCLC) is defined as the most universally diagnosed class of lung cancer. Cisplatin (DDP) is an effective drug for NSCLC, but tumors are prone to drug resistance. The current study set out to evaluate the regulatory effect of long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) in extracellular vesicles (EVs) derived from carcinoma-associated fibroblasts (CAFs) on DDP resistance in NSCLC cells. Firstly, NSCLC cells were treated with EVs, followed by detection of cell activity, IC50 values, cell proliferation and apoptosis, and Cy3-SNHG12. We observed that CAFs-EVs promoted IC50 values and cell proliferation and inhibited apoptosis. In addition, we learned that lncRNA SNHG12 carried by CAFs-EVs into NSCLC facilitated DDP resistance of NSCLC cells. Furthermore, ELAV like RNA binding protein 1 (HuR/ELAVL1) binding to lncRNA SNHG12 and X-linked inhibitor of apoptosis (XIAP) was verified and RNA stability of XIAP was also verified CAFs-EVs promoted RNA stability and transcription of XIAP, while silencing HuR could partially-reverse this promoting effect. Further joint experimentation showed that silencing XIAP partially inhibited DDP resistance in NSCLC cells. Additionally, the tumor growth and the positive rate of Ki67 and HuR were detected, which showed that CAFs-oe-EVs promoted the tumor and the positive rate of Ki67, as well as the levels of lncRNA SNHG12, HuR, and XIAP in vivo. Collectively, our findings indicated that lncRNA SNHG12 carried by CAFs-EVs into NSCLC cells promoted RNA stability and XIAP transcription by binding to HuR, thus augmenting DDP resistance in NSCLC cells.

Prospective Clinical Study of Postoperative Individualized Adjuvant Chemotherapy for Patients with Non-Small-Cell Lung Cancer Based on mRNA Expression of the Molecular Markers RRM1, TUBB3, and ERCC1.

OBJECTIVE: To investigate the clinical significance of the mRNA expression of RRM1, TUBB3, and ERCC1 in non-small-cell lung cancer (NSCLC) tissues for the selection of adjuvant/postoperative chemotherapy regimens. METHODS: Patients diagnosed with stage Ib-IIIa NSCLC were enrolled and randomly divided into a control group (undetected group) and an experimental group (detected group) after radical operation. The control group randomly received chemotherapy with gemcitabine plus cisplatin or paclitaxel plus cisplatin. The mRNA expression of RRM1, TUBB3, and ERCC1 was detected in the experimental group before chemotherapy, and based on the detected expression, the chemotherapy regimen of cisplatin plus gemcitabine or cisplatin plus paclitaxel was chosen. The disease-free survival (DFS) of the control group and experimental group was compared. RESULTS: Pathological type, stage, gene expression detection, and treatment method were not significantly correlated with DFS (P > 0.05). In the subgroups treated with gemcitabine, the median DFS was 17 months in the detected group and 10.5 months in the undetected group (hazard ratio = 0.2147, 95% confidence interval: 0.07909-0.5827, P=0.0025). Multivariate regression analysis was performed to analyse whether gene expression detection was independently correlated with DFS in the subgroups treated with gemcitabine (P=0.025). In the detected group, the prognosis of patients with low expression of RRM1 was better than that of patients with high expression of RRM1 after paclitaxel treatment (P=0.0039). CONCLUSIONS: The selection of chemotherapy regimen based on mRNA expression of the RRM1, TUBB3, and ERCC1 genes may improve selection of candidate patients to receive clinical chemotherapy. However, large-scale prospective clinical studies are needed for in-depth investigation.

Identification of a seven-long non-coding RNA signature associated with Jab1/CSN5 in predicting hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, accounting for over 700,000 deaths each year. The lack of predictive and prognostic biomarkers for HCC, with effective therapy, remains a significant challenge for HCC management. Long non-coding RNAs (lncRNAs) play a key role in tumorigenesis and have clinical value as potential biomarkers in the early diagnosis and prediction of HCC. Jun activation domain-binding protein 1 (Jab1, also known as COP9 signalosome subunit 5, CSN5) is a potential oncogene that plays a critical role in the occurrence of HCC. Here, we performed a comprehensive analysis for Jab1/CSN5-associated lncRNAs to predict the prognosis of HCC. The differentially expressed (DE) lncRNAs between in HCC were analyzed based on the TCGA RNA-seq data. We detected 1031 upregulated lncRNAs in 371 HCC tissues and identified a seven-lncRNA signature strongly correlated with Jab1/CSN5 (SNHG6, CTD3065J16.9, LINC01604, CTD3025N20.3, KB-1460A1.5, RP13-582O9.7, and RP11-29520.2). We further evaluated the prognostic significance of these lncRNAs by GEPIA ( http://gepia.cancer-pku.cn/ ). The expression data in 364 liver tumors indicated that this seven-lncRNA signature could better predict worse survival in HCC patients. Moreover, 35 clinical HCC samples were evaluated to assess the validity and reproducibility of the bioinformatic analysis. We found that the targeted lncRNAs were upregulated, with a strong association with Jab1/CSN5 and prognostic value in HCC. Functional enrichment analysis by Gene Ontology (GO) showed that these seven prognostic lncRNAs exhibit oncogenic properties and are associated with prominent hallmarks of cancer. Overall, our findings demonstrate the clinical implication of Jab1/CSN5 with the seven-lncRNAs in predicting survival for patients with HCC.

Application of 3D modeling and printing technology in accurate resection of complicated thoracic tumors.

BACKGROUND: To explore the application value of three-dimensional (3D) reconstruction and 3D printing in preoperative evaluation of precise resection of complicated thoracic tumors. METHODS: A retrospective analysis of 34 patients with complicated thoracic tumors who were treated by radical surgery from March 2016 to June 2019 was made. According to whether 3D reconstruction and 3D printing was used, the patients were divided into research group and control group. In the control group, preoperative evaluation was performed according to CT image data, and the operation plan was drawn up; in the research group, preoperative simulation and preoperative operation plan design were carried out according to 3D reconstruction and 3D printing technology. The operation time, change of operation approach, intraoperative blood loss, hospitalization time and postoperative complications were compared between the two groups. We also retrospectively reviewed additional 12 cases of unresectable complicated thoracic tumors. The above 34 patients who were treated by radical surgery were set as the resectable group. Three-dimensional reconstruction was performed for all cases. The tumor size, location, smoothness of tumor-vascular contact surface, close contact with adjacent organs were compared between these two groups. RESULTS: The 3D reconstruction and 3D printing model were successfully established. The indexes of operation time, change of incision approach and blood loss in the research group were lower than those in the control group (P<0.05). All the patients were followed up for 6 months, and there was no death, no tumor recurrence and metastasis in the two groups. In the unresectable group, the score of position and smoothness of tumor-vascular contact surface were significantly higher than that in the resectable group. CONCLUSIONS: 3D reconstruction and 3D printing can effectively help surgeons carry out accurate surgical treatment, reduce the operation time and bleeding, reduce the risk of surgery, and facilitate the postoperative rehabilitation of patients, which has the value of promotion and application.

Tumor-infiltrating immune cells in hepatocellular carcinoma: Tregs is correlated with poor overall survival.

Systematic interrogation of tumor-infiltrating immune cells (TIICs) is key to the prediction of clinical outcome and development of immunotherapies. However, little is known about the TIICs of hepatocellular carcinoma (HCC) and its impact on the prognosis of patients and potential for immunotherapy. We applied CIBERSORT of 1090 tumors to infer the infiltration of 22 subsets of TIICs using gene expression data. Unsupervised clustering analysis by 22 TIICs revealed 4 clusters of tumors, mainly defined by macrophages and T cells, with distinct prognosis and associations with immune checkpoint molecules, including PD-1, CD274, CTLA-4, LAG-3 and IFNG. We found tumors with decreased number of M1 macrophages or increased regulatory T cells were associated with poor prognosis. Based on the multivariate Cox analysis, a nomogram was also established for clinical application. In conclusion, composition of the TIICs in HCC was quite different, which is an important determinant of prognosis with great potential to identify candidates for immunotherapy.

Extracorporeal membrane oxygenation (ECMO) assisted mediastinal tumor resection and superior vena cava replacement are safe and feasible.

BACKGROUND: How to maximally improve the drainage of intracranial and upper body venous and to reduce neurological complications during thoracic tumor-causedsuperior vena cava replacement are still clinical problems to be solved. METHODS: We have innovatively used the bilateral jugular vein-left femoral vein ECMO shunting to perform mediastinal tumor resection and superior vena cava replacement in a 50-year-old woman. RESULTS: During the operation, this technique maintained the patient's hemodynamic stability, improved the cerebral oxygen saturation and reduced the cerebral ischemia, hypoxia as well as the neurological complications. CONCLUSION: It is indicated for patients with superior vena cava replacement who are unable to perform venous bypass (such as innominate vein to right atrial bypass) or venous shunting (such as differential pressure drainage from internal jugular vein to femoral vein).

miRNA 146a promotes chemotherapy resistance in lung cancer cells by targeting DNA damage inducible transcript 3 (CHOP).

The underlying molecular mechanism of lung cancer drug resistance is poorly understood. The mediator of endoplasmic reticulum stress CHOP (DNA damage inducible transcript 3) promotes stress-induced apoptosis and appears to function as a transcription factor in multiple diseases. However, its potential contributions to multidrug resistance in solid tumors is unknown. Here, we investigated CHOP expression in tumor tissues form 69 lung cancer patients, finding that deficient CHOP expression is associated with poor prognosis. Cisplatin-resistant lung cancer cells exhibited lower expression of CHOP compared to that in sensitive lung cancer cells, and silencing or augmenting CHOP expression enhanced or impaired cisplatin resistance, respectively. Mechanistic investigations revealed that CHOP is directly associated with the regulation of autophagy or apoptosis-regulatory genes including LC3-II, death receptor 5 (DR5), and telomere repeat-binding factor 3. Notably, CHOP was identified as a target of miR-146a, and increased miR-146a expression in lung cancer cells was suggested to be responsible for CHOP mRNA down-regulation. Further, animal models confirmed that abnormally expressed miR-146a in lung cancer cells does not affect growth, but rather alters chemotherapy sensitivity. Together, CHOP is a useful prognostic marker and miR-146a is a potential therapeutic target for multidrug-resistant lung cancer.

Trps1 is associated with the multidrug resistance of lung cancer cell by regulating MGMT gene expression.

Multidrug resistance (MDR) often leads to chemotherapy failure of lung cancer and has been linking to the cellular expression of several DNA transcription- and repair-related genes such as Trps1 and MGMT. However, their roles in the formation of MDR are largely unknown. In this study, overexpression/knockdown, luciferase assay and ChIP assay were performed to study the relationship between Trps1 and MGMT, as well as their roles in MDR formation. Our results demonstrated that Trps1 and MGMT expression both increased in drug-resistant lung cancer cell line (H446/CDDP). Silencing of Trps1 resulted in downregulation of MGMT expression and decrease in the multidrug sensitivity of H446/CDDP cells, while Trps1 overexpression exhibited the opposite effects in H446 cells. Ectopic expression of MGMT had no effect on Trps1 expression, but enhanced the IC50 values of H446 cells or rescued the IC50 values of Trps1-silenced H446/CDDP cells in treatment of multidrug. Our data further showed that, mechanistically, Trps1 acted as a transcription activator that directly induced MGMT transcription by binding to the MGMT promoter. Taken together, we consider that upregulation of Trps1 induces MGMT transcription contributing to the formation of MDR in lung cancer cells. Our findings proved potential targets for reversing MDR in clinical chemotherapy of lung cancer.

Long noncoding RNA H19 is up-regulated in esophageal squamous cell carcinoma and promotes cell proliferation and metastasis.

Long noncoding RNAs (lncRNAs) have been shown to play various roles in tumorigenesis, among which lncRNA H19 has been revealed as an ambivalent factor that acts as both an oncogene and a tumor suppressor in carcinogenesis. However, the exact biological role of H19 in esophageal squamous cell carcinoma (ESCC) remains to be determined. The aim of this study was to examine the expression pattern of H19 in ESCC and evaluate its biological role and clinical significance in the progression of ESCC. Expression of H19 was analyzed in 64 ESCC tissues and four ESCC cell lines by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Proliferation, cell cycle, migration, and invasion assays were performed in ESCC cell lines following knockdown of H19 to determine the biological function of H19 in the progression of ESCC both in vitro and in vivo. Western blot analysis was also performed to identify the potential mechanisms involved. H19 was highly expressed both in ESCC samples and cell lines compared with corresponding normal counterparts. The up-regulation of of H19 was significantly correlated with ESCC clinical stage and lymph node metastasis. Knockdown of H19 not only exerted inhibitory effect on tumor proliferation in vitro and in vivo, but also repressed the migratory and invasive capacity. G0/G1 phase arrest was also found in H19 knockdown cell lines. In addition, silencing of H19 up-regulated epithelial marker E-cadherin while down-regulating mesenchymal marker vimentin and metastasis-associated protein such as MMP-9. These findings indicate that H19 acts as an oncogene and promotes ESCC cell proliferation and metastasis, which may infer H19 as a marker of poor prognosis and, thus, a potential therapeutic target for treating ESCC patients.

Germline copy number loss of UGT2B28 and gain of PLEC contribute to increased human esophageal squamous cell carcinoma risk in Southwest China.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide with poor prognosis. Thus, identification of predictive biomarkers for early diagnosis and intervention is needed to improve patients' survival. Research shows that heritable mutations, such as single nucleotide polymorphisms (SNPs), contribute to human cancer susceptibility significantly. However, the association of copy number variations (CNVs), another major source of genetic variation, with ESCC risk remains poorly clarified. In this study, we aimed to identify ESCC risk-related CNVs based on candidate-gene strategy in a case-control study. A meta-analysis was first performed to identify the most variable chromosome regions of ESCC tissues. Bioinformatic analysis and dual-luciferase reporter assays were carried out to evaluate the properties of all recorded CNVs located on these regions. Six candidate CNVs located within well-known oncogenes and detoxification-associated enzymes were enrolled in the final analysis. A newly developed multiplex gene copy number quantitation method AccuCopy(TM) was employed to simultaneously genotype all six candidate sites in 404 ESCC patients and 402 cancer-free controls from Southwest China, and in 42 ESCC tissues. qRT-PCR was performed to measure UGT2B28 mRNA in cancerous and corresponding normal tissues. Unconditional logistic regression was applied to test association between germline CNV genotypes and ESCC risk. Relationship between germline copy number variation and somatic copy number alterations was further analyzed. Finally we found that copy number loss of UDP-glucuronosyltransferase family 2, polypeptide B28 (UGT2B28) and gain of plectin (PLEC) conferred increased ESCC risk (Adjusted OR = 2.085, 95% CI = 1.493-2.912, P < 0.001 for UGT2B28. Adjusted OR = 3.725, 95% CI = 1.026-13.533, P = 0.046 for PLEC). mRNA level was lower in UGT2B28 loss genotyped esophageal tissues than in two-copy tissues, indicating that UGT2B28 loss genotypes modify ESCC susceptibility perhaps by decreasing UGT2B28 expression level and enzyme activity. In addition, an association was drawn between germline copy number variations and somatic alterations for PLEC, UGT2B17 and UGT2B28, but not for other candidate loci.

Rho Guanine Nucleotide Exchange Factor 5 Increases Lung Cancer Cell Tumorigenesis via MMP-2 and Cyclin D1 Upregulation.

We sought to elucidate the role of Rho guanine nucleotide exchange factor 5 (ARHGEF5) in tumorigenesis of lung adenocarcinoma cells. ARHGEF5 protein levels were assessed in 91 human lung adenocarcinoma specimens, and A549 and NCI-H1650 cells, by IHC and Western blotting. In addition, ARHGEF5 mRNA expression was evaluated by quantitative reverse transcriptase-PCR. Furthermore, ARHGEF5 long and short isoform coexpression was detected by immunofluorescence. Finally, flow cytometry; CCK8 and wound-healing assays; cell invasion, migration and adhesion; and xenografts were used to evaluate the biologic significance of ARHGEF5. ARHGEF5 was significantly increased in lung adenocarcinoma tissues and cell lines. Interestingly, ARHGEF5 levels were significantly associated with tumor grade and pathologic stage, but not age, gender, T stage, or lymph node metastasis status. ARHGEF5 knockdown by RNAi resulted in dramatically reduced proliferation, adhesion, invasion, and migratory capability of A549 and NCI-H1650 cells. Likewise, protein levels of p-Src, p-Akt, and NF-κB were significantly decreased after ARHGEF5 knockdown. In parallel, increased S-phase population and MMP-2/cyclin D1 expression were observed in the cancer cells, which were not apoptotic. In addition, ARHGEF5 knockdown A549 and NCI-H1650 cells injected s.c. and i.v. into nude mice exhibited decreased xenograft volume and overtly reduced metastasis. Conversely, ARHGEF5 overexpression in A549 and NCI-H1650 cells increased their tumorigenicity in vitro. ARHGEF5 acts as a proto-oncogene in human lung adenocarcinoma cell tumorigenesis.

The auto-inhibitory state of Rho guanine nucleotide exchange factor ARHGEF5/TIM can be relieved by targeting its SH3 domain with rationally designed peptide aptamers.

The short isoform of Rho guanine nucleotide exchange factor ARHGEF5 is known as TIM, which plays diverse roles in, for example, tumorigenesis, neuronal development and Src-induced podosome formation through the activation of its substrates, the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a poly-proline sequence between the putative helix and the DH domain. In this study, we systematically investigated the structural basis, energetic landscape and biological implication underlying TIM auto-inhibition by using atomistic molecular dynamics simulations and binding free energy analysis. The computational study revealed that the binding of SH3 domain to poly-proline sequence is the prerequisite for the stabilization of TIM auto-inhibition. Thus, it is suggested that targeting SH3 domain with competitors of the poly-proline sequence would be a promising strategy to relieve the auto-inhibitory state of TIM. In this consideration, we rationally designed a number of peptide aptamers for competitively inhibiting the SH3 domain based on modeled TIM structure and computationally generated data. Peptide binding test and guanine nucleotide exchange analysis solidified that these designed peptides can both bind to the SH3 domain potently and activate TIM-catalyzed RhoA exchange reaction effectively. Interestingly, a positive correlation between the peptide affinity and induced exchange activity was observed. In addition, separate mutation of three conserved residues Pro49, Pro52 and Lys54 - they are required for peptide recognition by SH3 domain -- in a designed peptide to Ala would completely abolish the capability of this peptide activating TIM. All these come together to suggest an intrinsic relationship between peptide binding to SH3 domain and the activation of TIM.

Up-regulation of long noncoding RNA MALAT1 contributes to proliferation and metastasis in esophageal squamous cell carcinoma.

BACKGROUND: Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) has been demonstrated to be an important player in various human malignancies; it is thought to promote tumor growth by cell cycle regulating. However, the roles of MALAT1 in esophageal squamous cell carcinoma(ESCC), and the mechanisms involved in cell cycle regulation remain poorly understood. Moreover, the factors contributing to its up-regulation in tumor tissues are still largely unclear. METHODS: Expression of MALAT1 was determined from cell lines and clinical samples by qRT-PCR. The effects of MALAT1 knockdown on cell proliferation, cell cycle, apoptosis, migration, and invasion were evaluated by in vitro and in vivo assays. The potential protein expression changes were investigated by Western-blotting. The methylation status of the CpG island in the MALAT1 promoter was explored by bisulfite sequencing, while the copy numbers in tumor tissues and blood samples were detected by a well-established AccuCopy(TM) method. RESULTS: MALAT1 was over-expressed in 46.3% of ESCC tissues, mostly in the high-stage tumor samples. Enhanced MALAT1 expression levels were positively correlated with clinical stages, primary tumor size, and lymph node metastasis. Inhibition of MALAT1 suppressed tumor proliferation in vitro and in vivo, as well as the migratory and invasive capacity. MALAT1 depletion also induced G2/M phase arrest and increased the percentage of apoptotic cells. Western-blotting results implicated that the ATM-CHK2 pathway which is associated with G2/M arrest was phosphorylated by MALAT1 knockdown. No effects of CpG island methylation status on MALAT1 expression were found, whereas amplification of MALAT1 was found in 22.2% of tumor tissues, which correlated significantly with its over-expression. However, neither association between tissue copy number amplification and germline copy number variation, nor correlation between germline copy number variation and ESCC risk were identified in the case-control study. CONCLUSIONS: Our data suggest that MALAT1 serves as an oncogene in ESCC, and it regulates ESCC growth by modifying the ATM-CHK2 pathway. Moreover, amplification of MALAT1 in tumor tissues may play an important role for its up-regulation, and it seems that the gene amplification in tumor tissues emerges during ESCC progression, but is not derived from germline origins.