Engrailed 2 serves as a master regulator of the super-enhancer in the TNC gene locus in non-small cell lung cancer.

Engrailed 2 (EN2) is a homeodomain-containing protein that is dysregulated in many types of cancer. However, the role of EN2 in non-small cell lung cancer (NSCLC) and the mechanism underlying its biological function are largely unclear. Here, we showed that EN2 played an oncogenic function in NSCLC and greatly enhanced the malignant phenotype of NSCLC cells. Meanwhile, EN2 was able to boost the expression of a well-studied oncogenic Tenascin-C (TNC) gene, which in turn activated the AKT signaling pathway. Interestingly, we found that EN2 directly bound to the super-enhancer (SE) region in the TNC locus. The histone marker H3K27ac was also enriched in the region, indicating the activation of the SE. Treatment of the cells with JQ1, an inhibitor of SE activity, abrogated the effect of EN2 on the expression of TNC and phosphorylation of AKT-Ser473. Collectively, our work unveils a novel mode of EN2 function, in which EN2 governs the SE in the TNC locus, consequently activating the oncogenic TNC-AKT axis in NSCLC.

Engrailed 2 triggers the activation of multiple phosphorylation-induced signaling pathways in both transcription-dependent and -independent manners.

Homeodomain (HD)-containing proteins are typically recognized as transcription factors. Engrailed 2 (EN2) is an HD-containing protein that is highly expressed in various types of cancers, however, the mechanism underlying the biological function of EN2 is not fully understood. Here, we report a transcription-independent function of EN2 in addition to its role as a transcription factor. EN2 expression leads to the activation of multiple signaling pathways mediated by phosphorylation cascades. A phosphoproteomic analysis revealed that the phosphorylation status of numerous protein sites was altered after EN2 is expressed. Notably, EN2 was shown to interact with a myriad of proteins implicated in phosphorylation signaling cascades, as determined by immunoprecipitation-mass spectrometry (IP-MS). We validated the interaction between EN2 and B55α, the regulatory subunit of the PP2A-B55α complex, and confirmed that the phosphatase activity of the complex was suppressed by EN2 binding. To target EN2-induced malignancy, two kinds of small molecules were utilized to inhibit the EN2-activated NF-κB and AKT signaling pathways. A clear synergistic effect was observed when the activation of the two pathways was simultaneously blocked. Collectively, the data show that EN2 functions in a transcription-independent manner in addition to its role as a transcription factor. This finding may have therapeutic implications in treating esophageal squamous cell carcinoma (ESCC).

KDM2A plays a dual role in regulating the expression of malignancy-related genes in esophageal squamous cell carcinoma.

KDM2A is a histone demethylase, which primarily catalyzes the demethylation of H3K36me2. Abnormal expression of KDM2A is observed in many types of cancers; however, the molecular events connected to KDM2A expression remain unclear. We report that KDM2A performs an oncogenic function in esophageal squamous cell carcinoma (ESCC) and is robustly expressed in ESCC cells. ShRNA-mediated knockdown of KDM2A resulted in a significant inhibition of the malignant phenotype of ESCC cell lines, whereas ectopic expression of KDM2A showed the opposite effect. We also analyzed the function of KDM2A using a CRISPR-CAS9 depletion system and subsequent rescue experiment, which also indicated a cancerous role of KDM2A. Interestingly, analysis of the gene expression network controlled by KDM2A using RNA-seq revealed an unexpected feature: KDM2A could induce expression of a set of well-documented oncogenic genes, including IL6 and LAT2, while simultaneously suppressing another set of oncogenes, including MAT2A and HMGCS1. Targeted inhibition of the upregulated oncogene in the KDM2A-depleted cells led to a synergistic suppressive effect on the malignant phenotype of ESCC cells. Our results revealed the dual role of KDM2A in ESCC cells, which may have therapeutic implications.

Engrailed-2 promotes a malignant phenotype of esophageal squamous cell carcinoma through upregulating the expression of pro-oncogenic genes.

BACKGROUND: A number of homeobox genes have been implicated in the development of various cancers. However, the role of engrailed 2 (EN2), a member of the homeobox gene superfamily, in esophageal squamous cell carcinoma (ESCC) remains unknown. METHODS: The expression of EN2 was examined using quantitative real-time PCR and immunohistochemistry. A stable cell line was established to express exogenous EN2 using a lentivirus system. The malignant phenotype was analyzed with proliferation, clonogenicity, wound-healing and invasion assays. The CRISPR/Cas9 system was adopted to deplete endogenous EN2. RNA profiling was performed using gene expression microarray. The ShRNA-mediated method was used to knock down the expression of SPARC. The structure-function relationship was determined using site-directed mutagenesis. RESULTS: EN2 is highly expressed in ESCC. The malignant phenotype of the ESCC cell line was amplified by an overexpression of EN2 but was attenuated by a disruption of EN2. RNA profiling analysis revealed that distinct sets of genes were modulated by the expression of EN2 in various ESCC cell lines and oncogenes were among these. EN2 greatly increased the expression of SPARC in Eca109. Site-directed mutagenesis revealed that the induction of SPARC was closely correlated with the protumor function of EN2. ShRNA-mediated knockdown of SPARC attenuated the malignant phenotype of EN2-infected cells. These data suggest that SPARC is crucial for mediating the protumor function of EN2. DISCUSSION: EN2 has an oncogenic function in ESCC that is mediated by upregulating the expression of pro-oncogenic genes downstream. EN2 may potentially act as a diagnostic marker or therapeutic target for ESCC treatment in the future.

Transcriptomic and open chromatin atlas of high-resolution anatomical regions in the rhesus macaque brain.

The rhesus macaque is a prime model animal in neuroscience. A comprehensive transcriptomic and open chromatin atlas of the rhesus macaque brain is key to a deeper understanding of the brain. Here we characterize the transcriptome of 416 brain samples from 52 regions of 8 rhesus macaque brains. We identify gene modules associated with specific brain regions like the cerebral cortex, pituitary, and thalamus. In addition, we discover 9703 novel intergenic transcripts, including 1701 coding transcripts and 2845 lncRNAs. Most of the novel transcripts are only expressed in specific brain regions or cortical regions of specific individuals. We further survey the open chromatin regions in the hippocampal CA1 and several cerebral cortical regions of the rhesus macaque brain using ATAC-seq, revealing CA1- and cortex-specific open chromatin regions. Our results add to the growing body of knowledge regarding the baseline transcriptomic and open chromatin profiles in the brain of the rhesus macaque.

SIX4 acts as a master regulator of oncogenes that promotes tumorigenesis in non-small-cell lung cancer cells.

A number of homeobox genes are implicated in the malignancy of various cancers. Here, we investigated the role of the homeobox gene SIX4 in non-small-cell lung cancer (NSCLC). The sine oculis homeobox (SIX4) gene was found to be highly expressed at both mRNA and protein levels in NSCLC tumor tissues as compared with matching normal counterparts. In this study, the SIX4 gene of two human NSCLC cell lines (A549 and PC9) was overexpressed or silenced using the lentiviral system. We evaluated the malignancy-associated phenotype of transfected cells, which demonstrated that exogenous expression of the SIX4 gene greatly enhanced the proliferation, migration, and invasion of NSCLC cells. The opposite was true in the SIX4-silenced cells. Transcriptomic profiling analysis revealed that the SIX4 gene modulated the expression of hundreds of downstream target genes in a cell context-dependent manner. Most notably, the SIX4 gene controls the expression of crucial genes with evidently oncogenic function. We conclude that SIX4 plays an oncogenic role and may be potentially utilized as a diagnostic and therapeutic marker for NSCLC.

Evidence for an oncogenic role of HOXC6 in human non-small cell lung cancer.

BACKGROUND: Identification of specific biomarkers is important for the diagnosis and treatment of non-small cell lung cancer (NSCLC). HOXC6 is a homeodomain-containing transcription factor that is highly expressed in several human cancers; however, its role in NSCLC remains unknown. METHODS: The expression and protein levels of HOXC6 were assessed in NSCLC tissue samples by Quantitative real-time PCR (qRT-PCR) and immunohistochemistry, respectively. HOXC6 was transfected into the NSCLC cell lines A549 and PC9, and used to investigate its effect on proliferation, migration, and invasion using CFSE, wound healing, and Matrigel invasion assays. Next-generation sequencing was also used to identify downstream targets of HOXC6 and to gain insights into the molecular mechanisms underlying its biological function. RESULTS: HOXC6 expression was significantly increased in 66.6% (20/30) of NSCLC tumor samples in comparison to normal controls. HOXC6 promoted proliferation, migration, and invasion of NSCLC cells in vitro. RNA-seq analysis demonstrated the upregulation of 310 and 112 genes in A549-HOXC6 and PC9-HOXC6 cells, respectively, and the downregulation of 665 and 385 genes in A549-HOXC6 and PC9-HOXC6 cells, respectively. HOXC6 was also found to regulate the expression of genes such as CEACAM6, SPARC, WNT6, CST1, MMP2, and KRT13, which have documented pro-tumorigenic functions. DISCUSSION: HOXC6 is highly expressed in NSCLC, and it may enhance lung cancer progression by regulating the expression of pro-tumorigenic genes involved in proliferation, migration, and invasion. Our study highlighted the oncogenic potential of HOXC6, and suggests that it may be a novel biomarker for the diagnosis and treatment of NSCLC.

HOXC6 promotes migration, invasion and proliferation of esophageal squamous cell carcinoma cells via modulating expression of genes involved in malignant phenotypes.

BACKGROUND: HOXC6 is a member of the HOX gene family. The elevated expression of this gene occurs in prostate and breast cancers. However, the role of HOXC6 in esophageal squamous cell carcinoma (ESCC) remains largely uninvestigated. METHODS: The expression of HOXC6 was examined by immunohistochemistry, quantitative real-time PCR and immunoblotting assays. The lentivirus-mediated expression of HOXC6 was verified at mRNA and protein levels. Wound healing and Matrigel assays were performed to assess the effect of HOXC6 on the migration and invasion of cancer cells. The growth curving, CCK8, and colony formation assays were utilized to access the proliferation capacities. RNA-seq was performed to evaluate the downstream targets of HOXC6. Bioinformatic tool was used to analyze the gene expression. RESULTS: HOXC6 was highly expressed in ESCC tissues. HOXC6 overexpression promoted the migration, invasion, and proliferation of both Eca109 and TE10 cells. There were 2,155 up-regulated and 759 down-regulated genes in Eca109-HOXC6 cells and 95 up-regulated and 47 down-regulated genes in TE10-HOXC6 cells compared with the results of control. Interestingly, there were only 20 common genes, including 17 up-regulated and three down-regulated genes with similar changes upon HOXC6 transfection in both cell lines. HOXC6 activated several crucial genes implicated in the malignant phenotype of cancer cells. DISCUSSION: HOXC6 is highly expressed in ESCC and promotes malignant phenotype of ESCC cells. HOXC6 can be used as a new therapeutic target of ESCC.

Magnetic nanoparticle-loaded electrospun polymeric nanofibers for tissue engineering.

Magnetic nanoparticles have been one of the most attractive nanomaterials for various biomedical applications including magnetic resonance imaging (MRI), diagnostic contrast enhancement, magnetic cell separation, and targeted drug delivery. Three-dimensional (3-D) fibrous scaffolds have broad application prospects in the biomedical field, such as drug delivery and tissue engineering. In this work, a novel three-dimensional composite membrane composed of the tri-block copolymer poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL, PCEC) and magnetic iron oxide nanoparticles (Fe3O4 NPs) were fabricated using electrospinning technology. The physico-chemical properties of the PCEC/Fe3O4 membranes were investigated by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC). Morphological observation using scanning electron microscopy (SEM) showed that the composite fibers containing 5% Fe3O4 nanoparticles had a diameter of 250nm. In vitro cell culture of NIH 3T3 cells on the PCEC/Fe3O4 membranes showed that the PCEC/Fe3O4 fibers might be a suitable scaffold for cell adhesion. Moreover, MTT analysis also demonstrated that the membranes possessed lower cytotoxicity. Therefore, this study revealed that the magnetic PCEC/Fe3O4 fibers might have great potential for using in skin tissue engineering.

[Interaction between microRNAs and OCT4].

Octamer-binding transcription factor 4 (OCT4) belongs to the POU-homeodomain family of transcription factors and binds to an octamer motif, ATGCAAAT. OCT4 is the key transcription factor that is involved in the maintenance of pluripotency and self-renewal in undifferentiated embryonic stem (ES) cells. OCT4 has been reported to be overexpressed in various cancers including lung, germ cell tumors, breast, cervix, prostate, gastric, liver, and ovarian cancer. MicroRNAs (miRNAs), small non-coding RNAs, modulate mRNA expression through base pairing between seed sequences in miRNA and complementary sequences of the target mRNA, thereby destabilizing mRNA and/or inhibiting protein synthesis. Several miRNAs have been demonstrated to regulate stemness factors such as OCT4, NANOG, SOX2 and KLF4 in cancer cells, thereby modulating the proliferation, apoptosis, differentiation, drug resistance and immunity of cancer cells.

[Advances of lentiviral vectors].

Lentiviral vectors are currently very effective tools in molecular and cell experiment. Lentiviral vector, a kind of retroviral vectors, has a number of unique advantages in target gene transferation, for example, the ability of transfection to the dividing or nondividing cells, its high efficiency of transfection and a capacity of large target gene fragments. This paper describes the sources of lentiviral vectors, molecular characteristics, research progress, etc.

[A method for introducing mutations into large vectors].

BACKGROUND AND OBJECTIVE: In vitro site-directed mutagenesis is a routine technique in molecular biology labs. However, although there are numbers of related methods available, most of these methods are not suitable for introducing mutations into large vectors. METHODS: In this report, we describe a method which is highly effective for this purpose. Our method is based on the other site-directed method we recently reported. The basic protocol of our method is as follows: (1) Synthesize a pair of vector primers based on the sequences around the region to be mutated, each containing a suitable type IIs endonuclease restriction site; meanwhile, synthesize a pair of short complementary oligonucleotides which forms a mutagenic fragment after annealing; (2) Synthesize a pair of bridge primers which can specifically bind to a site in the vector sequence, each containing a suitable type IIs endonuclease restriction site; (3) Perform PCR reactions using these Vector primers and Bridge primers; (4) Digest the PCR products with the corresponding type IIs restriction enzyme; (5) Ligate the digested fragment with the mutagenic fragment to make the desired mutant. RESULTS: Using this protocol, we have introduced mutations into a vector larger than 9 kb. The results shows that the mutation rates are more that 90%. CONCLUSIONS: Our method provides a useful tool for performing site-directed mutagenesis experiment in large vector.

[A highly efficient in vitro site-directed mutagenesis protocol for introducing multiple-site mutations into target genes].

BACKGROUND: The methods for introducing point mutations into target genes are important for dissecting the relationship of gene structure and function. Up to date, there are numbers of protocols available for the purpose. However, many of them are suited for introducing single site mutation into the target gene. For introducing multiple-site mutations simultaneously into the target genes, it is required to further improve the related methods. METHODS: In this report, we describe an improvement on the type IIs restriction enzyme-dependent site-directed mutagenesis method and the improved protocol is highly efficient for multiple-site mutagenesis. In our method, a pair of mutagenic primers are synthesized for each desired site and each mutagenic primer contains a selected type IIs restriction site. The DNA fragments between two neighboring sites are amplified with PCR. All amplified fragments are then digested by the selected Type IIs restriction enzyme. The expected mutant is eventually generated by ligation of these digested DNA fragments. RESULTS: The improved protocol is very easy and can be achieved in just one day. As a proof of principle, we have introduced multiple-site, i.e., 3-site or 4-site mutations into the fusion gene of nm23 and EGFP (enhanced green fluorecence protein). The mutagenic frequencies are almost reached 100%. CONCLUSIONS: Our protocol provides a useful tool for gene function research.

nm23-H1 gene driven by hTERT promoter induces inhibition of invasive phenotype and metastasis of lung cancer xenograft in mice.

BACKGROUND: Lung cancer is the leading cause of cancer death in both men and women worldwide. Tumor metastasis is an essential aspect of lung cancer progression and patient death. The nm23-H1 gene has been extensively investigated as a metastasis suppressor gene. Our previous studies have revealed: that a significant relationship exists between the low-level expression nm23-H1 in primary non-small cell lung cancer (NSCLC) with increased metastasis and a poor prognosis; that L9981-nm23-H1 cells (a nm23-H1 transfactant cell) exhibited lower cell proliferation rates, more G0/G1 phase growth, and an increase in apoptosis with a dramatic decrease in the tumor cells' ability to invade than L9981 cells did; and that L9981- nm23-H1 cells also demonstrated a significantly reduced lymph node and distant metastatic capacity in vivo than L9981 cells did in nude mice. METHODS: In this study, we construct a plasmid containing the nm23-H1 gene, which was driven by the human telomerase reverse transcriptase (hTERT) promoter. We evaluated the anti-invasion and anti-metastatic effects of pGL3-hTP-nm23 on L9981, a human large cell lung cancer cell line with nm23-H1 negative expression, by transwell assay in vitro and bioluminescence in nude mice models. The toxicity of pGL3-hTP-nm23 and its effects on tumor growth were evaluated in nude mice models after gene therapy. The cell cycles, apoptosis, and proliferation of the nm23-H1 transfactant were also detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) and flow cytometry (FCM). RESULTS: The results showed that the hTERT-promoter dramatically drives nm23-H1 gene expression, and induces inhibition of cell growth and migration in L9981-luc cells and MRC-5 cells in vitro. nm23-H1 also significantly inhibited the tumorigenesis and distant metastasis of L9981-luc cell in vivo. Moreover, no obvious side effect was detected in normal mouse tissues after intratumoral injection of the vector. CONCLUSION: The treatment of the nm23-H1 gene driven by hTERT promoter appears to be a promising approach for the gene therapy of nm23-H1 low-expressed tumors.

Potential application of non-small cell lung cancer-associated autoantibodies to early cancer diagnosis.

To identify a panel of tumor associated autoantibodies which can potentially be used as biomarkers for the early diagnosis of non-small cell lung cancer (NSCLC). Thirty-five unique and in-frame expressed phage proteins were isolated. Based on the gene expression profiling, four proteins were selected for further study. Both receiver operating characteristic curve analysis and leave-one-out method revealed that combined measurements of four antibodies produced have better predictive accuracies than any single marker alone. Leave-one-out validation also showed significant relevance with all stages of NSCLC patients. The panel of autoantibodies has a high potential for detecting early stage NSCLC.

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning.

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.

[Methylation profile difference in human high-metastatic large cell lung cancer cell line L9981 and low-metastatic large cell lung cancer line NL9980].

BACKGROUND AND OBJECTIVE: Invasion and metastasis is one of malignant phenotype of lung cancer and the important cause of the death of lung cancer patients. The aim of this study is to explore the methylation profile difference in different metastatic potential cell lines. METHODS: To compare the DNA methylation profile of two large cell lung cancer cell lines with different metastatic potential by MeIP chip hybridization, hypermethylated and hypomethylated genes of L9981 cell lines were analyzed online in NIH-DAVID, KEGG and MILANO webside. RESULTS: Compared with NL9980 cell line, 735 genes are hypermethylated in L9981, including 656 known genes and 79 unknown genes; 809 gene are hypermethylated in L9981, including 698 known genes and 111 unknown genes; the different genes are involved in cell process, signal transduction, cell communication, cell adhesion, cell motility, and angiogenesis. CONCLUSION: Hypermethylation of suppressor genes and negative regulator of signal transduction and hopomethylation of oncogene and cell adhesion molecules (CAMs) in L9981 may promote metastasis of tumor cells.

[Interference of homologous sequences on the SNP study of CYP2A13 gene].

BACKGROUND AND OBJECTIVE: It has been proven that cytochrome P450 enzyme 2A13 (CYP2A13) played an important role in the association between single nucleotide polymorphisms (SNP) and human diseases. Cytochrome P450 enzymes are a group of isoenzymes, whose sequence homology may interfere with the study for SNP. The aim of this study is to explore the interference on the SNP study of CYP2A13 caused by homologous sequences. METHODS: Taqman probe was applied to detect distribution of rs8192789 sites in 573 subjects, and BLAST method was used to analyze the amplified sequences. Partial sequences of CYP2A13 were emplified by PCR from 60 cases. The emplified sequences were TA cloned and sequenced. RESULTS: For rs8192789 loci in 573 cases, only 3 cases were TT, while the rest were CT heterozygotes, which was caused by homologous sequences. There are a large number of overlapping peaks in identical sequences of 60 cases, and the SNP of 101 amino acid site reported in the SNP database is not found. The cloned sequences are 247 bp, 235 bp fragments. CONCLUSION: The homologous sequences may interfere the study for SNP of CYP2A13, and some SNP may not exist.

[Association between GSTM1 genetic polymorphism and lung cancer risk by SYBR green I real-time PCR assay].

BACKGROUND AND OBJECTIVE: Glutathione S-transferase M1 (GSTM1) is an important phase II metabolic enzyme gene which involves metabolism of various carcinogens in human body. Many studies showed that GSTM1 genetic polymorphism was associated with lung cancer risk. The aim of this study is to investigate the relationship between GSTM1 genetic polymorphism and lung cancer risk among Han nationality population in Tianjin district. METHODS: GSTM1 genetic polymorphism was detected by melting curve analysis of SYBR green I real-time PCR assay. Two hundred and sixty-five histological confirmed lung cancer patients and 307 health controls were recruited in this case-control study and the relationship between GSTM1 genetic polymorphism and lung cancer riskwas investigated. RESULTS: (1) The frequency of the GSTMI(-) in lung cancer and control groups was 56.6% and 57.0% respectively, and no significant difference was found between the distribution of the GSTM1 (-) genotype in the two groups (chi2 = 0.831, P = 0.362). (2) When considered the GSTM1(+) genotype as reference, there was no overall statistically increased lung cancer risk for carriers with the GSTM1(-) genotype adjusted by age, gender and smoking status (OR = 0.840, 95% CI: 0.578-1.221, P = 0.362). (3) The frequency of the GSTM1(-) genotype for squamous cell carcinoma, adenocarcinoma, SCLC and other histological types was 65.8%, 48.5%, 47.8% and 52.2% respectively, compared with the control group, no statistically increased lung cancer risk was observed (P > 0.05). CONCLUSION: No evidence is found between GSTMI genetic polymorphism and lung cancer risk among Han nationality population in Tianjin district.