Xingjun Jiang

Over-expression of BCAT1, a c-Myc target gene, induces cell proliferation, migration and invasion in nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but its molecular mechanisms of pathogenesis are poorly understood. Our previous work has demonstrated that BCAT1 mRNA is over expressed in NPC and knocking down its expression in 5-8F NPC cell line can potently inhibit cell cycle progression and cell proliferation. However, the mechanism of BCAT1 up-regulation and its functional role in NPC development remain to be elucidated yet.MethodsImmunohistochemistry (IHC) method was utilized to detect the expression of BCAT1 protein in NPC at different pathological stages. The roles of gene mutation, DNA amplification and transcription factor c-Myc in regulating BCAT1 expression were analyzed using PCR-sequencing, quantitative polymerase chain reaction (qPCR), IHC, ChIP and luciferase reporter system, respectively. The functions of BCAT1 in colony formation, cell migration and invasion properties were evaluated by RNA interference (RNAi).ResultsThe positive rates of BCAT1 protein expression in normal epithelia, low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues and NPC tissues were 23.6% (17/72), 75% (18/24 ), 88.9% (8/9) and 88.8% (71/80), respectively. Only one SNP site in exon1 was detected, and 42.4% (12/28) of the NPC tissues displayed the amplification of microsatellite loci in BCAT1. C-Myc could directly bind to the c-Myc binding site in promoter region of BCAT1 and up-regulate its expression. The mRNA and protein of c-Myc and BCAT1 were co-expressed in 53.6% (15/28) and 59.1% (13/22) of NPC tissues, respectively, and BCAT1 mRNA expression was also down-regulated in c-Myc knockdown cell lines. In addition, BCAT1 knockdown cells demonstrated reduced proliferation and decreased cell migration and invasion abilities.ConclusionsOur study indicates that gene amplification and c-Myc up-regulation are responsible for BCAT1 overexpression in primary NPC, and overexpression of BCAT1 induces cell proliferation, migration and invasion. The results suggest that BCAT1 may be a novel molecular target for the diagnosis and treatment of NPC.

Establishment and Applications of Epstein‐Barr Virus‐Based Episomal Vectors in Human Embryonic Stem Cells

Human embryonic stem (hES) cells are capable of unlimited cell proliferation yet maintain the potential to differentiate into many cell types. Here we reported an Epstein‐Barr virus (EBV)‐based vector system used to improve transfection efficiency in hES cells. Plasmids containing oriP, the latent replication origin of EBV, can be propagated stably as episomal DNA in human cells that express the EBV nuclear antigen 1 (EBNA1), which binds to oriP and functions as the trans‐acting replication initiator. It was reported that the EBV replicon could harbor a DNA fragment of up to 330 kilobase pairs. Plasmids containing an enhanced green fluorescent protein (EGFP)/puromycin resistance gene cassette along with or without oriP were used to transfect hES cells that stably express EBNA1. The presence of oriP moderately increased the transient transfection efficiency and more importantly it elevated the stable transfection efficiency by approximately 1,000‐fold as compared with oriP‐minus plasmids. The oriP plasmid as episomal DNA and green fluorescent protein expression in hES cells was maintained for months in the presence of drug selection and gradually lost (2%–4% per cell doubling) in the absence of selection. The presence of EBNA1 did not interfere with the hES cell properties or differentiation we tested and could maintain stable EGFP expression during differentiation. In addition to transgene expression, the EBV vector system could effectively enhance the RNA interference efficiency in hES cells. Thus, the EBV vector system that allows a large DNA insert and sustained expression of transgene or small hairpin RNA will enhance basic and translational research using hES cells.

Underlying mechanisms for LTF inactivation and its functional analysis in nasopharyngeal carcinoma cell lines

The lactoferrin (LTF) gene, located at 3p21.3, behaves like a tumor suppressor gene in diverse tumors. To elucidate the exact role of LTF in NPC, we first detected its expression level in seven NPC cell lines by semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). The results showed the mRNA level of LTF was nearly undetectable in all the seven NPC cell lines, while it could be detected in chronic nasopharyngitis tissues. Subsequently, we used methylation‐specific PCR (MSP), microsatellite assay, PCR‐single‐strand conformation polymorphism (PCR‐SSCP) and sequencing methods to examine the promoter methylation, loss of heterozygosity (LOH) and gene mutation of LTF in NPC cell lines respectively. Consequently, we found that 100% (7 of 7) of NPC cell lines were methylated in LTF promoter, only one cell line (14%, 1 of 7) had LOH and gene mutation of LTF, respectively, while LTF exhibited re‐expression in all cell lines after 5‐aza‐dC treatment, indicating promoter methylation should be the key mechanism causing LTF downregulation in NPC cell lines. Furthermore, patched methylation assay confirmed that promoter methylation could down‐regulate LTF gene expression in NPC cells. Finally, we investigated the function of LTF in NPC cell lines by gene transfection. Restoration of LTF expression in NPC cells resulted in blockage of cell cycle progression, significant inhibition of cell growth and a reduced colony‐formation capacity in vitro and obviously weaker tumor formation potential in vivo. In conclusion, our data indicate LTF may participate in NPC carcinogenesis as a negative effector, that is, a tumor suppressor gene. J. Cell. Biochem. 112: 1832–1843, 2011.

Transplantation of hUC-MSCs seeded collagen scaffolds reduces scar formation and promotes functional recovery in canines with chronic spinal cord injury.

Spinal cord injury (SCI) can lead to locomotor deficits, and the repair of chronic SCI is considered one of the most challenging clinical problems. Although extensive studies have evaluated treatments for acute SCI in small animals, comparatively fewer studies have been conducted on large-animal SCI in the chronic phase, which is more clinically relevant. Here, we used a collagen-based biomaterial, named the NeuroRegen scaffold, loaded with human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in a canine chronic SCI model. To generate chronic SCI, the T8 spinal cord segment was removed by complete transection of the spinal cord. Two months later, glial scar tissue was removed and a NeuroRegen scaffold was transplanted into the lesion area. Functionalized NeuroRegen scaffold implantation promoted both locomotor recovery and endogenous neurogenesis in the lesion area. Moreover, some newly generated neurons successfully matured into 5-HT-positive neurons at 1 year post-injury. In addition, many regenerated axon fibers in the lesion area exhibited remyelination and synapse formation at 1 year post-injury in the functionalized NeuroRegen scaffold group. In conclusion, the NeuroRegen scaffold functionalized with hUC-MSCs is a promising potential therapeutic approach to chronic SCI that promotes neuronal regeneration, reduces glial scar formation, and ultimately improves locomotor recovery.

Analysis of Serum microRNA Profile by Solexa Sequencing in Women With Endometriosis

Objective(s): The potential roles of serum microRNAs (miRNAs), as biomarkers, in noninvasive diagnosis of endometriosis have been reported by microarray analysis. However, microarray analysis cannot perform well in outcome accuracy and repeatability and is not suitable to be used for exploring new targets. Here, Solexa sequencing, a wide and precise method, was adopted to further analyze the serum miRNAs profile in endometriosis, which may offer more evidence to apply serum miRNAs as biomarkers in diagnosis of endometriosis. Materials and Methods: Serum samples were collected from 30 patients with minimal–mild endometriosis and 20 women without endometriosis as control. Expression of serum miRNAs was measured by Solexa sequencing and validated by quantitative real-time polymerase chain reaction (qPCR). Results: Solexa sequencing showed 93.63% clean readouts for all small RNAs in the serum of patients with endometriosis and controls. A total of 108 miRNAs were found to be differentially expressed in the serum of patients with endometriosis by deep sequencing, compared to controls. Among them, 98 miRNAs were significantly downregulated, while 10 miRNAs were significantly upregulated. Only 21 of 98 significantly downregulated miRNAs, and none of significantly upregulated miRNAs were reported in published literatures, which may be due to the differences in samples and analytical methods. The Solexa sequencing results were consequently validated by qPCR in additional samples. Some miRNAs were identified to be promising diagnostic markers of endometriosis. The functional annotation of target genes revealed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that a majority of differential miRNAs might be involved in endometriosis. Conclusion: Circulating miRNAs may be useful as detection biomarkers for the early diagnosis of minimal–mild endometriosis.

CD109 is identified as a potential nasopharyngeal carcinoma biomarker using aptamer selected by cell-SELEX

Nasopharyngeal carcinoma (NPC) is one of the most prevailing cancers in southern China and southern Asia. Because of the nonspecific symptoms and lack of effective biomarker, most patients are diagnosed at advanced stages, resulting in poor 5-year survival rate. To identify a novel NPC biomarker facilitating early detection and effective therapy of NPC, a two-step strategy consisting of cancer cell-Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) procedure and aptamer-based purification approach was developed. Using cell-SELEX procedure, four aptamers (S3, S5, S12 and S27) differentiating the molecular differences between NPC cells and NP cells were successfully screened. Then, using aptamer-based protein purification, membrane protein CD109 was identified as the target of aptamer S3. CD109 protein was further identified to be over-expressed in NPC cell lines and clinic tissues, but not or low in NP cell line and clinic NP tissues, detected by western blot and immunohistochemistry experiments. Our study demonstrated that CD109 identified by cell-SELEX and aptamer-based purification strategy might be used as a potential NPC biomarker for early diagnosis and targeted therapy.

Promoter hypermethylation along with LOH, but not mutation, contributes to inactivation of DLC‐1 in nasopharyngeal carcinoma

Previous studies have shown that promoter hypermethylation plays a key role in DLC‐1 inactivation in nasopharyngeal carcinoma (NPC). However, DLC‐1 mutation in NPC has not been reported, and there remain some discrepancies in methods and results between different groups. Here, we examined the mRNA and protein expression of DLC‐1 in chronic nasopharyngitis (CN) and NPC tissues by reverse transcription‐polymerase chain reaction/qPCR and immunohistochemistry, respectively. DLC‐1 mRNA was undetectable in all the seven widely used NPC cell lines and absent or significantly down‐regulated in 70% of NPC tissues. DLC‐1 protein level was reduced in 74.3% of NPCs when compared with CN tissues, and significantly lower in NPC samples at advanced clinical stages than that at early stages. Then, we purified the same batch of specimens by microdissection and analyzed the possible mechanisms of DLC‐1 downregulation with mutation and allelic loss analysis, methylation‐specific PCR and bisulfite genomic sequencing. Only one mutation was detected at codon 693 of exon 8 in 3.3% of NPCs and five single nucleotide polymorphisms (SNPs) were identified. Loss of DLC‐1 was detected in 23.3% of NPC tissues. The 100% of NPC cell lines, 80% of primary NPC and 22.2% of CN tissues showed methylation in DLC‐1 promoter, while DLC‐1 expression was recovered in seven NPC cell lines after 5‐aza‐dC treatment. Patched methylation assay confirmed that promoter methylation could repress DLC‐1 expression. This report demonstrates that DLC‐1 is negatively associated with NPC carcinogenesis, and promoter hypermethylation along with loss of heterozygosity, but not mutation, contributes to inactivation of DLC‐1 in NPC.

DLC-1, a candidate tumor suppressor gene, inhibits the proliferation, migration and tumorigenicity of human nasopharyngeal carcinoma cells

In our previous study we demonstrated the downregulation or loss of deleted in liver cancer‑1 (DLC-1) gene expression in nasopharyngeal carcinoma (NPC). In this study, we report the effects of the DLC-1 gene on NPC cells and its mechanisms of action. DLC-1 expression was restored in the 5-8F NPC cell line, which lacks DLC-1 expression, and the biological characteristics of 5-8F-DLC‑1 cells were analyzed by MTT assay, colony formation assay, flow cytometry (FCM), tumorigenesis analysis in nude mice, as well as invasion and migration assay. Differentially expressed genes in response to DLC-1 expression were screened using microarray analysis and identified by RT-PCR. The re-expression of DLC-1 in the NPC cells attenuated the proliferation and colony formation ability of the cells in vitro, blocked NPC cells at the G0/G1 phase, reduced tumorigenicity potential in vivo, inhibited the invasion and migration ability of NPC cells and resulted in the reorganization of the actin cytoskeleton. DLC-1 altered the gene expression profile in 5-8F cells. Some tumor suppressor genes (TSGs) were upregulated and some oncogenes were downregulated. These results demonstrate that DLC-1 gene can partially reverse the malignant phenotype of NPC cells by changing the tumor-related gene expression profile, and may be a candidate tumor suppressor gene and a promising diagnostic and therapeutic target in NPC.

Identification of ABCG2+ cells in nasopharyngeal carcinoma cells

Tumor stem cells are a small subset of tumor cells with the ability of self-renewal and differentiation and are regarded as a cause of tumor growth and recurrence. Previously we have shown that stem-like label-retaining cells (LRCs) can be detected in nasopharynx, tongue, esophagus and xenograft tumors formed by nasopharyngeal carcinoma (NPC) cell lines (5–8F, 6–10B and TMNE). The present study aimed to identify ABCG2+ cells in 5–8F NPC cells and compare their tumorigenic potential with ABCG2− cells, expecting that we can obtain insight into the mechanism of the differential phenotypes of ABCG2+ and ABCG2− cells. By using magnetic cell sorting (MACS) method, we isolated ABCG2+ cells and ABCG2− cells from 5–8F cells. Among these two subpopulations and unsorted 5–8F cells, the rate of ABCG2+ cells at G1 phase was highest, while the rate of ABCG2− cells at S phase was highest, indicating that ABCG2+ cells were mostly quiescent. However, ABCG2+ cells showed lower cloning efficiency and tumorigenicity than ABCG2− cells. We also used Affymetrix U133 plus 2.0 human whole genome expression chip to identify the gene expression profile of ABCG2+ and ABCG2− cells and found that both subpopulations expressed some stem cell associated genes, e.g., PSCA, ABCG2 and ALPI were expressed in ABCG2+ cells, and K19, integrin α6, integrin β4, CD44 and K14 were expressed in ABCG2− cells, suggesting there were stem cells in both ABCG2+ and ABCG2− cells. Our data demonstrated that there exist ABCG2+ cells in NPC cells, but ABCG2 alone is not sufficient for isolating cancer stem cells in 5–8F NPC cells.

HESRG: a novel biomarker for intracranial germinoma and embryonal carcinoma

The novel stem cell-related gene, HESRG, was first identified by our group, and its expression pattern in human tumors remains unknown. In this study, we used RT-PCR to systematically investigate the expression of HESRG in various types of intracranial tumors and found that HESRG was expressed only in germinoma and embryonal carcinoma, but hardly at all in other types of brain tumors. Real-time PCR results further confirmed this expression pattern. Subsequently, we tested 134 intracranial non-germ cell tumors and 64 intracranial germ cell tumors by immunohistochemistry. Our results showed that HESRG was expressed strongly and diffusively in the nuclei of tumor cells in intracranial germinoma and embryonal carcinoma as well as in human embryonic stem cells. No positive staining signal was observed in any other type of intracranial tumors. In germinomas, 25 of 31 showed intensive (3+) expression, four cases showed moderate (2+) immunostaining and the remaining 2 cases showed weak (1+) immunostaining. In embryonal carcinoma, 6 of 9 showed intensive (3+) immunostaining and 3 of 9 showed moderate (2+) immunostaining. These results suggest that HESRG is a novel, sensitive and specific biomarker for intracranial germinoma and embryonal carcinoma.