TCRP1 promotes radioresistance of oral squamous cell carcinoma cells via Akt signal pathway

Tongue cancer resistance-associated protein 1 (TCRP1) is a novel gene located on human chromosome 11q13.4 which has been reported as a candidate related to chemotherapeutic resistance to cisplatin. Results suggest that TCRP also contribute to radioresistance in oral squamous cell carcinoma (OSCC) cells. We previously established exogenous overexpression of TCRP1 cell line Tca8113/TCRP1 and TCRP1 knockdown cell line Tca8113/PYM-siRNA and paired control cell lines, which provides a cell model system to investigate the roles and mechanisms of TCRP1-mediated radioresponse in OSCC. In this study, we first compared the radiosensitivity of up/down-regulating expression of TCRP1 cell lines and paired control cell lines by a clonogenic survival assay, Hoechst 33258 staining, cell growth assay, and comet assay. The results indicated that TCRP1 played a significant role in mediating OSCC radioresistance through decreased cells apoptosis and increased cellular proliferation and long-term survival. The further study found that TCRP1 function by up-regulating Akt activity and levels and then elevating the level of NF-κB. In summary, these results provided strong evidence for the linkage between TCRP1 and radiation sensitivity and may provide theoretical base of TCRP1 as a potential molecular mark of estimating the response for irradiation in OSCC.     

Purification and biochemical characterization of a novel protein-tongue cancer chemotherapy resistance-associated protein1 (TCRP1)

Multidrug resistance is a major obstacle to successful treatment of oral squamous cell carcinoma (OSCC). Lately, we found a novel human gene named tongue cancer chemotherapy resistance-associated protein1 (TCRP1) in the tongue cancer multi-drug resistance cell line (Tca8113/PYM) established by us. In this study, we focus on recombinant expression, purification, and biochemical characterization of TCRP1. After molecular cloning and purification of the gene encoding the 24-kDa protein, a mouse polyclonal antibody against TCRP1 was prepared, and the specialty of the antibody was confirmed by Western blot. The cell proliferation was evaluated by MTS assay and DNA damage was determined by comet assay, the results indicated that this protein especially mediated the cell's resistance to cisplatin; it was associated with its role of providing protection against DNA damage. We also found that TCRP1 expression was increased in cisplatin-resistant carcinoma cell lines (Tca/PYM and A549/DDP), but not in cisplatin-sensitive MDR cell lines (MCF-7/5-Fu), compared with their parental counterparts by Western blot analysis. Immunofluorescence and immunohistochemical analysis showed TCRP1 is mainly expression in cytoplasmic, the Mann-Whitney U test exhibited that TCRP1 positive patients predicted the worst sensitive with cisplatin of OSCC patients. All these findings suggest that TCRP1 is a novel cisplatin-resistant protein which is mainly localized in the cytoplasm and can mediate cisplatin resistance against DNA damage; the expression level of TCRP1 in patients with OSCC may be useful as an indicator of therapeutic efficacy of the sensitivity to cisplatin.     

Identification of carbonic anhydrase 9 as a contributor to pingyangmycin-induced drug resistance in human tongue cancer cells

Drug resistance is the major obstacle to successful cancer treatment. To understand the mechanisms responsible for drug resistance in tongue cancer, Tca8113 cells derived from moderately differentiated human tongue squamous cell carcinoma were exposed to stepwise escalated concentrations of pingyangmycin (PYM) to develop the resistant cell line called Tca8113/PYM, which showed over 18.78-fold increased resistance to PYM as compared with Tca8113 cells, and cross-resistance to cisplatin, pirarubicin, paclitaxel, adriamycin, and mitomycin. We found that the resistance was not associated with multidrug resistance transporter 1 (p170, p-gp), multidrug resistance-associated protein 1 and breast cancer resistance protein overexpression, so we hypothesized that Tca8113/PYM cells must have some other resistance mechanism selected by PYM. To test this hypothesis, the global gene expression profiles between Tca8113 and Tca8113/PYM cells were compared by cDNA microarray. Eighty-nine genes and thirteen expressed sequence tags with differential expression levels between the two cell lines were identified. Some differential expression levels were validated with real-time PCR and western blot. Furthermore, the functional validation showed that both carbonic anhydrase (CA) inhibitor acetazolamide application and CA9 silencing with CA9 antisense oligonucleotides contribute to the medium pH increase of Tca8113/PYM cells and enhanced PYM chemosensitivity. Moreover, both acetazolamide and CA9 antisense oligonucleotides significantly increased PYM-induced caspase 3 activation in Tca8113/PYM cells. Thus, our study suggests that the resistance of Tca8113/PYM cells is probably associated with CA9 and other differential expression molecules, and that CA9 may be an important marker for prediction of PYM responsiveness in tongue cancer chemotherapy.     

Proteomic Analyses of Sirt1-Mediated Cisplatin Resistance in OSCC Cell Line

Oral squamous cell carcinoma (OSCC) accounts for about 90% of malignant oral lesions, and is recognized as the third most common cancer in developing nations and the sixth most common cancer worldwide. While chemotherapy remains the primary treatment for both resectable and advanced OSCC, most OSCC are naturally resistant to anticancer drugs, rendering new therapeutic avenues in dire need. Sirt1, a class III histone deacetylase, was linked to cisplatin resistance in several cancer types; however, the underlying mechanism is still unclear. Here, we demonstrated that overexpression of Sirt1 survived OSCC cell line Tca8113 under cisplatin treatment. Notably, BML-210, a chemical inhibitor of class III histone deacetylase, significantly abolished Sirt1-mediated cisplatin resistance in Tca8113 cells. Further, inactivation of endogenic Sirt1 by nicotinamide markedly increased chemo-sensitivity in cisplatin resistant sub-cell line Tca8113/CDDP. Proteomic strategy was applied to profile the differentially expressed proteins between pcDNA3.1-Sirt1- and mock vector-treated Tca8113 cells. Among 54 spots identified, 31 proteins were up-regulated and 23 proteins were down-regulated upon Sirt1 expression. Expression of four proteins with most significant alteration, including Annexin A4, Stathmin, SOD2 and thioredoxin, were validated by both RT-PCR and Western blot. Finally, we showed that Sirt1 could prevent cisplatin-induced ROS accumulation in Tca8113 cells. Our findings are considered as a significant step toward a better understanding of Sirt1-mediated cisplatin resistance.     

Microarray-Assisted Pathway Analysis Identifies MT1X & NFκB as Mediators of TCRP1-Associated Resistance to Cisplatin in Oral Squamous Cell Carcinoma

We recently reported that TCRP1, a novel multidrug-resistance associated human gene, can mediate cisplatin resistance in OSCC cells. However, the molecular mechanism underlying this role of TCRP1 remained to be elucidated. In this study, by using Human Toxicology and Drug Resistance Microarray, we identified 30 genes with significantly different expression levels between Tca/PYM and TCRP1 knockdown cell lines. Co-immunoprecipitation experiments and GST-pull down assays showed that metallothionein1X (MT1X) and Akt interact with TCRP1. siRNA-mediated knockdown of TCRP1 and MT1X was found to sensitize cells to cisplatin, leading to increased apoptosis and inhibition of cell proliferation. These functions of TCRP1 may be caused at least in part via activation of the PI3K/Akt/NF-κB signaling pathway. Taken together, our findings indicate that TCRP1 may be an important drug target for improvement of the treatment and survival of patients with oral squamous cell carcinoma.     

Fat-1 基因真核表达载体的构建及其对人口腔鳞癌细胞 脂肪酸含量的影响

目的:构建真核表达载体pcDNA3.1-Fat1,线性化稳定转染人口腔鳞癌细胞株Tca8113,检测其细胞内脂肪酸含量变化。方法:通过重叠延伸PCR方法人工合成利于真核表达的Fat-1基因,用基因重组技术构建真核表达载体pcDNA3.1-Fat-1,用脂质体转染真核细胞的方法转染人口腔鳞癌细胞株Tca8113,用气相色谱仪检测脂肪酸的变化情况。结果:测序及酶切鉴定成功合成真核偏好表达的Fat-1基因。与对照组相比,转染Fat-1基因的口腔癌细胞的n-3脂肪酸明显增多,n-6/n-3明显下降。结论:成功构建真核表达载体pcDNA3.1-Fat1,并对口腔鳞癌细胞内脂肪酸含量产生明显影响,为进一步研究Fat-1基因在口腔鳞癌中的生物学功能奠定了基础。     

Fat-1基因真核表达载体的构建及其对人口腔鳞癌细胞脂肪酸含量的影响

目的：构建真核表达载体pcDNA3．1-Fat1,线性化稳定转染人口腔鳞癌细胞株Tca8113,检测其细胞内脂肪酸含量变化。方法：通过重叠延伸PCR方法人工合成利于真核表达的Fat-1基因,用基因重组技术构建真核表达载体pcDNA3．1-Fat—1,用脂质体转染真核细胞的方法转染人1：／腔鳞癌细胞株Tca8113,用气相色谱仪检测脂肪酸的变化情况。结果：测序及酶切鉴定成功合成真核偏好表达的Fat-1基因。与对照组相比,转染Fat—1基N的口腔癌细胞的n-3脂肪酸明显增多,n-6／n-3明显下降。结论：成功构建真核表达载体pcDNA3．1-Fat1,并对口腔鳞癌细胞内脂肪酸含量产生明显影响,为进一步研究Fat-1基因在口腔鳞癌中的生物学功能奠定了基础。     

miR-21 downregulates the tumor suppressor P12 CDK2AP1 and stimulates cell proliferation and invasion

The present study was undertaken to investigate the regulation of P12(CDK2AP1) by miRNAs. A conserved target site for miR-21 within the CDK2AP1-3'-UTR at nt 349-370 was predicted by bioinformatics software and an inverse correlation of miR-21 and CDK2AP1 protein was observed. Highly specific amplification and quantification of miR-21 was achieved using real-time RT-PCR. Transfection of HaCaT cells with pre-miR-21 significantly suppressed a luciferase reporter including the CDK2AP1-3'-UTR, whereas transfection of Tca8113 with anti-miR-21 increased activity of this reporter. This was abolished when a construct mutated at the miR-21/nt 349-370 target site was used instead. Anti-miR-21-transfected Tca8113 cells showed an increase of CDK2AP1 protein and reduced proliferation and invasion. Resected primary tumors and tumor-free surgical margins of 18 patients with head and neck squamous cell carcinomas demonstrated an inverse correlation between miR-21 and P12(CDK2AP1). This study shows that P12(CDK2AP1) is downregulated by miR-21 and that miR-21 promotes proliferation and invasion in cultured cells.     

HuR stabilizes lnc-Sox5 mRNA to promote tongue carcinogenesis

Long noncoding RNAs (lncRNAs) have been recently regarded as systemic regulators in multiple biological processes including tumorigenesis. In this study, we report an ultra-highly expressed lncRNA, lnc-Sox5, in tongue tumor tissues. The results imply that lnc-Sox5 may play vital role in tongue carcinoma progression. We observed that the growth of Tca8113 cells was suppressed by lnc-Sox5 downregulation. Additionally, lnc-Sox5 knockdown simultaneously increased Tca8113 cell apoptosis, but the cell cycle was arrested. RNA immunoprecipitation suggested that HuR directly bound to and stabilized lnc-Sox5 RNA. Consistently, HuR knockdown reduced the level of lnc-Sox5 in Tca8113 cells. However, overexpression of HuR induced more lnc-Sox5 in Tca8113 cells. Both lnc-Sox5 knockdown and HuR knockdown suppressed Tca8113 cell tumorigenesis in xenograft models. These results suggest that lnc-Sox5, which was stabilized by HuR, could regulate carcinogenesis of tongue cancer and may serve as a predicted target for tongue carcinoma therapies.     

Bmi1 Essentially Mediates Podocalyxin-Enhanced Cisplatin Chemoresistance in Oral Tongue Squamous Cell Carcinoma

Oral tongue squamous cell carcinoma (OTSCC) is one of the most common head and neck cancers. Innate or acquired resistance to cisplatin, a standard chemotherapy agent for OTSCC, is common in patients with OTSCC. Understanding the molecular basis for cisplatin chemoresistance in OTSCC cells may serve as a basis for identification of novel therapeutic targets. Podocalyxin (PODXL) has been found critical for malignant progression in a variety of cancers. Bmi1 has recently been found to induce cell apoptosis and cisplatin chemosensitivity in OTSCC cells. In this study, we explored the interaction between PODXL and Bmi1 in OTSCC cells, and assessed its impact on OTSCC cell chemoresistance to cisplatin. PODXL and/or Bmi1 were stably overexpressed or knocked down in SCC-4 and Tca8113 human OTSCC cells. Overexpression of PODXL in both cell lines markedly elevated the expression level of Bmi1 and the half maximal inhibitory concentration (IC50) of cisplain and reduced cisplatin-induced cell apoptosis, which was abolished by knockdown of Bmi1 or a selective focal adhesion kinase (FAK) inhibitor. On the other hand, knockdown of PODXL significantly decreased the Bmi1 expression level and cisplatin IC50 and increased cisplatin-induced cell apoptosis, which was completely reversed by overexpression of Bmi1. While overexpression and knockdown of PODXL respectively increased and decreased the FAK activity, Bmi1 showed no significant effect on the FAK activity in OTSCC cells. In addition, overexpression of PODXL markedly elevated the stability of Bmi1 mRNA, which was abolished by a selective FAK inhibitor. In conclusion, this study provides the first evidence that PODXL up-regulates the expression level of Bmi1 in OTSCC cells by increasing the stability of Bmi1 mRNA through a FAK-dependent mechanism; this effect leads to enhanced cisplatin chemoresistance in OTSCC cells. This study adds new insights into the molecular mechanisms underlying OTSCC chemoresistance.     

miR-21 modulates chemosensitivity of tongue squamous cell carcinoma cells to cisplatin by targeting PDCD4

Chemoresistance is a challenge for clinician in management of tongue cancer. Therefore, it is necessary to explore alternative therapeutic methods to overcome drug resistance. miRNAs are endogenous ?22nt RNAs that play important regulatory roles by targeting mRNAs. miR-21, an essential oncogenic molecule, is associated with chemosensitivity of several human cancer cells to anticancer agents. In this study, we investigated the effects and molecular mechanisms of miR-21 in chemosensitivity of tongue squamous cell carcinoma cells (TSCC) to cisplatin. miR-21 expression was detected in tongue cancer tissue using RT-PCR and PDCD4 protein expression was measured using immunohistochemistry. miR-21 and(or) PDCD4 depleted cell lines were generated using miR-21 inhibitor and(or) siRNA. The viabilities of treated cells were analyzed using MTT assay. RT-PCR was used to detect miR-21 expression and immunoblotting was used to detect protein levels. Cell cycle and apoptosis were analyzed using propidium iodide (PI) staining and Annexin V/PI staining, respectively. The expression of miR-21 in tumorous tissue was significantly higher compared with adjacent normal tissue and loss of PDCD4 expression was observed in TSCCs. Transfection of miR-21 inhibitor induced sensitivity of TSCC cells (Tca8113 and CAL-27) to cisplatin. TSCC cells transfected with PDCD4 siRNA became more resistant to cisplatin therapy. We found an increase PDCD4 protein level following the transfection of miR-21 inhibitor using Western blot analysis. In addition, the enhanced growth-inhibitory effect by miR-21 inhibitor was weakened after the addition of PDCD4 siRNA. Suppression of miR-21 or PDCD4 could significantly promote or reduce cisplatin-induced apoptosis, respectively. Our data suggest that miR-21 could modulate chemosensitivity of TSCC cells to cisplatin by targeting PDCD4, and miR-21 may serve as a potential target for TSCC therapy.     

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (P < 0.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G₁ arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC. Xiumei Wang, Wenjing Sun, and Jing Bai contributed equally to this paper.     

Overexpression of BLCAP induces S phase arrest and apoptosis independent of p53 and NF-κB in human tongue carcinoma

Bladder cancer-associated protein gene (BLCAP) is a novel candidate tumor suppressor gene identified from the human bladder carcinoma. Our previous studies have shown that BLCAP overexpression could inhibit cell growth by inducing apoptosis in HeLa cells [Zuo Z, Zhao M, Liu J, Gao G, Wu X: Tumor Biol 27: 221–226, 2006]. Such evidence suggests the alterations in BLCAP may play an important role in tumorigenesis. To further study the biological function of the BLCAP gene, we constructed a recombinant retroviral vector encoding BLCAP cDNA. Overexpressed BLCAP, via stable infection of exogenous BLCAP, resulted in growth inhibition of the human tongue cancer cell line Tca8113 in vitro, accompanied by S phase cell cycle arrest and apoptosis. The growth inhibition was correlated with up-regulation of p21^WAF1/CIP1 expression and down-regulation of Bcl-XL and Bcl-2 expressions. However, p53 expression and NF-κB activity remained unchanged post infection. Furthermore, no changes in p53 phosphorylation at Ser46 and nuclear localization, which are critical to p53 function, were observed in BLCAP-overexpressed cells. Taken together, BLCAP may play a role not only in regulating cell proliferation but also in coordinating apoptosis and cell cycle via a novel way independent of p53 and NF-κB. Jun Yao and Li Duan contributed equally to this work.     

CXCR4 promotes oral squamous cell carcinoma migration and invasion through inducing expression of MMP-9 and MMP-13 via the ERK signaling pathway

The increased migration and invasion of oral squamous cell carcinoma cells are key events in the development of metastasis to the lymph nodes and distant organs. Although the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1α, have been found to play an important role in tumor invasion, its precise role and potential underlying mechanisms remain largely unknown. In this study, we showed that knockdown of CXCR4 significantly decreased Tca8113 cells migration and invasion, accompanied with the reduction of MMP-9 and MMP-13 expression. Inhibition of ligand binding to CXCR4 by a specific antagonist TN14003, also led to reduced cancer cell migration and invasion. Because the degradation of the extracellular matrix and the basement membrane by proteases, such as matrix metalloproteinases (MMP) is critical for migration and invasion of cancer cells, we investigated the expression of several MMPs and found that the expression of functional MMP-9 and MMP-13 was selectively decreased in CXCR4 knockdown cells. More importantly, decreased cell migration and invasion of CXCR4 knockdown cells were completely rescued by exogenous expression of MMP-9 or MMP-13, indicating that the two MMPs are downstream targets of CXCR4-mediated signaling. Furthermore, we found the level of phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased in CXCR4-silenced cells, suggesting that ERK may be a potential mediator of CXCR4-regulated MMP-9 and MMP-13 expression in Tca8113 cells. Taken together, our results strongly suggest the underlying mechanism of CXCR4 promoting Tca8113 migration and invasion by regulating MMP-9 and MMP-13 expression perhaps via activation of the ERK signaling pathway.     

Long noncoding RNA LINC00961 inhibited cell proliferation and invasion through regulating the Wnt/β-catenin signaling pathway in tongue squamous cell carcinoma

Tongue squamous cell carcinoma (TSCC) is the most frequent style of oral squamous cell carcinoma. However, the molecular mechanisms and function of LINC00961 in the TSCC progression remain unknown. In this study, we proved that LINC00961 expression was downregulated in TSCC cells (Tca8113, SCC1, SCC-4, and SCC-15) compared with normal tissue. In addition, we showed that LINC00961 expression was downregulated in TSCC samples compared with matched normal tissues. Moreover, ectopic expression of LINC00961 decreased TSCC cell growth and invasion and suppressed epithelial-mesenchymal transition in TSCC cell. Furthermore, we indicated that overexpression of LINC00961 decreased β-catenin expression. Knockdown of LINC00961 promoted cell proliferation and invasion partly via promoting the Wnt/β-catenin signaling pathway. These results suggested that LINC00961 was downregulated in TSCC tissues and acted as a tumor suppressor gene in the development of TSCC.     

Growth suppression induced by Notch1 activation involves Wnt-beta-catenin down-regulation in human tongue carcinoma cells

BACKGROUND INFORMATION: Involvement of Notch1 signalling in several cancers is well known, but its role in human tongue squamous cell carcinoma, one of the most common carcinomas of the human oral cavity, remains poorly characterized. RESULTS: Our studies demonstrated that constitutively over-expressed active Notch1, via stable transfection of exogenous ICN (intracellular fragment of Notch), resulted in growth suppression of the human tongue cancer cell line Tca8113 in vitro and in vivo, accompanied by G(0)-G(1) cell cycle arrest and apoptosis. Moreover, down-regulation of beta-catenin protein expression was observed in Tca8113 cells stably expressing active Notch1. Activated Notch1 also led to dramatic increase in p21(WAF1/CIP1) and p53 expression with decreases in Skp2 (S-phase kinase-associated protein 2) and Bcl-2 (B-cell lymphocytic-leukaemia proto-oncogene 2) expression, which may participate in the induction of apoptosis and cell cycle arrest. CONCLUSIONS: Since the effects of the Notch1 pathway are cell-type specific and context-dependent in cell types where Notch1 has an anti-proliferative effect, down-regulation of Wnt/beta-catenin signalling may be one of the mechanisms which induces apoptosis and cell cycle arrest.     

microRNA-21对人舌鳞癌细胞的增殖和凋亡的影响

目的:探讨micro RNA-21(mi R-21)对人舌鳞癌细胞增殖和凋亡的影响。方法:选取8例舌鳞癌组织和4例癌旁组织为研究材料,采用实时荧光定量聚合酶链式反应(q RT-PCR)法对舌鳞癌及癌旁组织中的mi R-21相对表达量进行检测,利用人工合成的mi R-21mimic对人舌鳞癌Tca8113细胞进行瞬时转染,采用q RT-PCR法对Tca8113细胞中mi R-21相对表达量进行检测,采用四唑盐比色法(MTT)法对Tca8113细胞增殖情况进行检测,采用流式细胞术对Tca8113细胞周期与凋亡情况进行检测。结果:舌鳞癌组织中mi R-21的相对表达量(3.502±0.674),高于癌旁组织(0.998±0.192),差异有统计学意义(P0.05)。mi R-21mimic导致了Tca8113细胞中的mi R-21相对表达量上调(6.864±1.324),明显高于对照scramble组[(0.997±0.187),P0.05],对Tca8113细胞的增殖发挥了促进作用(P0.05)。经mi R-21mimic转染之后,Tca8113细胞进入S期的细胞出现了明显的增加[(27.4±5.1)%vs(48.6±8.7)%,P0.05],处于G1期的细胞出现了显著的减少[(56.3±9.6)%vs(36.2±7.2)%,P0.05],细胞凋亡数量出现了显著减少[(9.4±2.3)%vs(18.6±3.9)%,P0.05]。结论:mi R-21在舌鳞癌组织中高表达,过表达mi R-21有效促进了Tca8113细胞的增殖,抑制细胞的凋亡,mi R-21在舌鳞癌诊断和治疗可能具有一定的新型靶点价值。     

Genes expressed in sugarcane maturing internodal tissue

To explore gene expression during sugarcane culm maturation, we performed a partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases, while 111 matched uncharacterised plant expressed sequence tags (ESTs) only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. This research has provided a framework for functional gene analysis in sugarcane sucrose-accumulating tissues.     

应用大规模测序技术和生物信息学研究造血干/祖细胞的基因表达及新基因的识别和克隆

从新生儿脐血和成人骨髓中分选出造血干／祖细胞（ＨＳＣ／ＨＰＣ）,构建成ｃＤＮＡ文库,对其进行大规模表达序列标签（ＥＳＴ）测序,通过生物信息学等手段分析基因表达谱,并进行新基因的全长ｃＤＮＡ克隆。在所测的１０５１２条可分析ＥＳＴ序列中,有９８６６条来自脐血ＣＤ３４＋细胞,其中４６９７条（４７６％）为已知基因,２６０３条（２６４％）为已知ＥＳＴ,１４１５条（１４３％）代表未知ＥＳＴ。在已知基因中,８２％基因与造血相关,２２７％涉及细胞代谢、结构和迁移,１３０％与细胞分裂和防御相关,２６２％与ＲＮＡ、蛋白质的合成相关,１０６％和细胞信号传递有关。对一些已知和未知的ＥＳＴ,综合测序、生物信息学等方法,进行全长克隆,已获得２３个新基因的全长ｃＤＮＡ。