Fengjie Guo

Autoantibodies as potential biomarkers for nasopharyngeal carcinoma

Autoantibody signatures, as new biomarkers, may improve the early detection of nasopharyngeal carcinoma (NPC). We constructed a T7 phage cDNA library from mixed NPC tissues, and we isolated 31 tumor‐associated proteins using biopan enrichment techniques with sera from NPC patients and from healthy population. DNA sequence analysis showed that among 31 phage‐displayed proteins, 22 have sequence identity with known or putative tumor‐associated proteins. The results of immunochemical reactivity of patients sera with phage‐expressed proteins showed enrichment in the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in the sera of patients but not in the sera of healthy donors. The autoantibody against phage‐expressed protein MAGE, HSP70, Fibronectin, and CD44 measured by ELISA had greater predictive value than that against EBNA‐1, respectively. The antibody levels against MAGE in sera positively correlated with the clinical stages of NPC, and the antibody levels against other three proteins partly correlated with the clinical stages of NPC. Our studies suggested that the autoantibodies against tumor‐associated antigens in the sera of NPC patients could be used as a screening test for NPC. Studies of the corresponding proteins may have significances in tumor biology, novel drug development, and immunotherapy.

Stanniocalcin1 (STC1) Inhibits Cell Proliferation and Invasion of Cervical Cancer Cells

STC1 is a glycoprotein hormone involved in calcium/phosphate (Pi) homeostasis. There is mounting evidence that STC1 is tightly associated with the development of cancer. But the function of STC1 in cancer is not fully understood. Here, we found that STC1 is down-regulated in Clinical tissues of cervical cancer compared to the adjacent normal cervical tissues (15 cases). Subsequently, the expression of STC1 was knocked down by RNA interference in cervical cancer CaSki cells and the low expression promoted cell growth, migration and invasion. We also found that STC1 overexpression inhibited cell proliferation and invasion of cervical cancer cells. Moreover, STC1 overexpression sensitized CaSki cells to drugs. Further, we showed that NF-κB p65 protein directly bound to STC1 promoter and activated the expression of STC1 in cervical cancer cells. Thus, these results provided evidence that STC1 inhibited cell proliferation and invasion through NF-κB p65 activation in cervical cancer.

Identification of genes associated with tumor development in CaSki cells in the cosmic space

It is important to understand the mechanisms of tumor development for curing cervical cancer. However, the molecular basis determining the different characteristics of tumor remains unclear. Space environment as a special study model can expand the study field of tumor development. To approach this, after human cervical carcinoma CaSki cells were flown on “Shen Zhou IV” space shuttle mission, the cell morphology and proliferation was investigated after flying to ground. We found that the growth of 48A9 CaSki cell (flight group) became slow compared with ground groups. Observation of cells by light microscopy revealed differences in cell morphology between ground controls and flight groups, and the flight group exhibited morphologic differences, characterized by rounder, smoother, decreased, smaller and low-adhension cells. Transmission electron microscope images showed the structure of the ultrastructural characteristics of 48A9 CaSki cells were clearly distinct from those of the ground CaSki cells in aspects of mitochondrion, cytoplasm, nucleus and ribosomes. MTT and soft agar assay showed that 48A9 CaSki cells grew slowly compared to ground control. Furthermore, suppression subtractive hybridization combining with reverse Northern blot was used to identify differently expression genes between flight and ground groups. These differentially expressed genes included cytoskeleton, cell differentiation, cell apoptosis, signal transduction, DNA repair, protein synthesis, substance metabolism, and antigen presentation. The identification of differently expressed genes which is likely to increase our understanding of the molecular processes underlying tumor development will provide new insight into tumor development mechanisms, and may facilitate the development of new anticancer strategies.

Gammaaminobutyric Acid A Receptor Alpha 3 Subunit is Overexpressed in Lung Cancer

The identification of tumor-associated antigens, which are specifically expressed in cancer tissues, is very important for immunotherapy of lung cancer. We have combined the in silico screening and experimental verifying to identify genes that are differently expressed in cancers compared with their corresponding normal tissues. Using these methods, we have identified that GABRA3 gene was overexpressed in lung cancer and rarely expressed in other cancers. Furthermore, GABRA3 protein expression was significantly higher in the lower grade of lung cancer. It may compose functional GABA-gated channel with other subunits. This study demonstrated GABRA3 could be a potential biomarker for diagnosis of lung cancer, and GABAA receptors may play an important role in cancer differentiation.

BMI-1 autoantibody in serum as a new potential biomarker of nasopharyngeal carcinoma.

BMI-1 is overexpressed in a variety of cancers, which can activate the immune system to produce antibodies in tumor tissues. In this study, we isolated phage expressing BMI-1 protein by screening of a mixture of nasopharyngeal carcinoma (NPC) cDNA T7 phage library and found that the antibody against BMI-1 was elevated in the sera from NPC patients. BMI-1 mRNA was over-expressed at different levels in seven NPC cell lines compared with normal nasopharyngeal epithelial cell line NP69. Histochemistry showed that patient sera were more reactive with BMI-1 than normal sera. Antibody affinity assay using sera from 40 NPC patients and 54 controls showed that BMI-1 antibody was significantly greater in patient sera than in normal controls (patient 0.791 ± 0.025 and normal 0.488 ± 0.042; P < 0.001) and the BMI-1 autoantibody be significantly related with the progress of NPC (Benign versus LNPC P=0.001; LNPC versus MNPC P=0.047). Analysis of the results with logistic regression and receiver operating characteristics (ROC) curves showed that BMI-1 antibody was a modest marker for NPC (sensitivity 0.74 and specificity 0.73; AUC = 0.8044). The showed that BMI-1 antibody as a potential marker of NPC may be rational, and could have diagnostic and prognostic value.

Inhibition of ADP-ribosylation factor-like 6 interacting protein 1 suppresses proliferation and reduces tumor cell invasion in CaSki human cervical cancer cells

ADP-ribosylation factor-like 6 interacting protein 1 (ARL6IP1) is an apoptotic regulator. To investigate the role of ARL6IP1 in human cervical cancer progression, we designed and used short hairpin RNA (shRNA) to inhibit ARL6IP1 expression in CaSki cells and validated its effect on cell proliferation and invasion. Changes in gene expression were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) or western blot. Down-regulation of ARL6IP1 expression by infection with ARL6IP1-specific RNAi-expressing vector inhibited CaSki cell proliferation and colony formation. In addition, down-regulation of ARL6IP1 expression arrested CaSki cell cycling at the G0/G1 phase and mitigated CaSki cell migration, determined by wound healing assays. ARL6IP1 was involved in cervical cancer cell growth, cell cycle progression, and invasion through regulation of gene expression, such as Caspase-3, Caspase-9, p53, TAp63, NF-κB, MAPK, Bcl-2, and Bcl-xL, suggesting that ARL6IP1 could have important implications in cervical cancer biology. Our findings illustrate the biological significance of ARL6IP1 in cervical cancer progression, and provide novel evidence that ARL6IP1 may serve as a therapeutic target in the prevention of human cervical cancer.

Identification of HTA as a novel-specific marker for human hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common malignancy in the world, especially in China. Early diagnosis of new and recurrent hepatocellular carcinoma, followed by timely treatment, will help decrease mortality. Currently biomarkers are not satisfactory. Better diagnostic methods are highly demanded.MethodsIn this study, we have used in silico identification and RT-PCR test and discovered a hepatoma associated gene (HTA). Knockdown of endogenous HTA expression was performed by small interfering RNA in malignant hepatocyte HepG2. Then we tested the cell proliferative ability of these cells in vitro and in vivo.ResultsHTA was expressed specifically in some kinds of tumors, but not detected in any normal tissues. It was expressed especially high in hepatocellular carcinoma. Knockdown of endogenous HTA expression in HepG2 by small interfering RNA attenuated HCC cell growth.ConclusionsHCA is a very good marker for tumors, especially for HCC. It could play important roles in HCC development and progression and can be a promising molecular target for the development of new diagnostic and therapeutic strategies for HCC.

Identification of Rho GTPase Activating Protein 6 Isoform 1 Variant as a New Molecular Marker in Human Colorectal Tumors

The early diagnosis of colorectal cancer (CRC) is important because it is one of the most readily curable of all cancers, if detected early. However, the sensitivity of current markers is low. Immunostaining intensity for the monoclonal antibody Hb3 in CRC cell lines and tissues was stronger than in controls. Interestingly, this was associated with a low level of tumor differentiation. We used Hb3-coupled affinity chromatography to search for a corresponding Hb3 antigen as a candidate biomarker for early detection, and identified a Rho GTPase activating protein 6 (RhoGAP6) isoform 1 variant as an Hb3 antigen by mass spectrometry. Using reverse transcription polymerase chain reaction and western blot analysis, we confirmed that the expression levels of this variant were elevated in aberrant cells and tissues. Thus, the RhoGAP6 isoform 1 variant might serve as a biomarker for the development and progression of CRC.

The ability of human bispecific anti-idiotype antibody to elicit humoral and cellular immune responses in mice

Our goal is to compare the immunogenicity and the extent of immunologic reactivity between bispecific and mono anti-idiotype vaccines. We previously obtained two human anti-Id antibody fragments fuse5-G22, fuse5-I50 by phage display technology which were mimics of the antigens from nasopharyngeal carcinoma cell line (HNE2). In this study, we developed and characterized a bispecific anti-Id antibody vaccine G22-I50 and its parent monovalent antibody vaccines G22 and I50. The efficacy of G22-I50, G22, and I50 as tumor vaccines was evaluated in Balb/c mice with three injections of these vaccines adjuvanted with Freunds adjuvant. Mice immunized with G22-I50 exhibited comparable levels of antibody titers and stronger binding inhibition capabilities. Spleen cells from G22-I50-immunized mice gave a significant proliferative response and higher expression level of IFN-gamma and IL-2.These results suggested that bispecific anti-Id antibody vaccine was able to induce more powerful humoral and cell-mediated immune responses, which might make it to be a potential vaccine candidate for the therapy of nasopharyngeal carcinoma.

Preparation of human scFv antibody against nasopharyngeal carcinoma and identification of its specificity.

Abstract Conclusion: The selected scFv antibody could specifically recognize and target nasopharyngeal carcinoma (NPC), and could be applied to clinical diagnosis and therapy. Objective: The aim was to construct and screen fully human anti-NPC single chain Fv fusion phage libraries, and to identify the specificity of the scFv antibody. Methods: Peripheral blood mononuclear cells of patients with NPC were immunized in vitro by NPC cells and transformed by Epstein-Barr virus. The total RNAwas used to construct the scFv libraries. By means of ELISA and immunochemistry, the positively bound scFv was selected and identified. The positive scFv was fused to EGFP, and was then expressed in E. coli strain BL21 (DE3) and purified. Furthermore, we observed the binding bioactivity. Results: The fusion protein has the biological activity of binding the NPC cells and emitting green fluorescence. In targeting experiments in vivo, the results showed that the fusion protein can successfully target the NPC.