FAM122A Is Required for Mesendodermal and Cardiac Differentiation of Embryonic Stem Cells.

Mesendodermal specification and cardiac differentiation are key issues for developmental biology and heart regeneration medicine. Previously, we demonstrated that FAM122A, a highly conserved housekeeping gene, is an endogenous inhibitor of protein phosphatase 2A (PP2A) and participates in multifaceted physiological and pathological processes. However, the in vivo function of FAM122A is largely unknown. In this study, we observed that Fam122 deletion resulted in embryonic lethality with severe defects of cardiovascular developments and significantly attenuated cardiac functions in conditional cardiac-specific knockout mice. More importantly, Fam122a deficiency impaired mesendodermal specification and cardiac differentiation from mouse embryonic stem cells but showed no influence on pluripotent identity. Mechanical investigation revealed that the impaired differentiation potential was caused by the dysregulation of histone modification and Wnt and Hippo signaling pathways through modulation of PP2A activity. These findings suggest that FAM122A is a novel and critical regulator in mesendodermal specification and cardiac differentiation. This research not only significantly extends our understanding of the regulatory network of mesendodermal/cardiac differentiation but also proposes the potential significance of FAM122A in cardiac regeneration.

FAM122A supports the growth of hepatocellular carcinoma cells and its deletion enhances Doxorubicin-induced cytotoxicity.

FAM122A is a highly conserved protein in mammals, however its function is still largely unknown so far. In this study, we investigated the potential role of FAM122A in hepatocellular carcinoma (HCC). By analyzing HCC patient cohorts from RNA sequencing datasets, we found the expression level of FAM122A mRNA is significantly upregulated in HCC patients. Moreover, this abnormally higher expression pattern of FAM122A protein was also found in partial HCC tumor tissues, compared with the normal parts. Further, we demonstrated that CRISPR/Cas9-mediated FAM122A knockout significantly inhibits the growth, clonogenic potential and xenografts of HCC cells, induces cell cycle arrest and reduces the expression of proliferation-related genes. Interestingly, FAM122A deletion significantly enhances the cytotoxicity effect of Doxorubicin (Dox), a drug used in standard chemotherapy in HCC patients. In contrary, overexpression of FAM122A not only promotes HCC cell growth, but also inhibits Dox-induced DNA damage and cell death. Considering that FAM122A is previously identified as an endogenous inhibitor of PP2A, we asked whether FAM122A regulating HCC cell growth is associated with PP2A. The results showed FAM122A can also modulate PP2A activity in HCC cells although the modulated effect is relatively slight, however, treatment with a PP2A inhibitor okadaic acid did not rescue the inhibitory effects of cell growth and proliferation in FAM122A deletion cells, indicating that FAM122A may support HCC cell growth independent of its ability to modulate PP2A. Collectively, these results suggest that FAM122A is required for maintaining HCC cell growth, and its elimination combined with chemotherapy may represent a potential novel therapeutic strategy for HCC patients.

Low serum gastrin associated with ER+ breast cancer development via inactivation of CCKBR/ERK/P65 signaling.

BACKGROUND: Gastrin is an important gastrointestinal hormone produced primarily by G-cells in the antrum of the stomach. It normally regulates gastric acid secretion and is implicated in a number of human disease states, but how its function affects breast cancer (BC) development is not documented. The current study investigated the suppressive effects of gastrin on BC and its underlying mechanisms. METHODS: Serum levels of gastrin were measured by enzyme-linked immunosorbent assay (ELISA) and correlation between gastrin level and development of BC was analyzed by chi-square test. Inhibitory effects of gastrin on BC were investigated by CCK-8 assay and nude mice models. Expressions of CCKBR/ERK/P65 in BC patients were determined through immunohistochemistry (IHC) and Western blot. Survival analysis was performed using the log-rank test. RESULTS: The results indicated that the serum level of gastrin in BC patients was lower compared with normal control. Cellular and molecular experiments indicated that reduction of gastrin is associated with inactivation of cholecystokinin B receptor (CCKBR)/ERK/P65 signaling in BC cells which is corresponding to molecular type of estrogen receptor (ER) positive BC. Furthermore, we found that low expression of gastrin/CCKBR/ERK /P65 was correlated to worse prognosis in BC patients. Gastrin or ERK/P65 activators inhibited ER+ BC through CCKBR-mediated activation of ERK/P65. Moreover, combination treatment with gastrin and tamoxifen more efficiently inhibited ER+ BC than tamoxifen alone. CONCLUSIONS: We concluded that low serum gastrin is related to increased risk of ER+ BC development. The results also established that CCKBR/ERK/P65 signaling function is generally tumor suppressive in ER+ BC, indicating therapies should focus on restoring, not inhibiting, CCKBR/ERK/P65 pathway activity.

[The Analysis of Element and Measure Analysis of NMR Spectrum and XRD Spectrum for High Purity 3,4,9,10 Perylenetetracarboxylic Dianhydride-PTCDA].

vacuum sublimation method was used to purify the homegrown 3,4,9,10 perylenetetracarboxylic dianhydride(PTCDA)powder with a purity of 98% in its sublimation point of 450 ℃. With Bill's law and ultraviolet-visible spectrophotometer testing analysis, its purity reached to 99.8%. Meanwhile, the contents of C and H elements in the pre-and post-purified molecules were also measured by using elemental analyzer. The measured results indicate that the contents of C and H elements in the post-purified the molecules are very close to the theoretical value. H element in the molecular structure was investigated with nuclear magnetic resonance (NMR) spectroscopy and the results demonstrated that there are an equal number of H atoms in two different chemical environments and it can only be located on the aromatic ring. By discussing the chemical bond formation of PTCDA molecules, the C, H and O atoms in high purity PTCDA molecules are mainly covalent bonds. The crystalline state and crystal structure of this organic material were tested and analyzed by X-ray diffractometer. The results suggest that the post-purified PTCDA power existed α-PTCDA and ß-PTCDA two phases, in which α-PTCDA phase is major component while ß-PTCDA phase accounts for about one five of the total ingredients. Besides, the crystal cell belonged to bottom-centered monoclinal structure. Meanwhile, the crystal state, grain size and band structure of PTCDA single crystal thin films formed on the surface of p-type silicon in its sublimation point are investigated in detail. During the high-purity α-PTCDA forming organic single thin film on the surface of p-type single silicon, the π-electron cloud covered on the top, bottom and two sides of its thin film's molecular layer plane. Due to the formation of delocalized bond that attributed to the overlap of the outermost valence electron orbital of C, H, O atom, the valence electrons generate co-movement and the energy level splitting for the band. The energy difference between valence band and the first tight binding is 2.2 eV which lead to this organic material possessing the properties of semiconductor conduction. In addition, this organic material with the intrinsic carrier concentration for 1014 cm-3 belong to weak p-type organic semiconductor material. This organic material combines with the surface of p-type silicon to form hetehomo-type heterojunction which is provided with excellent response for visible light to near infrared wavelengths of light.

[Purification of PTCDA by Vacuum Sublimation and Spectral Test and Analysis].

The organic semiconductor 3, 4, 9, 10 perylenetetracarboxylic dianhydride (PTCDA) with the purity of 97.5% was purified by sublimation to 99.9%. The high-purity PTCDA material was measured by mass spectra, infrared spectrum and X-ray photoelectron spectroscopy (XPS). Detailed analysis revealed its molecular structure, the forming of chemical bond, the vibration modes of atoms in equilibrium lattice position, electronic configuration and the shift of binding energy of atoms. Based on the infrared spectrum analysis, the molecular structure of PTCDA is consisting of perylene core group with five C rings and two anhydrides located at both ends of perylene core, which is mainly bonded with covalent bond. The stretching vibration of C atoms in the crystal lattice dominates in their equilibrium positions. The PTCDA molecules have a large number of π electrons which can move freely; the intermolecular delocalized π bond overlap determines the conductivity of PTCDA. Based on XPS analysis, it can be found that there exist two kinds of C atoms with different binding energy: 285.3 and 288.7 eV, respectively, corresponding to the C atoms in the perylene ring and anhydride. In addition, there are two kinds of O atoms, i. e. C==0 and C--O--C, whose bonding energy is 531.3 and 533.1 eV, respectively.

Chloroquine enhances cobalt chloride-induced leukemic cell differentiation via the suppression of autophagy at the late phase.

We previously reported that moderate hypoxia and hypoxia-mimetic agents including cobalt chloride (CoCl(2)) induce differentiation of human acute myeloid leukemia (AML) cells through hypoxia-inducible factor-1 α (HIF-1 α), which interacts with and enhances transcriptional activity of CCAAT-enhancer binding factor alpha and Runx1/AML1, two important transcriptional factors for hematopoietic cell differentiation. Here, we show that autophagy inhibitor chloroquine (CQ) increases HIF-1 α accumulation, thus potentiating CoCl(2)-induced growth arrest and differentiation of leukemic cells. Furthermore, the increased effect of CQ on differentiation induction is dependent of the inhibition of autophagosome maturation and degradation, since this sensitization could be mimicked by the suppression of expression of both lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2). These findings not only provide the evidence that CQ is a sensitizer for CoCl(2)-induced differentiation of leukemic cells but also possibly propose the new therapeutic strategy for differentiation induction of AML.

Comprehensive Mutation Profiling of Colorectal Cancer Patients With Lung or Liver Metastasis by Targeted Next-Generation Sequencing.

OBJECTIVES: Primary tumor tissue is often analyzed to search for predictive biomarkers and DNA-guided personalized therapies, but there is an incomplete understanding of the discrepancies in the genomic profiles between primary tumors and metastases, such as liver and lung metastases. METHODS: We performed in-depth targeted next-generation sequencing of 520 key cancer-associated genes for 47 matched primary and metastatic tumor samples which were retrospectively collected. RESULTS: A total of 699 mutations were detected in the 47 samples. The coincidence rate of primary tumors and metastases was 51.8% (n = 362), and compared to patients with liver metastases, patients with lung metastases had a significantly greater coincidence rate (P = .021). The number of specific mutations for the primary tumors and liver and lung metastases was 186 (26.6%), 122 (17.5%), and 29 (4.1%), respectively. Analysis of a patient with all three occurrences, including a primary tumor, liver metastasis, and lung metastasis, indicated a possible polyclonal seeding mechanism for liver metastases. Remarkably, multiple samples from patients with primary and metastatic tumors supported a mechanism of synchronous parallel dissemination from primary tumors to metastatic tumors that were not mediated through pre-metastatic tumors. We also found that the PI3K-Akt signaling pathway significantly altered lung metastases compared to matched primary tumors (P = .001). In addition, patients with mutations in CTCF, PIK3CA, or TP53 and LRP1B, AURKA, FGFR1, ATRX, DNMT3B, or GNAS had larger primary tumor sizes and metastases, especially patients with both LRP1B and AURKA mutations. Interestingly, CRC patients with TP53-disruptive mutations were more likely to have liver metastases (P = .016). CONCLUSION: In this study, we demonstrate significant differences in the genomic landscapes of colorectal cancer patients based on the site of metastasis. Notably, we observe a larger genomic variation between primary tumors and liver metastasis compared to primary tumors and lung metastasis. These findings can be used for tailoring treatments based on the specific metastatic site.

DDX54 Plays a Cancerous Role Through Activating P65 and AKT Signaling Pathway in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most malignant cancers, and its incidence is still steadily increasing. The DDX RNA helicase family members have been found to play a role in various cancers; however, the role of DDX54 in colorectal cancer is still unclear and needed to be defined. Here, we found DDX54 was overexpressed in CRC tissues by the label-free mass spectrum, which was also verified in tissue microarray of colon cancer, as well as the CRC cell lines and TCGA database. High DDX54 level was correlated with tumor stage and distant metastasis, which always indicated a poor prognosis to the CRC patients. DDX54 could promote the proliferation and mobility of CRC cells through increasing the phosphorylation level p65 and AKT leading to the tumorigenesis. Here, we have preliminarily studied the function of DDX54 in CRC, which would improve our understanding of the underlying biology of CRC and provide the new insight that could be translated into novel therapeutic approaches.

[Evaluation of liquid based cytology test (LCT) in avoiding medical tangles].

OBJECTIVE: To evaluate of liquid based cytology test (LCT) in avoiding medical tangles. METHODS: One thousand five hundred five thirty one cases, which were collected from out-patients of precancerous lesions of uterine cervix, were randomly divided into three groups based on different smear preparation: LCT method was used in two groups (one with ThinPrep kit and one with ArtoBrain kit), conventional Papauicolaou smear (PS) was used in one group. All cases of abnormal cervical smears were identified by cytologic test underwent colposcopic examination and colopscopically multiple biopsy. Results of test were analyzed by software SPSS 11.0. RESULTS: Significant diference were found between LCT method and PS method compared by index of satifacation, sensitivity, specificity, accuracy, false negative rate and erroneous diagnosis rate (P < 0.05, but no difference were found between two LCT groups (ThinPrep kit and ArtoBrain kit). CONCLUSION: LCT method can improve diagnostic level of precancerous lesions of uterine cervix either tested by ThinPrep kit or ArtoBrain kit, so have the powerfull value to avoid medical tangles.

FAM122A, a new endogenous inhibitor of protein phosphatase 2A.

The regulation of the ubiquitously expressed protein phosphatase 2A (PP2A) is essential for various cellular functions such as cell proliferation, transformation, and fate determination. In this study, we demonstrate that the highly conserved protein in mammals, designated FAM122A, directly interacts with PP2A-Aα and B55α rather than B56α subunits, and inhibits the phosphatase activity of PP2A-Aα/B55α/Cα complex. Further, FAM122A potentiates the degradation of catalytic subunit PP2A-Cα with the increased poly-ubiquitination. In agreement, FAM122A silencing inhibits while its overexpression enhances cell growth and colony-forming ability. Collectively, we identify FAM122A as a new endogenous PP2A inhibitor and its physiological and pathophysiological significances warrant to be further investigated.

Protective effect of sodium nitroprusside on the rat small intestine transplanted mucosa.

The intestinal mucosal epithelium is extremely susceptible to even brief periods of ischemia. Mucosal barrier damage, which is associated with ischemia/reperfusion (I/R) injury and consequently bacterial translocation, remains a major obstacle for clinically successful small bowel transplantation (SBT). Previous studies have demonstrated a protective effect of nitric oxide (NO) on other transplanted organs and NO mediated intestinal protection has also been reported in vitro. The aim of this study was to evaluate the effect of sodium nitroprusside (SNP), NO donor, on graft mucosal histology and molecular markers of function after SBT in rats. We used SNP in different period of heterotopic SBT rats. The groups consisted of SBT, pre-SNP group, and post-SNP group. Interestingly, the pre-SNP graft samples exhibited less damage compared to the SBT and post-SNP samples. In addition, mucosal samples from the pre-SNP group showed higher Na(+)-K(+)-ATPase activity and higher levels of laminin expression compared to the SBT and post-SNP samples. The findings of the present study reveal that SNP given before graft ischemia/reperfusion injury has a protective effect on mucosal histology and molecular markers of function in the transplanted small intestine.

In situ analysis of p16/INK4 promoter hypermethylation in esophageal carcinoma and gastric carcinoma.

OBJECTIVE: Inactivation of the tumor suppressor gene by CpG hypermethylation is a common event in a variety of tumors. The present study was designed to be a comprehensive analysis of p16/INK4 methylation in carcinomas of the upper digestive tract. METHODS: Series of esophageal carcinomas (34 cases) and gastric carcinomas (25 cases) were examined for CpG methylation in p16/INK4 using methylation-specific PCR (MSP). The tissue sections underwent MSP in situ and were then examined microscopically. Immunohistochemical detection of the expression of p16 in the tumor specimens was also performed. RESULTS: Immunohistochemistry detected positive p16 expression in 8 cases of esophageal squamous cell carcinoma and 15 cases of gastric carcinoma. In esophageal carcinoma, hypermethylation of the p16/INK4 promoter region was detected in 5 cases without statistical correlation with its loss of expression, whereas in the gastric carcinomas, p16 expression was positively correlated with the T-classification (r = 0.488, P = 0.01); p16/INK4 methylation was identified in 8 cases. In addition, p16 expression was lower in the methylated samples than in the non-methylated samples (25% vs 76.47%, P = 0.03). Analysis of the MSP-in-situ sections showed that the distribution of methylated cells in esophageal carcinoma differed from that in gastric carcinoma. CONCLUSION: The role of DNA methylation in the silence of p16/INK4 may different between these two types of upper digestive tract tumor.

Phosphorylation of epidermal growth factor receptor and chromosome 7 polysomy in gastric adenocarcinoma.

OBJECTIVE: To investigate the phosphorylation of epidermal growth factor receptor (EGFR) and its potentially associated chromosomal aberrations in gastric adenocarcinoma. METHODS: Phosphorylated EGFR (pEGFR) was detected by immunohistochemistry on 145 specimens including 60 tumoral, 60 non-tumoral, 12 tumor-adjacent intramucosal dysplasia from patients with gastric adenocarcinoma and 13 mucosae from cancer-free patients. EGFR gene amplification and chromosome 7 (Chr-7) polysomy were detected by fluorescence in situ hybridization. RESULTS: Positivity of pEGFR was found in 50 tumoral (83.3%) and 42 non-tumoral specimens (70.0%). There was an association between tumoral and non-tumoral zones on immunostains of pEGFR (r = 0.353, P = 0.006). Nuclear pEGFR usually presented in mucosae with Helicobacter pylori infection, stromal reaction or vascular invasion. Cytoplasmic pEGFR was correlated with local cancer extension (r = 0.337, P = 0.014) and inversely related with gastrokine 2, which had been previously detected in the same specimens. Eleven intramucosal dysplastic specimens were also positive for pEGFR while 13 mucosae from cancer-free patients were all negative. No EGFR gene amplification was observed. However, seven tumor specimens showed Chr-7 polysomy (11.7%) in which 5 were strongly positive for pEGFR. CONCLUSIONS: EGFR phosphorylation may be one of the mechanisms that promote tumor initiation and expansion in gastric adenocarcinoma. Detection of pEGFR with analysis of its nuclear or cytoplasmic patterns could be clinicopathologically valuable. Chr-7 polysomy may partially contribute to EGFR activation in gastric adenocarcinoma, although its role does not predominate.

Gastrin inhibits a novel, pathological colon cancer signaling pathway involving EGR1, AE2, and P-ERK.

Human anion exchanger 2 (AE2) is a plasma membrane protein that regulates intracellular pH and cell volume. AE2 contributes to transepithelial transport of chloride and bicarbonate in normal colon and other epithelial tissues. We now report that AE2 overexpression in colon cancer cells is correlated with expression of the nuclear proliferation marker, Ki67. Survival analysis of 24 patients with colon cancer in early stage or 33 patients with tubular adenocarcinoma demonstrated that expression of AE2 is correlated with poor prognosis. Cellular and molecular experiments indicated that AE2 expression promoted proliferation of colon cancer cells. In addition, we found that transcription factor EGR1 underlies AE2 upregulation and the AE2 sequester p16INK4a (P16) in the cytoplasm of colon cancer cells. Cytoplasmic P16 enhanced ERK phosphorylation and promoted proliferation of colon cancer cells. Gastrin inhibited proliferation of colon cancer cells by suppressing expression of EGR1 and AE2 and by blocking ERK phosphorylation. Taken together, our data describe a novel EGR1/AE2/P16/P-ERK signaling pathway in colon carcinogenesis, with implications for pathologic prognosis and for novel therapeutic approaches.

PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway.

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.

Detection of ß-catenin, gastrokine-2 and embryonic stem cell expressed ras in gastric cancers.

UNLABELLED: ERas activation and GKN2 reduction in gastric cancer has raised some notices in recent years, while nuclear beta-catenin positivity is considered as a tumoral marker. In this study, we compared immunohistochemistry of beta-catenin, GKN2 and ERas on tumoral and non-tumoral mucosae of 50 gastric carcinomas and 13 gastric samples of cancer-free patients. Nuclear positivity of beta-catenin was strong in 31 non-tumoral mucosae (62%) and 29 tumoral mucosae (58%). It was absent in samples of cancer-free patients. There was a correlation between non-tumoral and tumoral zones for nuclear beta-catenin positivity (P=0.013). ERas was positive in 35 non-tumoral tissues (70%) and 31 tumoral tissues (62%) but negatvie in samples of cancer-free patients. It was weak and spotty in non-tumoral mucosae but strong and diffuse in tumors. Positivity of ERas was age-related (P=0.028). However it had background staining effect. GKN2 was expressed in 33 non-tumoral mucosae (66%) and 35 tumoral mucosae (70%). Though GKN2 staining was moderate to strong in non-tumoral tissues and was comparatively weaker in tumors, their difference was minimal and difficult to discern. CONCLUSIONS: Beta-catenin nuclear location could be considered as a paraneoplastic pattern which is considerably tumor-related. ERas may be a potential biomarker for gastric cancer, but advanced studies are wanted. GKN2 reduction is indiscernible by immunostaining.

Expression of anion exchanger 1 is associated with tumor progress in human gastric cancer.

PURPOSE: Anion exchanger 1 (AE1) is a transmembrane glycoprotein which is abundantly expressed in erythrocyte plasma membrane and mediates the electroneutral exchange of Cl(-) and HCO(3) (-). We previously reported that the AE1 protein was unexpectedly expressed in the gastric and colonic cancer and take part in the carcinogenesis of the cancer cells. The aim of the present study is to determine the potential clinical implications of AE1 expression in gastric carcinoma. METHODS: Immunohistochemistry assay was used to determine the expression of AE1 protein. The expression of AE1 in normal and malignant tissues from 286 patients with early and advanced gastric carcinoma was examined. The correlations of AE1 expression with clinicopathological parameters, including age, tumor size, location and subtypes, expression frequency, survival period and lymph metastasis were assessed by Chi-squared test and t test analysis. RESULTS: AE1 immunoreactivity was negative in normal gastric tissue. Positive immunostaining of AE1 was detected in gastric carcinoma regardless of the location. AE1 was most frequently expressed in the gastric antrum carcinoma compared with gastric body cancer (P = 0.034). Expression of AE1 was significantly associated with bigger tumor size, deeper invasion, shorter survival period, and non-lymph metastasis. In para-cancer tissues of intestinal-type gastric cancer, the expression frequency of AE1 was higher than that in diffuse-type (P = 0.011). CONCLUSION: The results showed a strong association of AE1 expression with the onset and progression of the gastric cancer and that may be helpful for improving the tumor classification and the treatment of cancer.

Expression of cytoplasmic p16 and anion exchanger 1 is associated with the invasion and absence of lymph metastasis in gastric carcinoma.

The tumor suppressor p16 is a negative regulator of the cell cycle, commonly believed to act in the nucleus. We recently found that p16 protein is expressed in the cytoplasm of gastric cancer cells, concomitantly with anion exchanger 1 (AE1). The aim of this study was to analyze the significance of cytoplasmic p16 and its relationship to AE1 in the progression of gastric cancer. Expression of p16 and AE1 was examined by immunohistochemical analysis in 196 patients; 98 with early gastric cancer and 98 with advanced gastric cancer. The relationship between cytoplasmic p16 and clinicopathological features, and the relationship between cytoplasmic p16 and AE1, were analyzed statistically. Expression of p16 was observed in the nucleus in early stage gastric cancer, but was located mainly in the cytoplasm in advanced cancer cells. Furthermore, cytoplasmic expression of p16 was correlated with AE1 expression, and both were associated with the absence of lymph metastasis in gastric cancer. In conclusion, cytoplasmic immunoreactivity of p16 appears to be a good prognostic indicator in advanced gastric cancer. Co-localization of p16 and AE1 predicts a lack of metastasis in gastric cancer. The role of cytoplasmic p16 and AE1, and the mechanisms involved in the progression of gastric cancer, warrant further investigation.

[Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].

OBJECTIVE: To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. METHODS: The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. RESULTS: Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. CONCLUSIONS: The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.