Objective To investigate cholecystokinin (CCK),carbachol, and vasoactive intestinal peptide(VIP)stimulating peptide chain elongation in mouse pancreatic acini in vitro and its molecular mechanism. Methods ~3H-lecucine incorporation assay was used to measure the basal and secretagogues-stimulated pancreatic acini elongation rates. Western blot was applied to analyse the effect of phosphorylation of the elongation factor 2 (eEF2) and the eEF2 kinase. MEK inhibitor (PD98059), SAPK/p38 inhibitor (SB202190), and mTOR inhibitor (rapamycin) were used to respectively block MEK, SAPK/p38, and mTOR intracellular pathways or the phosphatase inhibitor (calyculin A) pretreatment before CCK treatment. Results All secretagogues except VIP increased the peptide chain elongation in mouse pancreatic acini in vitro. All secretagogues except VIP inhibited the phosphorylation level of eEF2 on Thr-56 and increased the phosphorylation level of eEF2K on Ser-366, which might correlate with their activation status. MEK inhibitor PD98059 partially reversed the dephosphorylation of eEF2 induced by CCK, as did treatment p38 MAPK inhibitor SB202190, mTOR inhibitor rapamycin, and the phosphatase inhibitor calyculin A.Conclusion CCK increases peptide chain elongation via inducement of dephosphorylation of eEF2 and eEF2 kinase phosphorylation in pancreatic acini in vitro. CCK-induced dephosphorylation of eEF2 in pancreatic acinar cells involves MEK, SAPK/p38, and mTOR, the three intracellular pathways.

Objective In order to investigate the effect of SH2B1 on leptin signal transduction JAK2/IRS2 and its biological function.Methods Vitro kinase assay and Western blot were used to analyse tyrosine phosphorylatin of key molecule JAK2 and insulin receptor substrate-2 (IRS2). ELISA was used to measure the plasma leptin levels in mice. The postnatal growth of mice was monitored over 27 weeks. Results SH2B1 dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and IRS2 in HEK293 cells stably expressing LRb (HEK239~(LRb)). Leptin-stimulated activation of hypothalamic JAK2 and phosphorylation of hyphothalamic IRS2 were significantly impaired in SH2B1~(-/-) mice. The deletion of SH2B1 led to leptin resistance,and fasting and randomly fed plasma leptin levels were respectively 3.2 times and 5.1 times higher in SH2B1~(-/-) males than wild-type littermates at 15 weeks of age. SH2B1~(-/-) males gained body weight rapidly and exceeded wild-type littermates from 5~(th) week. SH2B1(-/-) (at 21 weeks) was approximately twice heavier than wild-type littermates.Conclusion SH2B1 is an endogenous enhancer of leptin sensitivity and required for maintaining normal bodyweight in mice via leptin JAK2/IRS2 pathway.

The molecular techniques were used to analyse tyrosine phosphorylation of JAK2 and STAT3 in leptin receptor overpression cell lines and SH2-Bβ knockout (SH2-Bβ-/-) mice. The serum level of leptin in SH2-Bβ mice was measured by ELISA. The results showed that SH2-Bβ dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and STAT3 in vitro. Leptin-stimulated activation of JAK2 and phosphorylation of STAT3 were significantly impaired in hypothalamus of SH2-Bβ-/- mice. The fasting and postprandial serum levels of leptin and body weight were markedly increased in SH2-Bβ-/- mice. Therefore, SH2-Bβ is an endogenous enhancer of leptin sensitivity and regulates body weight via leptin/ JAK2/STAT3 pathway.

Objective To identify lung adenocarcinoma-associated antigens by using Serologic Proteome Analysis(SERPA),an approach which combined conventional proteome analysis with serological screening.Methods SERPA of four human lung adenocarcinoma tissues were performed.The Western blot imaging films which reacted with autologous patient serum and with control normal serum were obtained and the differential reacting protein spots were recognized.Results Well-resolved,reproducible 2-DE Western blot imaging films of human lung adenocarcinoma reacted with autologous patient sera and the control sera were obtained.Totally(27?5) differentially expressed proteins which only were reactive with lung adenocarcinoma patient sera were found.Some differentially expressed proteins were identified by peptide mass fingerprint(PMF).Some of the proteins were the products of oncogenes,and others were involved in the regulation of cell cycle and signal transduction.Conclusion These results will provide scientific foundation on screening the molecular biomarker for the diagnosis,treatment,and prognosis of the lung adenocarcinoma.

Objective To observe the expression and influence of SH2-B in hepatocarcinoma,and to investigate the molecular mechanisms of canceration in hepatocarcinoma.Methods By using SABC imunohis-tochemistry,the expressions of SH2-B were detected in 27 cases of hepatitis,29 cases of hepatocirrhosis and 47 cases of hepatocarcinoma.Hepatocarcinoma cell (HepG)2 with a low-expressed SH2-B was selected using immunofluorescence assay.There were 3 groups:the transfected group (transfected with pcDNA3.1 -SH2-B), the vector group (transfected with pcDNA3.1 )and the blank group (without transfection).After gene transfec-tion,SH2-B expression was detected by Western blotting;cell proliferation was measured by MTT assay;cell colony was counted by colony formation test;and cell cycle was analyzed by flowcy tometer.Results The posi-tive rate of SH2-B in hepatocarcinoma (95.7%)was significantly higher than 55.2% in hepatocirrhosis (χ2 =1 8.64,P <0.01 )and 25.9% in hepatitis (χ2 =40.01 ,P <0.01 ).After being transfected with pcDNA 3.1 -SH2-B,SH2-B expression dramatically increased in HepG2 cells.After cultured for 48 h,the average optical density value of the transfected group was 1 .1 2 ±0.1 9,obviously higher than 0.45 ±0.1 1 in the vector group (t =-31 .55,P <0.01 ),which indicated that cells proliferation was significantly enhanced after being trans-fected with SH2-B.The cell colony numbers of the transfected group was 1 66 ±1 4,significantly higher than
82 ±8 in the vector group (t =-20.33,P <0.01 )and 78 ±9 in the blank group (t =-1 9.64,P <0.01 ), which indicated that the cell colony numbers increased after being transfected with SH2-B.The S stage cells of the transfected group was (45.7 ±5.8)%,significantly higher than (1 9.4 ±4.7)% in the vector group (t =-20.33,P <0.01 )and (20.5 ±5.1 )% in the blank group (t =-34.69,P <0.01 ),which indicated that SH2-B could enhance promote cell cycle of HepG2 cells.Conclusion The expression of SH2-B in hepatocar-cinoma is high,and it may be involved in the canceration of hepatocarcinoma though promoting cell cycle,cell proliferation and cell transformation.

OBJECTIVE: To explore the expression of SH2B1 adaptor protein in oesophageal cancer and its clinical significance. METHODS: SH2B1 expression in tissue specimens of 120 primary oesophageal cancers, tissues of 120 paired adjacent non-cancer and another 120 normal tissues was analyzed by immunohistochemical SABC staining and Western blot. SH2B1 expression in the oesophageal cancer tissues was analyzed with clinicopathological parameters. SH2B1 expression of normal human esophageal epithelial cells (HEEC) and 2 oesophageal cancer cell lines, TE-1 and Eca109, were evaluated by RT-PCR and Western blot. RESULTS: SH2B1 expression in the normal oesophageal tissues, adjacent non-cancer tissues and cancer tissues was gradually increased (P<0.05), and significantly associated with the depth of invasion, clinical TNM stage, lymph node metastasis (P<0.05), but it did not have significant association with gender, age, drinking, type and grade of tumor (P>0.05). SH2B1 expression was detectable in all cell lines by RT-PCR and Western blot, but the expression in the two oesophageal cancer cell lines was significantly higher than that in the normal HEEC. CONCLUSIONS Over-expression of SH2B1 might play an important role in the occurrence and development of human oesophageal cancer and closely correlate with malignant progression of invasion and metastasis of oesophageal cancer.
Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Tumor Cells, Cultured

To screen for serum biomarkers for lung squamous carcinoma, two-dimensional gel electrophoresis (2-DE) was performed to separate serum proteins from healthy individuals and stage 1 lung squamous carcinoma(LSC) patients, respectively. PDquest software was used to analyze 2-DE images, and the differential serum protein spots between the healthy individuals and LSC patients were identified by ESI-Q-TOF MS/MS. Then Western blot and immunohistochemistry were used to detect the expression levels of haptoglobin-2(HP-2), one of the differential proteins, in the sera and tumor tissues in the patients with LSC, respectively. 2-DE maps of serum proteins from healthy individuals and stage 1 LSC patients were established. Ten differential serum protein spots were detected, four proteins of which were identified by MS/MS. Western blot showed that the serum level of HP-2 in the LSC patients was significantly higher than that in healthy individuals, but was not associated with LSC staging. Immunohistochemistry showed that the expression level of HP-2 in the LSC tissues was significantly higher than that in the normal bronchial epithelial tissues adjacent to tumors. The results indicated that serum HP-2 protein is a candidate biomarker for LSC, and might be useful for diagnosis of LSC. Up-regulation of HP-2 in the LSC tissues may contribute to the high serum level of HP-2 in the patients.

OBJECTIVE: To investigate the expression pattern of adapter protein with a Src-homology 2 domain (SH2B1), the suppressor of cytokine signaling-3 (SOCS3), protein-tyrosine phosphatase 1B (PTP1B) and neturopetide Y (NPY) in obese and normal mice hypothalamus and its relation with serum leptin and insulin levels. METHODS: The obesity animal model was prepared with healthy C57/bl6 mice. Lee's index and Homeostasis model assessment-insulin resistance (HOMA-IR) were calculated. The mRNA levels of SH2B1, SOCS3, PTP1B and NPY were measured by fluorescent quantitation RT-PCR. The SH2B1 and NPY protein expressions were detected by Western blot. RESULTS: Compared with the normal mice of the same age, SH2B1 mRNA expression in the obese mice hypothalamus decreased. SOCS3 and PTP1B mRNA expression increased. Western blot showed that SH2B1 protein expression decreased, while NPY protein expression increased in the obese mice. Linear correlation analysis showed that the serum leptin and fasting insulin levels were negatively correlated with SH2B1mRNA expression and positively correlated with SOCS3 and PTP1B mRNA expression. CONCLUSION SH2B1, SOCS3, PTP1B and NPY are key factors for obesity development.
Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Hypothalamus ; metabolism ; Insulin ; blood ; Insulin Resistance ; Leptin ; blood ; Mice ; Mice, Inbred C57BL ; Neuropeptide Y ; metabolism ; Obesity ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; metabolism ; RNA, Messenger ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; metabolism

Objective To investigate the expression of long non-coding RNA NONHSA1254644 in lung adenocarcinoma and its clinical significance.Methods 99 cases of lung adenocarcinoma and the adjacent tissues as well as clinical data was collected.The expression level was detected by quantitative realtime polymerase chain reaction (qRT-PCR).Then the relationship between the expression level and the clinical parameters was analyzed.Cell counting kit-8 (CCK8) was used to investigate its effect of lung adenocarcinoma cell line A549 on proliferation.Results The expression of NONHSA1254644 was down-regulated in lung adenocarcinoma,and its expression was positively correlated with the differentiation of patients.The overexpression of NONHSA1254644 could inhibit the proliferation of A549 cells.Conclusions NONHSA1254644 is involved in the development and progression of lung adenocarcinoma.

OBJECTIVE: To explore two transcript variants expression of long noncoding RNA C6orf176 in non-small cell lung cancer (NSCLC) and the clinical pathological significance. 
 METHODS: The expressions of transcript variant 1 (TV1) and transcript variant 2 (TV2) of long noncoding RNA C6orf176 in 57 NSCLC and adjacent cancerous tissues were examined by qPCR with β-actin as internal control.
 RESULTS: Based on the results of qPCR, for C6orf176-TV1, 42 cases were down-regulated and 15 cases were up-regulated. The C6orf176-TV1 level was correlated to the grade of differentiation (P<0.05) but it was not correlated with gender, age, smoking history, tumor type and TNM stage. The expression of C6orf176-TV1 had a potential value in diagnosis of NSCLC by receiver operator characteristic (ROC) curve. Area under curve (AUC) of ROC curve was 0.708 (95% CI 0.615 to 0. 802). The sensitivity and specificity were 51% and 88%, respectively. For C6orf176-TV2, 39 cases were down-regulated and 18 cases were up-regulated. The C6orf176-TV2 level was correlated with the grade of differentiation (P<0.05) but it was not correlated with gender, age, tumor size, smoking history and TNM stage. C6orf176-TV2 level had value in diagnosis of NSCLC by ROC curve. AUC of ROC curve was 0.64 (95% CI 0.531 to 0.749). The sensitivity and specificity were 49% and 75%, respectively. Of the 57 specimens, 53 cases were simultaneous up or down-regulation of C6orf176-TV1 and C6orf176-TV2. The correlation coefficient was 0.99.
 CONCLUSION The expression of C6orf176-TV1 or C6orf176-TV2 is down-regulated in NSCLC and it is correlated with the grade of differentiation. It may act as a diagnosis indicator for NSCLC patients.
Actins ; Area Under Curve ; Carcinoma, Non-Small-Cell Lung ; Cell Differentiation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; RNA, Long Noncoding ; ROC Curve ; Real-Time Polymerase Chain Reaction ; Smoking ; Transcription, Genetic ; Up-Regulation