OBJECTIVES: Liver cancer is the sixth most common malignant tumor in the world. Hepatocellular carcinoma (HCC) accounts for 85%-90% of all patients with liver cancer. It possesses the characteristics of insidious onset, rapid progression, early recurrence, easy drug resistance, and poor prognosis. NIMA related kinase 2 (NEK2) is a cell cycle regulating kinases, which regulates cell cycle in mitosis. Cellular senescence is a complex heterogeneous process, and is a stable form of cell cycle arrest that limits the proliferative potential of cells. This study aims to investigate the relationship between the expression level of NEK2 and the senescence in hepatoma cells, and to explore the effect of NEK2 expression on hepatoma cell senescence and the underlying molecular mechanism. METHODS: A total of 581 senescence-relevant genes were obtained from the GenAge website. The gene expression data of tumor tissues of 370 HCC patients were downloaded from the Cancer Genome Atlas database. The co-expression of NEK2 and aging-related genes was analyzed by R-package. KEGG was used to analyze the significant gene enrichment pathway of differentially expressed genes in NEK2 overexpression HEK293. The stable transfected cell lines with overexpression and knockdown of NEK2 were constructed in hepatoma cell line SMMC-7721 and HepG2, and senescence-associated β-galactosidase (SA-β-gal) staining was used to detect senescence, the cell proliferation was detected by CCK-8 method and clone formation experiment, the cell cycle was analyzed by flow cytometry, and the expression of proteins related to p53/p21, p16/Rb, and phosphatase and tensin homolog deleted on chromosome ten (PTEN)/Akt signal transduction pathway was detected by Western blotting. RESULTS: There were 320 senescence related genes co-expressed with NEK2. KEGG analysis showed that the senescence signaling pathway was significantly enriched in HEK293 cells with overexpression of NEK2.Compared with SMMC-7721 or HepG2 without knockdown of NEK2, the senescent cells of SMMC-7721 and HepG2 with knockdown of NEK2 were increased, cell proliferation and clone formation were decreased significantly, the percentage of cells in G₀/G₁ phase was increased, the expression levels of phospho-Akt (p-Akt) and phospho-Rb (p-Rb) protein were decreased significantly, and the expression level of p16 protein was increased significantly (all P<0.05). Compared with SMMC-7721 or HepG2 transfected with blank plasmid, the senescent cells of SMMC-7721 and HepG2 overexpressing NEK2 were decreased, the cell proliferation and clone formation were increased significantly, the percentage of cells in G₀/G₁ phase were decreased, the expression levels of p-Akt and p-Rb protein were increased significantly, and the expression level of p16 protein was decreased significantly (all P<0.05). CONCLUSIONS NEK2 may mediate the anti-aging effect of hepatoma cells through p16/Rb and PTEN/Akt signal transduction pathways, which provides a new theoretical basis for NEK2 to promote the progress of liver cancer and a new idea for the targeting treatment for liver cancer.
Carcinoma, Hepatocellular/pathology* ; Cell Line, Tumor ; Cell Proliferation/physiology* ; Cellular Senescence/genetics* ; HEK293 Cells ; Humans ; Liver Neoplasms/pathology* ; NIMA-Related Kinases/genetics* ; Proto-Oncogene Proteins c-akt/metabolism*

PURPOSE: C-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor that is overexpressed in many cancers. CtBP1 transcriptionally represses a broad array of tumor suppressors, which promotes cancer cell proliferation, migration, invasion, and resistance to apoptosis. Recent studies have demonstrated that CtBP1 is a potential target for cancer therapy. This study was designed to screen for compounds that potentially target CtBP1. METHODS: Using a structure-based virtual screening for CtBP1 inhibitors, we found protocatechuic aldehyde (PA), a natural compound found in the root of a traditional Chinese herb, Salvia miltiorrhiza, that directly binds to CtBP1. Microscale thermophoresis assay was performed to determine whether PA and CtBP1 directly bind to each other. Further, clustered regularly interspaced short palindromic repeats associated Cas9 nuclease-mediated CtBP1 knockout in breast cancer cells was used to validate the CtBP1 targeting specificity of PA. RESULTS: Functional studies showed that PA repressed the proliferation and migration of breast cancer cells. Furthermore, PA elevated the expression of the downstream targets of CtBP1, p21 and E-cadherin, and decreased CtBP1 binding affinity for the promoter regions of p21 and E-cadherin in breast cancer cells. However, PA did not affect the expression of p21 and E-cadherin in the CtBP1 knockout breast cancer cells. In addition, the CtBP1 knockout breast cancer cells showed resistance to PA-induced repression of proliferation and migration. CONCLUSION Our findings demonstrated that PA directly bound to CtBP1 and inhibited the growth and migration of breast cancer cells through CtBP1 inhibition. Structural modifications of PA are further required to enhance its binding affinity and selectivity for CtBP1.

Objective To evaluate perioperative nutritional support for patients with acute abdomen in enhanced recovery after surgery (ERAS) programme.Methods A total of 490 patients with acute abdominal disease were collected,287 in ERAS group and 203 in conservative perioperative management (CPM) group.Biochemical and clinical markers of the 2 groups were compared.Results ERAS group had higher plasma albumin level,quicker bowel function recovery,lower postoperative complications,shorter hospital stay,and a lower WHO pain rating scale (all P ＜ 0.05).Conclusion With perioperative nutritional support,ERAS programme can accelerate recovery after emergency surgery,reduce the rate of overall complications,promote bowel function recovery,and decrease morbidity in the perioperative period for patients with acute abdominal disease.

OBJECTIVE: To examine the expression of Twist1 in cervical cancer and to explore its biological function in the progression of cervical cancer.
 METHODS: The expressions of Twist1 in 32 cervical cancers and matched normal tissues were examined by immunohistochemistry (IHC). Cell invasive ability and the expression of invasion-related genes were determined in RNAi-based Twist1-silencing HeLa cells. The relationship between Twist1 and microRNA-33a (miR-33a) in cervical cancer was studied by Pearson correlation analysis, and the roles of miR-33a in regulation of Twist1 and cell invasiveness were studied.
 RESULTS: The positive expression rate of Twist1 was 75.0% (24/32) and 21.9% (7/32) in the cervical cancer and the matched normal tissues, respectively, with significant difference between them (P<0.05). Twist1 shRNA significantly decreased the invasiveness of HeLa cells (P<0.05). Compared with the matched normal tissues, the expression of miR-33a was increased in the cervical cancer tissues, which was negatively correlated with Twist1 (r=-0.661, P<0.05). Overexpression of miR-33a could significantly suppress Twist1 expression as well as cell invasiveness (P<0.05).
 CONCLUSION Twist1 is critical for the invasiveness of cervical cancer cells; miR-33a, as a tumor suppressor gene, functions as an upstream regulator of Twist1 and is involved in the invasiveness of cervical cancer cell.
Female ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; HeLa Cells ; Humans ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; RNA Interference ; RNA, Small Interfering ; Twist-Related Protein 1 ; metabolism ; Uterine Cervical Neoplasms ; pathology

Objective To explore the clinical value of high-frequency color Doppler ultra-sonography in identifying axillary metastatic and benignant lymph nodes of breast cancer.Methods 106 axillary enlarged lymph nodes of 78 patient with breast cancer were retrospectively analyzed by high-frequency two-dimensional ultrasonography combined with color Doppler flow imaging (CD-FI).The sonograms and characteristics of blood flow were compared between 56 axillary metastatic enlarged lymph nodes and 50 axillary benignant enlarged lymph nodes.Results There were signifi-cant differences of L /S,thickness-increased characteristics of skin texture,presence or absence of confluent shape and calcified plaque,distribution characteristics of blood flow signals and degrees of blood flow between axillary metastatic and benignant enlarged lymph nodes of breast cancer.Conclu-sion Axillary metastatic enlarged lymph nodes of breast cancer are greatly different from axillary benignant enlarged lymph nodes in the shape,internal echo and blood flow patterns.High-frequen-cy color Doppler ultrasonography is a useful method with higher clinical application value for identi-fying axillary metastatic and benignant enlarged lymph nodes of breast cancer.

Objective To explore the clinical value of high-frequency color Doppler ultra-sonography in identifying axillary metastatic and benignant lymph nodes of breast cancer.Methods 106 axillary enlarged lymph nodes of 78 patient with breast cancer were retrospectively analyzed by high-frequency two-dimensional ultrasonography combined with color Doppler flow imaging (CD-FI).The sonograms and characteristics of blood flow were compared between 56 axillary metastatic enlarged lymph nodes and 50 axillary benignant enlarged lymph nodes.Results There were signifi-cant differences of L /S,thickness-increased characteristics of skin texture,presence or absence of confluent shape and calcified plaque,distribution characteristics of blood flow signals and degrees of blood flow between axillary metastatic and benignant enlarged lymph nodes of breast cancer.Conclu-sion Axillary metastatic enlarged lymph nodes of breast cancer are greatly different from axillary benignant enlarged lymph nodes in the shape,internal echo and blood flow patterns.High-frequen-cy color Doppler ultrasonography is a useful method with higher clinical application value for identi-fying axillary metastatic and benignant enlarged lymph nodes of breast cancer.

OBJECTIVE: To explore the relation between human papillomavirus (HPV16) infection and expression of Toll-like receptor 4 (TLR4) in cervical cell lines and cervical lesion tissues and to investigate the effect of TLR4 on cervical cancer progression. METHODS: Expression of HPV16 E6 mRNA was detected by RT-PCR. Western blot and immunohistochemistry were used to detect the expression of TLR4 in H8, SiHa, Caski cell lines and formalin-fixed and paraffin-embedded cervical tissue specimens with cervicitis, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinama (CSCC). DNA was extracted from paraffin-embedded cervical cancer tissues and HPV16 genes were detected. RESULTS: The differentiation expression of HPV16 E6 mRNA and TLR4 in SiHa and Caski was significantly higher than that of normal cervical cell H8 (P<0.05). The positive expression rates of TLR4 and HPV16 in chronic cervicitis, CIN, and cervical cancer were 32.0%, 59.4%, and 77.8% (P<0.01) and 8.0%, 48.4%, and 81.0% (P<0.01), respectively. Up-regulation of TLR4 was correlated with tumor differentiation (P<0.01), but not with FIGO stages or lymph node metastasis (P>0.05). The expression of TLR4 was significantly correlated with HPV16 infection in CIN and CSCC (r=0.303, P<0.05, r=0.633, P<0.05). CONCLUSION High expression of TLR4 may play important roles in the development and progression of CIN and CSCC, and the expression of TLR4 can be up-regulated by HPV16 infection.
Carcinoma, Squamous Cell ; metabolism ; virology ; Cell Line, Tumor ; Cervical Intraepithelial Neoplasia ; metabolism ; virology ; Female ; Human papillomavirus 16 ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Oncogene Proteins, Viral ; metabolism ; Papillomavirus Infections ; metabolism ; RNA, Messenger ; Repressor Proteins ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; metabolism ; virology

Objective:To obtain the full length cDNA sequences of hepatoma associated gene HTA, analyze its alternative splicing, detect the expression pattern of 2 HTA gene transcripts in different hepatic cell lines, and to establish a base for further study of HTA gene function in hepatocellular carcinoma (HCC) occurence and development. Methods:The full length cDNA of HTA gene was cloned by rapid amplification of cDNA 3' ends (3'-RACE), rapid ampliifcation of cDNA 5' ends (5'-RACE) and DNA sequencing. The gene structure and alternative splicing were analysed. Northern blot assay was performed to detect the expression pattern of 2 HTA gene transcripts in different hepatic cell lines. Results:The full length of HTA gene was 1414 bp, composed of 3 exons and 2 introns, and the second intron could be retained in mRNA. Northern blot assay showed that 2 transcripts of HTA mRNA(1.4 kb and 1.7 kb) could express in the HCC cell lines HepG2 and QGY-7703, but not in the non-malignant cell line L-02 and HUVEC. The expression level of 1.4 kb transcript was much higher than 1.7 kb one. Conclusion:This study successfully has obtained the full length cDNA of HTA gene, and analysed the gene sequence and alternative splicing, 2 transcripts of HTA mRNA specifically expressed in HCC cell lines. As a hepatoma associated gene, HTA deserves further investigation.

Objective To express and purify the human scFv antibody,SA3,against the hepatoma fused to enhanced green fluorecsent protein,and to observe the targeted capacity of fusion protein EGFP-SA3 in vivo.Methods SA3 and EGFP genes were cloned into plasmid pET-25b( + )to construct the recombinant plasmid EGFP-SA3/pET-25b ( + ),followed by DNA sequencing.Then it was transformed into E.coli BL21 ( DE3 ) and induced for fusion expression of EGFP-SA3with IPTG.The expressed fusion protein EGFP-SA3 was purified and detected with SDS-PAGE.HepG2 cells were incubated with the fusion protein EGFP-SA3 in vitro,and the binding bioactivity was observed under the fluorecsent microscope.Further more,we injected the EGFP-SA3 by caudal vein into nude mice planted by hepatoma and observed the whole body fluorescence image of EGFP.Results SA3 and EGFP genes were successfully cloned into pET-25b( + ),which was confirmed by restriction enzyme Nco I-Xho I or Nco I-Eco RI.A band migrated at the position 750 bp,same to EGFP gene,emerged when recombinant plasmid was digested by restriction enzyme Nco I-Eco RI.Similarly,a band,about 1 500 bp,emerged when digested by Nco I-Xho I.The open-reading frame was confirmed by DNA sequencing.Fusion protein EGFP-SA3 was expressed as inclusion body.After purification and refolding,the result of immunofluorecsence detection verified that EGFP-SA3could specifically bind to HepG2 cells and maximum tumor penetration was at 24 h after the injection.Conclusion The purified fusion protein EGFP-SA3 has strong binding capacity to HepG2cells,indicating the scFv SA3 has a potential value as a targeting molecule for diagnosis and targeted therapy for liver cancer.

OBJECTIVE: To recombinant express hepatoma associated gene(HTA) and pre-test the function of HTA to determine the role of HTA in the development of liver cancer. METHODS: HTA338-616 was amplified from HepG2 cells and cloned into the prokaryotic expression vector pET21a(+)-MBP. The proteins MBP and MBP-HTA were induced, purified by His-tag magnetic bead purification kit and identified by Western blot and ELISA. HepG2 cells were stimulated with MBP or MBP-HTA proteins. MTT assay and colony formation assay were employed to examine the proliferation of these cells and the changes of cell cycle distribution were determined by flow cytometry. RESULTS: The prokaryotic expression plasmid pET21a(+)-MBP-HTA was successfully constructed. We got a 52 kD purified purpose protein.The proliferation of HepG2 cells stimulated with MBP-HTA was significantly higher than those stimulated with MBP and negative controls. HepG2 cells stimulated with MBP-HTA showed significant decrease fraction in G1 phase and increase fraction in S phase, and the cell proliferation was enhanced. CONCLUSION HTA protein can significantly promote the proliferation of HepG2 cells, which may be related to the promotion of G1 phase to S phase.
Base Sequence ; Cell Proliferation ; drug effects ; Escherichia coli ; genetics ; metabolism ; Genes, Neoplasm ; physiology ; Genetic Vectors ; Hep G2 Cells ; Humans ; Maltose-Binding Proteins ; biosynthesis ; genetics ; pharmacology ; Molecular Sequence Data ; Neoplasm Proteins ; biosynthesis ; genetics ; pharmacology ; Open Reading Frames ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology