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Objective: To investigate the role of Rho GTPase-activating protein 21 (ARHGAP21) in regulating the migration and metastasis of non-small cell lung cancer (NSCLC) cells., Methods: TCGA, CPTAC database were used to analyze the correlation of ARHGAP21 expression level in NSCLC and the patients' prognosis. The expression of ARHGAP21 in clinical specimens of NSCLC tissues was examined using Western blotting and immunohistochemistry. The effect of ARHGAP21 knockdown on migration ability of lung cancer cell lines was examined using Transwell assay and wound healing assay. A nude mouse model with injection of lung cancer H1299 cells via the tail vein was used to examine the effect of ARHGAP21 knockdown on the metastatic ability of the tumor cells. The possible mechanism of ARHGAP21 was predicted by bioinformatics analysis and verified using Western blotting., Results: A low ARHGAP21 expression was associated with poor prognosis of patients with NSCLC ( P < 0.05). ARHGAP21 expression was significantly downregulated in lung cancer tissues as compared with the adjacent tissues ( P < 0.001). In cultured lung cancer cells, ARHGAP21 knockdown obviously promoted the migration ability of the cells ( P < 0.001). In the nude mouse models, injection of H1299 cells with ARHGAP21 knockdown, as compared with the negative control cells, resulted in a greater number of metastatic lung cancer nodules ( P < 0.05), which expressed higher levels of N-cadherin and vimentin. Bioinformatic analysis showed a close correlation of ARHGAP21 with APC, GSK3β, and Axin ( P < 0.001). Western blotting showed that ARHGAP21 knockdown significantly decreased ubiquitination of β-catenin, upregulated N-cadherin and activated the WNT signaling pathway in the lung cancer cells., Conclusion: ARHGAP21 downregulation can significantly promote the migration and metastatic ability of NSCLC possibly as a result of WNT signaling pathway activation, which reduces the ubiquitination of β-catenin by affecting the expressions of APC, GSK3β, and Axin.

Background and objective Axin is an important negative regulator of Wnt signaling pathway. It can induce the phosphorylation and degradation of β-catenin. The reduced expression of axin or high expression of β-catenin and TCF-4 were associated with malignant proliferation in many tumors. The aim of this study is to examinethe relationships among the expressions and locations of axin, β-catenin and other relevant molecules, and the roles of axin on proliferation, invasive ability and apoptosis of lung cancer cells. Methods The axin cDNA was transfected into lung cancer BE1 cell line which has very low axin expression. The levels of expression and location of axin, β-catenin and TCF-4 before and after transfection were detected using immunofluorescence. The mRNA levels of expression of axin, β-catenin and TCF-4 were examined using reverse transcription-polymerase chain reaction (RT-PCR). The apoptosis,proliferation and invasive ability of lung cancer cells before and after transfection were examined with flow cytometry, MTTand transwell methods. Results After transfection of axin gene into BE1 cells (BE1-axin cells), axin mRNA and protein were overexpressed significantly. Meanwhile, the protein expression of β-catenin and mRNA expression of TCF-4 were decreased significantly in BE1-axin cells than that in BE1 or vector control cells. The flow cytometry revealed that the apoptosis rate of BE1-axin cells was enhanced, but MTT and Transwell assay indicated that the proliferation andinvasive ability were decreased significantly in BE1-axin cells than those in BE1 or vector control cells. Conclusion The overexpression of axin could down-regulate the protein expression of β-catenin and the transcription of TCF-4, and inhibit the proliferation and invasive ability of lung cancer cells.

Objective To construct the axis inhibition protein 1 (AXIN1) gene-knockout ACT-1 human undifferentiated thyroid cancer single clone cell line. Methods Molecular cloning technology and clustered regularly interspaced short palindromic repeats/Cas9 nuclease (CRISPR/Cas9) were used to construct AXIN1 gene-knockout single clone cell lines. Real-time quantitative PCR and Western blotting were used to detect AXIN1 mRNA and protein levels of ACT-1 cells, respectively. Results T7 detection results showed two effective single guide RNAs (sgRNAs) Cr3 and Cr5 were successfully constructed; enzyme digestion identification and sequencing showed AXIN1-targeted sgRNA viral vectors carrying green fluorescent protein (GFP) were successfully constructed. We successfully obtained 4 monoclonal ACT-1 undifferentiated thyroid cancer cell lines. AXIN1 mRNA and protein levels in the gene-knockout group were significantly reduced. Conclusion The ACT-1 undifferentiated thyroid cancer cell line with AXIN1 gene knockout has been successfully constructed using CRISPR/Cas9.

Objective: To investigate the mRNA and protein expression of axin inhibitor (AXIN), β-chain protein (β-catenin), matrix metalloproteinase-7 (MMP-7) and matrix metalloproteinase-9 (MMP-9), and their relationship in lymphoma cells., Methods: The expressions of MMP-7, MMP-9, β-catenin and AXIN in lymphoma cell lines were detected by Western blot and RT-PCR. Moreover, the lymphoma cells with relatively low expression of AXIN were grouped and were transiently transfected by using pcDNA5-His-β-catenin and pCMV5-HA-AXIN; the protein and mRNA expression of MMP-7, MMP-9 and β-catenin in lymphoma cells was detected by Western blot and RT-PCR, respectively; the cell infiltration and migration ability in group with stable ligh expression of AXIN, group of interfering stable high expression of AXIN and blank control group were analyzed by transwell experiment., Results: The AXIN negatively correlated with MMP-7, MMP-9 and β-catenin expression in lymphoma cell lines. After the up-regulation of AXIN, the mRNA expression of MMP7, MMP-9 and β-catenin in Raji cells all not significantly changed, while the pratein expression of MMP-7, MMP-9 and β-catenin all significantly decreased (P<0.05); after the up-regulation of β-catenin, the mRNA and protein expression of MMP-7, MMP-9 was also up-regulated significantly (P<0.05). After interfering the AXIN, the mRNA expression of MMP-7, MMP-9 and β-catenin in group with stable high expression of AXIN all not changed significantly, while protein expression of MMP-7, MMP-9 and β-catenin was down-regulated significantly (P<0.05); after interfering the β-catenin, the protein and mRNA expression of MMP-7 and MMP-9 in group with stable high expression of AXIN all were down-regulated significantly(P<0.05)., Conclusion: The up-regulation of AXIN expression in lymphoma cells can lead to decrease of β-catenin expression and the resuts in significant decrease of MMP-7 and MMP-9 expression, there by plays a role to block the infiltration and migration of lymphoma cells.

In recent years, Wnt/beta-catenin signaling has been identified as a key player in embryogenesis and human diseases. Canonical Wnt signaling pathway is controlled by a variety of classic molecules like Wnt, beta-catenin, Axin, APC, GSK-3beta and CK1, which interact and coordinate to regulate the expressions of cell signaling molecules. The latest evidences suggest that some components of the Wnt/beta-catenin signaling, like APC, GSK-3beta, CK1, Dkk2 and WISE, play dual roles different from what they have been thought previously. Here we reviewed some recent discoveries on the canonical Wnt/beta-catenin signaling pathway to provide some new ideas and principles for signaling transduction studies.

Objective: To investigate the association of Axis inhibitor-2 (AXIN2) gene rs2240308, rs8081536 and rs9913621 single nucleotide polymorphisms (SNPs) with Hirschsprung disease(HSCR)., Methods: DNA was extracted from 120 HSCR patients and 120 healthy controls. The AXIN2 gene exon2-rs2240308, exon5- rs8081536 and exon6-rs9913621 were amplified by polymerase chain reaction (PCR). SNPs of AXIN2 gene were analyzed by restrictive endonuclease digestion with CviJI, DdeI and BstNI and DNA sequencing. The allele and genotype frequencies and risk factors of HSCR and control group were analyzed by Chi-square test., Results: No significant differences were found in genotype frequencies of CC and CT in AXIN2 rs8081536 between HSCR patients and the control group (P> 0.05). The frequencies of genotypes GG, AG and AA as well as alleles A and G genotypes in AXIN2 gene rs2240308 locus were found to be associated with HSCR (P< 0.05). The disease risk of genotypes GG and AA and allele G with was 2.091, 0.846 and 1.703, respectively. The frequencies of genotypes CC, CT and TT as well as alleles C and T in AXIN2 gene rs9913621 locus were also associated with HSCR (P< 0.05). The disease risk of the genotypes CC and TT and the allele T was 0.535, 1.113 and 1.569, respectively. Heterozygote mutation for rs2240308 was found in the HSCR patients, i.e. the GCA to CCA mutation at position 301. Heterozygosity for rs9913621 was observed in the HSCR patients, i.e. the CAC to CAG mutation at position 199., Conclusion: The rs8081536 allelic variation in AXIN2 gene does not contribute to the susceptibility of HSCR in the patients. AXIN2 rs2240308 and rs9913621 allelic variation might be related to HSCR. Individuals having allele G and T in these loci are at relatively high risk for HSCR.

Background & Objective: Axin2 gene which negatively regulates the Wnt signaling pathway was cloned recently. A truncated mutation of Axin2 (mtAxin2) is the most common mutation pattern in colorectal cancer (CRC) and could enhance the luciferase activity of T-cell factor (TCF). This study was to explore the mechanism of Wnt signaling pathway regulation by mtAxin2 in CRC., Methods: The expression of beta-catenin in a mtAxin2-positive and a mtAxin2-negative CRC specimens was detected by immunohistochemistry. Plasmids containing wild-type or mutated Axin2 (pCMV-Flag-wtAxin2 and pCMV-Flag-mtAxin2) were constructed and co-transfected into 293 cells. The interactions of wtAxin2 and mtAxin2 with core components in Wnt signaling pathway was tested by co-immunoprecipitation (IP) and Western blot. TNT T3/T7 in vitro transcription and translation system were used to produce mtAxin2 and wtAxin2 proteins to verify the homodimerization of mtAxin2. Firefly and Renilla activities of mtAxin2 were detected with dual-luciferase report assay. Retinoid X receptor (RXR) and ecodysone receptor (ECR) plasmids were prepared for the experiment of restoration of TCF activity of mtAxin2., Results: Immunohistochemistry showed that beta-catenin accumulated in the nuclei of mtAxin2-positive CRC cells and in cytoplasm of mtAxin2-negative CRC cells. Western blot showed that mtAxin2 bound to GSK-3beta, APC, beta-catenin, DVL-1 and PP2A as wtAxin2 did. After co-transfection of wtAxin2 and mtAxin2 in 293 cells, mtAxin2 competitively bound to beta-catenin. TNT T3/T7 experiment showed that wtAxin2 formed oligomers or dimers with Flag-wtAxin2, but not with Flag-mtAxin2. TCF activity in 293 cells was increased after transfection of pCMV-mtAxin2-RXR, but reduced after transfection of pCMV-mtAxin2-ECR or co-transfection of pCMV-mtAxin2-RXR and pCMV-mtAxin2-ECR, indicating the fusion of mtAxin2 and RXR formed dimers and restored its inhibitory effect on TCF activity., Conclusion: Because of loss of DIX domain, mtAxin2 can not form dimmers, disturbs the degradation of beta-catenin, and leads to the nuclear accumulation of beta-catenin and activation of Wnt signaling pathway.

Background & Objective: Previous researches showed that down-regulating the expression of cyclin B1 in tumor cells by RNA interference may inhibit tumorigenesis, but the mechanism remains to be clarified. This study was to reveal the molecular mechanism of antisense cyclin B1 in tumorigenesis inhibition by comparative proteomic technique., Methods: A recombinant plasmid containing the full-length antisense cDNA of mouse cyclin B1 was transfected into mouse colon carcinoma cell line CT26. Total proteins of transfected cells and control cells were extracted and separated by two-dimensional gel electrophoresis (2-DE). The differential expression proteins were analyzed with PDQuest software, and identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and Mascot database searching. The 2 differential proteins with the highest confidence of the peptides were selected and verified by Western blot., Results: Seven differentially expressed proteins were identified: Axin2, CCTtheta, DR5, and HPCM27 were up-regulated in transfected cells, while RFP17, mKIAA1195, and LOC77035 were down-regulated. The expression abundance differences of Axin2 and DR5, with the highest confidence, were verified by Western blot., Conclusions: Several proteins expressed differentially in CT26 cells after transfection of antisense cyclin B1, which take part in some signal pathways in cell proliferation, differentiation, migration, apoptosis, and transcriptional control. The antitumor effect of antisense cyclin B1 may relate to the interplay of the above proteins.

Objective: To investigate the protein expression of Axin and beta-catenin, the exon 3 mutation status of beta-catenin and their clinicopathological correlations in non-small cell lung cancer (NSCLC)., Methods: A total of 100 NSCLC samples and their corresponding normal lung tissues were obtained from the patients undergoing surgery in the First Affiliated Hospital of China Medical University between 2001 and 2003. Protein expressions of Axin and beta-catenin were detected by immunohistochemistry. DNA sequence alterations of exon 3 of beta-catenin were investigated by polymerase chain reaction (PCR) and direct sequencing., Results: A reduced membranous expression rate of beta-catenin was observed in 80.0% of the cases (80/100) along with a nuclear expression rate of 26.0% (26/100). There was a significant difference in beta-catenin expression between well and poorly differentiated NSCLCs. Well to moderately differentiated NSCLCs showed a reduced expression rate of 70.0% (35/50), in contrast to 90.0% (45/ 50) in poorly differentiated tumors (P = 0.012). Reduced beta-catenin expression rate was 87.3% (48/55) in cases with lymph node metastasis, in contrast to 71.1% (32/45) in cases without lymph node metastasis (P = 0.044). The positive expression rate of Axin was 48.0% (48/100). Well to moderately differentiated NSCLCs demonstrated a 60.0% positive expression rate of Axin (30/50), much higher than poorly differentiated tumors [36.0% (18/50), P = 0.016]. The positive expression rate of Axin in beta-catenin nuclear expressed NSCLCs was 15.4% (4/26), much lower than cases without beta-catenin nuclear expression [59.5% (44/74), P < 0.001]. Axin nuclear expression was found in two cases in this study, suggesting that it may function as a nuclear-cytoplasmic shuttling protein. PCR and direct sequencing failed to reveal any exon 3 mutation of beta-catenin gene., Conclusions: The reduced membranous expression of beta-catenin is associated with poorly differentiated and lymph node positive NSCLCs. The expression of Axin is inversely correlated with the degree of tumor differentiation and nuclear expression of beta-catenin. The exon 3 mutations do not contribute to the abnormal protein expression of beta-catenin in NSCLCs.

Aim: To construct the eukaryotic expression vector pIRES2-EGFP-Axin, and to express Axin in C6 glioma cells., Methods: The Axin gene was amplified by PCR using pCMV5-HA-Axin as a template, and confirmed by DNA sequencing. The eukaryotic expression vector pIRES2-EGFP-Axin was constructed by introducing Axin DNA fragment into the sites of Nhe I and Sal I of pIRES2-EGFP vector. The plasmid was transfected into the C6 cells using lipofectamine. The expressed EGFP was observed under fluorescent microscope and the Axin protein expression was detected by immunostaining using anti-Axin antibody., Results: The eukaryotic expression vector pIRES2-EGFP-Axin was constructed and transfected successfully into C6 glioma cells. The green fluorescence of EGFP was observed in the plasma and nuclei of transfected cells, and Axin protein was only found in the plasma., Conclusion: The recombinant expression vector pIRES2-EGFP-Axin was constructed, and the EGFP and Axin gene could be co-expressed in the C6 cells. This study laid a foundation for the further research of the function of Axin in cell differentiation, growth and tumorigenesis.