Aurora A Kinase Plays a Key Role in Mitosis Skip during Senescence Induced by Ionizing Radiation / 生物医学与环境科学（英文）

OBJECTIVE@#To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation (IR).@*METHODS@#Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237 (MLN) and/or p21 depletion by small interfering RNA (siRNA). Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator (FUCCI) system combined with histone H3 phosphorylation at Ser10 (pS10 H3) detection. Senescence was assessed using senescence-associated-β-galactosidase (SA-β-Gal), Ki67, and γH2AX staining. Protein expression levels were determined using western blotting.@*RESULTS@#Tumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment. The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells, ultimately leading to senescence in G1. During this process, the p53/p21 pathway is hyperactivated. Accompanying p21 accumulation, Aurora A kinase levels declined sharply. MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction.@*CONCLUSION@#Persistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation, leading to senescence via mitotic skipping.

Molecular Mechanism of Aurora Kinase A Regulating the Meiosis of Oocyte / 中国医学科学院学报

Aurora kinase A (AURKA),a family member of aurora kinases,is involved in mitotic entry,maturation and separation of centrosome,assembly and stabilization of bipolar spindle,and condensation and separation of chromosome.Studies have demonstrated that AURKA plays a similar role in meiosis,while the specific mechanism and the similarities and differences in its role between meiosis and mitosis remain unclear.Therefore,we reviewed the studies about the localization and activation of AURKA in oocyte meiosis,and compared the role of AURKA in regulating spindle formation,activating spindle assembly checkpoint,and correcting the kinetochore-microtubule attachment between the meiosis of oocytes and the mitosis of somatic cells.This review will lay a theoretical foundation for revealing the mechanism of AURKA in the regulation of cell division and for the clinical research related to cancer and reproduction.

Investigação das quinases Aurora A e Aurora B na tumorigenicidade mediada pelo oncogene KRAS / Investigation of Aurora kinases A and B in KRAS-induced lung tumorigenesis

O câncer de pulmão é a principal causa de morte relacionada ao câncer no mundo. Mutações em KRAS são altamente prevalentes no câncer e têm sido diretamente associadas ao processo tumorigênico. Apesar disso, até hoje todas as terapias visando inibir KRAS diretamente falharam e a caracterização de alvos indiretos, importantes para a oncogênese mediada por KRAS, é fundamental para o desenvolvimento de novas terapias contra o câncer de pulmão. Nós mostramos previamente que as quinases Aurora A (AURKA) e B (AURKB) são alvos a jusante de KRAS, importantes para o crescimento, viabilidade e oncogenicidade de linhagens celulares derivadas de tumores pulmonares mediados por KRAS. Aqui, nós aprofundamos os nossos estudos para melhor caracterizar AURKA e AURKB como potenciais alvos terapêuticos no câncer de pulmão. Os objetivos deste trabalho foram (1) investigar o mecanismo de perda de viabilidade induzido pela inibição de AURKA e/ou AURKB; (2) avaliar como a inibição de AURKA e/ou AURKB afeta propriedades oncogênicas relacionadas à agressividade tumoral; e (3) como a inibição destas quinases afeta o crescimento tumoral in vivo. Para tanto, nós utilizamos dois modelos celulares: (1) células A549 e H358, que apresentam mutações em KRAS, geneticamente modificadas para a expressão estável e induzível de shRNAs contra AURKA ou AURKB, e (2) células tumorais H1703, que não apresentam mutações em KRAS, geneticamente modificadas para a expressão induzível de KRASG12V, tratadas ou não com inibidores farmacológicos das quinases Aurora. A inibição farmacológica ou por interferência de RNA de AURKA e/ou AURKB em células H358 e A549 reduziu a proliferação celular, sendo esta inibição acompanhada de anomalias mitóticas, além de aneuploidia e poliploidia. A inibição destas quinases também induziu morte celular in vitro, tanto em mitose, quanto em interfase. Mais interessantemente, a inibição farmacológica dual de AURKA e AURKB induziu morte celular in vitro em células H1703, somente na presença de KRASG12V, indicando que a inibição das quinases Aurora afeta preferencialmente células portadoras de mutações em KRAS. Além disso, a inibição de AURKA e/ou AURKB reduziu propriedades malignas celulares relacionadas à agressividade tumoral, como migração, invasão e adesão. Finalmente, a inibição de AURKA por RNA de interferência em células A549 também reduziu a formação de tumores in vivo. Entretanto, como a inibição destas quinases levou a anomalias mitóticas e à instabilidade genética, nós resolvemos investigar se a inibição de TPX2, um substrato e ativador de AURKA, poderia ser uma abordagem alternativa para inibir esta via em câncer de pulmão induzido por KRAS. Primeiramente, nós observamos nos nossos modelos celulares que KRAS regula positivamente a expressão de TPX2. Além disso, a inibição de TPX2 em células pulmonares portadoras de KRAS oncogênica reduziu a viabilidade e proliferação celulares e induziu morte celular. Mais interessantemente, esses efeitos ocorreram preferencialmente em células que expressam KRAS oncogênica. Em conclusão, nossos resultados apoiam a hipótese de que a ativação de AURKA/TPX2 e AURKB por KRAS são eventos importantes no câncer de pulmão e sugerem a inibição destas vias, possivelmente em combinação com outras terapias citotóxicas, como uma nova abordagem terapêutica para o câncer de pulmão induzido por KRAS

Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS mutations are widespread in lung cancer and have been causally linked to tumorigenesis. Nonetheless, therapies targeting KRAS directly have so far failed and characterization of indirect KRAS targets, which play important roles in KRAS-mediated oncogenesis, is crucial for the development of new therapies for lung cancer. We have previously shown that mitotic kinases Aurora A (AURKA) and B (AURKB) are downstream targets of oncogenic KRAS, important for the growth, viability, and oncogenicity of KRAS-transformed lung cancer cell lines. Here, we studied these kinases more in depth in order to better characterize them as potential therapeutical targets for KRAS-induced lung cancer. The aims of this study were (1) to investigate the mechanism leading to loss of viability upon AURKA and/or AURKB targeting; (2) to evaluate how AURKA and/or AURKB inhibition affects malignant properties associated with tumor aggressiveness; and (3) to determine whether AURKA and/or AURKB inhibition reduces KRAS-induced tumor growth in vivo. For that purpose, we used two cell-based models: (1) KRAS mutant A549 and H358 cells with stable and inducible shRNA-mediated knockdown of AURKA or AURKB, and (2) KRAS wildtype H1703 tumor cell lines, genetically engineered to inducibly express oncogenic KRASG12V treated or not with Aurora kinase pharmacological inhibitors. Targeting AURKA and/or AURKB pharmacologically or by RNA interference in H358 and A549 cells led to decreased cell proliferation, which was accompanied by mitotic abnormalities, leading to aneuploidy and hyperploidy. Aurora kinase targeting also induced cell death in vitro, both during mitosis and interphase. More importantly, AURKA and AURKB inhibition with a dual pharmacological inhibitor in H1703 cells induced cell death in vitro, but only in the presence of KRASG12V, indicating that Aurora kinase targeting affects preferentially lung cells harboring oncogenic KRAS. Furthermore, AURKA and/or AURKB targeting reduced malignant properties associated with tumor aggressiveness, such as cell migration, invasion and adhesion. Finally, AURKA targeting by RNA interference in A549 cells also reduced growth of xenograft tumors in vivo. Nonetheless, since Aurora targeting was associated with mitotic abnormalities and genetic instability, we decided to investigate if targeting TPX2, a substrate and an activator of AURKA, could constitute an alternative approach to targeting this pathway in KRAS-induced lung cancer. First, using our cell-based models, we determined that KRAS positively regulates TPX2 expression. In addition, TPX2 inhibition by RNA interference in KRAS-positive lung cells reduced cell viability and proliferation and induced cell death. Finally, these effects occurred preferentially in cells harboring oncogenic KRAS. In conclusion, our results support the hypothesis that activation of AURKA/TPX2 and AURKB by KRAS are important events in lung cancer and suggest inhibition of these pathways, possibly in combination with other cytotoxic therapies, as a new approach for KRAS-induced lung cancer therapy

Characterization of the Indirubin Derivative LDD970 as a Small Molecule Aurora Kinase A Inhibitor in Human Colorectal Cancer Cells

Aurora kinase A plays an essential role in mitosis including chromosome separation and cytokinesis. Aberrant expression and activity of Aurora kinase A is associated with numerous malignancies including colorectal cancer followed by poor prognosis. The aim of this study is to determine the inhibitory effects of LDD970, an indirubin derivative, on Aurora kinase A in HT29 colorectal cancer cells. In vitro kinase assay revealed that, LDD970 inhibited levels of activated Aurora kinase A (IC₅₀=0.37 mM). The inhibitory effects of LDD970 on Aurora kinase A, autophosphorylation and phosphorylation of histone H3 (Ser10), were confirmed by immunoblot analysis. Moreover, LDD970 inhibited migration of HT29 cells and upregulated apoptosis-related protein cleaved PARP. In cell viability assay, LDD970 was observed to suppress HT29 cell growth (GI₅₀=4.22 µM). Although further studies are required, results of the present study suggest that LDD970 provide a valuable insight into small molecule indirubin derivative for therapeutic potential in human colorectal cancer.

Aurora Kinase A Is a Prognostic Marker in Colorectal Adenocarcinoma

BACKGROUND: Aurora kinase A (AURKA), or STK15/BTAK, is a member of the serine/threonine kinase family and plays important roles in mitosis and chromosome stability. This study investigated the clinical significance of AURKA expression in colorectal cancer patients in Korea. METHODS: AURKA protein expression was evaluated by immunohistochemistry in 151 patients with colorectal adenocarcinoma using tissue microarray blocks. We analyzed the relationship between clinicopathological characteristics and AURKA expression. In addition, the prognostic significance of various clinicopathological data for progression-free survival (PFS) was assessed. Also we evaluated copy number variations by array comparative genomic hybridization and AURKA gene amplification using fluorescence in situ hybridization in colorectal carcinoma tissues. RESULTS: AURKA gene amplification was found more frequently in the 20q13.2–13.33 gain-positive group than the group with no significant gain on the AURKA-containing locus. AURKA protein expression was detected in 45% of the cases (68/151). Positive staining for AURKA was observed more often in male patients (p = .035) and distally located tumors (p = .021). PFS was shorter in patients with AURKA expression compared to those with low-level AURKA expression (p < .001). Univariate analysis revealed that AURKA expression (p = .001), age (p = .034), lymphatic invasion (p = .001), perineural invasion (p = .002), and TNM stage (p = .013) significantly affected PFS. In a multivariate analysis of PFS, a Cox proportional hazard model confirmed that AURKA expression was an independent and significant prognostic factor in colorectal adenocarcinoma (hazard ratio, 3.944; p < .001). CONCLUSIONS: AURKA could serve as an independent factor to predict a poor prognosis in Korean colorectal adenocarcinoma patients.

The anti-tumor activity and molecular mechanisms of an Aurora kinase inhibitor ZLJ213 in suppressing colon cancer growth / 药学学报

The aim of this study is to evaluate anti-tumor activities and mechanism of a novel kinase inhibitor ZLJ213 which targeted Aurora A and vascular endothelial growth factor receptor (VEGFR) in vitro and in vivo against human colon cancer. Results showed that ZLJ213 inhibited cell proliferation and induced cell cycle arrest and apoptosis of HCT1 16 and SW48 cell lines. In HCT116-derived xenograft, ZLJ213 dosed at 100 mg · kg(-1) inhibited tumor growth by 73.24%. The IC50 of ZLJ213 on the expression of p-Aurora A was 0.258 µmol · L(-1) analyzed by ELISA. Under the concentration of 0.08 µmol · L(-1), ZLJ213 could inhibit the activities of Aurora A, Histone H3 and VEGFR of HCT116 and SW48 cell lines. Simultaneously, ZLJ213 induced activation of Caspase 3 and PARP cleavage. Above data suggested that ZLJ213 had the ability to inhibit cell proliferation and induce cell apoptosis both in vitro and in vivo in colon cancer, and down-regulate the expression of p-Aurora A and p-VEGFR. ZLJ213 might be a potential therapeutic agent against colon cancer.

Prognostic value of combined expression of Aurora A, p53 and p21 WAF1 in patients after curative resection of non-small cell lung cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The aim of this study was to investigate the prognostic value of combined expression of Aurora A, Ki-67, p53 and p21 WAF1 in patients after curative resection of non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>Expressions of Aurora A, Ki-67, p53 and p21 WAF1 in 58 tumor samples from resected primary NSCLCs were detected by immunohistochemistry. The correlation of proteins, survival and clinicopathological characteristics was analyzed.</p><p><b>RESULTS</b>The positive rates of Aurora A, Ki-67, p53 and p21 WAF1 expression were 89.7% (52/58), 53.4% (31/58), 46.6% (27/58) and 34.5% (20/58), respectively. Aurora A expression was positively correlated with nodal metastasis (69.2% vs. 37.8%, P = 0.045). The univariable analysis showed that the overall survival (OS) was 75.0%in patients with low Aurora A expression and 46.0% in patients with high Aurora A expression (P = 0.039). The 3-year survival rate was 40.0% in patients with positive expression of Aurora A and p53, 65.0% in the patients with positive expression of Aurora A or p53, and 82.1% in the patients with negative expression of Aurora A and p53 (P = 0.039). The Cox regression model showed that combined expression of Aurora and p53 is an independent factor affecting the prognosis of NSCLC patients (P = 0.015).</p><p><b>CONCLUSIONS</b>Our findings suggest that the positive expression of Aurora A, Ki-67 and p53 proteins is an unfavorable factor affecting the prognosis for NSCLC patients, and the overexpression of Aurora A is an independent unfavorable factor association with shorter OS in NSCLC patients. Detection of positive Aurora A and p53 expression may be a useful predictive prognostic indicator for NSCLC patients.</p>

Effect of aurora inhibitor VX-680 on proliferation and apoptosis of CML cells / 中国实验血液学杂志

This study was aimed to explore the effect of VX-680, an aurora inhibitor, on proliferation and apoptosis of K562, KCL22 cell lines and CD34⁺ cells from chronic myeloid leukemia (CML) patients in vitro. The proliferation of K562 and KCL22 cell was detected by CCK-8 method. Apoptosis of cells was detected by Annexin V-PI labeling and flow cytometry. The colony forming ability of bone marrow CD34⁺ cells derived from CML patients and donors was determined by the colony forming test. The results showed that the treatment of K562, KCL22 and CML CD34⁺ cells with VX-680 of 20-100 nmol/L for 3 days could obviously inhibit the cell proliferation in a concentration-dependent manner (P < 0.01). VX-680 treatment significantly induced apoptosis of K562 and KCL22 cells. Compared to bone marrow CD34⁺ cells derived from the healthy donors, the colony forming ability of CML CD34⁺ cells derived from bone marrow of CML patients was remarkably reduced (P < 0.01). It is concluded that VX-680, an aurora inhibitor, can inhibit the proliferation and induce apoptosis of CML cells in vitro.

Elevated Aurora Kinase A Protein Expression in Diabetic Skin Tissue

BACKGROUND: Aurora kinase A (Aurora-A) plays an important role in the regulation of mitosis and cytokinesis. Dysregulated Aurora-A leads to mitotic faults and results in pathological conditions. No studies on Aurora-A expression in human diabetic skin tissue have been reported. In light of this, we explored the expression of Aurora-A in human diabetic skin tissue. METHODS: Aurora-A protein was evaluated by western blotting in 6 human diabetic skin tissue and 6 normal skin specimens. RESULTS: Increased expression of Aurora-A protein was detected in all diabetic skin tissue samples in both western blot analysis and immunohistochemical staining. However, in the case of the normal skin tissue, no bands of Aurora-A protein were detected in either the western blotting analysis or the immunohistochemical staining. CONCLUSIONS: Thus far, there have been no studies on the expression of Aurora-A in diabetic skin tissue. However, we believe that oxidative DNA damage related to the expression of Aurora-A protein and Aurora-A could be involved inhuman diabetic skin tissue.

Suppression of Aurora-A by RNA interference inhibits laryngeal cancer Hep-2 cell growth / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of knockdown of Aurora-A by RNA interference on laryngeal cancer Hep-2 cell growth in vitro and in vivo.</p><p><b>METHODS</b>A plasmid containing siRNA against Aurora-A was constructed and transfected into human laryngeal cancer cell line Hep-2. Measurements included the CCK-8 assay for viability and proliferation, Transwell assay for invasion, colony formation assay for cell anchorage-independent growth. Western blot and immunohistochemistry assay for protein expression. Tumorigenicity was observed in vivo.</p><p><b>RESULTS</b>In Hep-2 cells transfected by Aurora-A siRNA (designated as siRNA-3), protein expression of Aurora-A was suppressed by 52%. In CCK-8 assay, absorbance value of siRNA-3 cells (3.268 ± 0.106, (x(-) ± s)) was lower than that of Hep-2 cells (3.722 ± 0.152, F = 17.634, P < 0.001). In Transwell assay, the average invasive cells per field in siRNA-3 cells (110.0 ± 18.0) was less than that in Hep-2 cells (236.0 ± 26.0, F = 26.462, P < 0.01). In colony formation assay, the average colony number of siRNA-3 cells (31.0 ± 6.6) was lower than that of Hep-2 cells (104.0 ± 14.0). The average tumor size in siRNA-3 group was (127.77 ± 174.83) mm(3), which was less than Hep-2 cell group (837.26 ± 101.80) mm(3), (F = 28.187, P < 0.001). Silencing of Aurora-A decreased the expression of focal adhesion kinase (FAK) and matrix metalloproteinase-2 (MMP-2), key regulators in cell adhesion and invasion.</p><p><b>CONCLUSIONS</b>The knockdown of Aurora-A inhibits the growth and invasiveness of Hep-2 cells in vitro and in vivo, which may be a promising therapeutic strategy for LSCC.</p>

The over-expression of serine/threonine kinase 15 protein in oral carcinogenesis / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the expression of STK15 and P53 proteins in oral precancerous lesions and oral squamous cell carcinoma (OSCC) and elucidate the possible role of P53/STK15 switch activation-independent pathway in oral carcinogenesis.</p><p><b>METHODS</b>Formalin-fixed, paraffin-embedded tissues of 8 cases of normal oral epithelium, 27 cases of dysplasia with different degree epithelium dysplasia and 43 cases of OSCC with different differentiation were investigated for the expression of STK15 and P53 proteins by using immunohistochemistry. The clinical and pathological significance of STK15 over-expression in oral carcinogenesis were statistically analyzed by SPSS 12.0.</p><p><b>RESULTS</b>STK15 protein was not detectable in normal oral epithelium and significantly altered from mild-dysplasia to OSCC. The percentage of STK15 over-expression were 40.74% (11/27) in dysplasia and 67.44% (29/43) in OSCC (P < 0.05). The percentage of STK15 over-expression in OSCC with positive P53 staining was significantly higher than that in OSCC with negative P53 staining (P < 0.05). STK15 over-expression was significantly associated with regional lymph node involvement (P < 0.05), while no correlation was found for STK15 over-expression and tumor differentiation, as well as TNM stages.</p><p><b>CONCLUSION</b>STK15 up-regulation was an early event in oral carcinogenesis. The up-regulation of STK15 protein in OSCC may partly result from p53 mutations, which probably contribute a role in lymph node metastasis of OSCC as well. P53/STK15 switch activation-independent pathway may play some roles in oral carcinogenesis.</p>

Genetic polymorphisms in STK15 and MMP-2 associated susceptibility to esophageal cancer in Mongolian population / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the frequencies of alleles and the association with risk of esophageal cancer in a Mongolian population, and to compare the allele frequencies of these polymorphisms between the two populations and the susceptibility to esophageal cancer.</p><p><b>METHODS</b>A case-control study was conducted, and 8 single nucleotide polymorphisms (SNP), including FAS - 670G/A, FAS - 1377G/A, FASL -844T/C, COX-2 - 1290A/G, COX-2 - 1195G/A, STK15 Phe31Ile, MMP-2 - 1306C/T and MMP -2 -735C/T, were detected by polymerase chain reaction-based restriction fragment length polymorphism assay (PCR-RFLP) in 188 esophageal cancer cases and 324 normal controls of Mongolian. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by unconditional logistic regression. The results were then compared with the reported data of the Han ethnic Chinese population.</p><p><b>RESULTS</b>In Mongolian, as compared with the STK15 31Ile/Ile genotype, the STK15 31Phe/Phe genotype carriers had an increased risk of esophageal cancer (adjusted OR = 2.20, 95% CI: 1.12-4.31), and the subjects with MMP-2 - 735TT genotype had an increased risk of esophageal cancer as compared with those with the MMP-2 - 735CC genotype (adjusted OR =4.82, 95% CI: 1.59 - 14.60). However, the rest of SNPs were not associated with the susceptibility to esophageal cancer. The allele frequencies of FASL - 844 T/C [0.264(171/648)/0.736 (477/648), 0.323(418/1296)/0.677(878/1296)], COX-2 - 1195G/A [0.431(279/648)/0.569(369/ 648), 0.492(1250/2540)/0.508(1290/2540)], MMP-2 - 1306C/T [0.869(563/648)/0.131(85/ 648), 0.835(1298/1554)/0.165(256/1554)] and MMP-2 - 735C/T [0.789(511/648)/0.211(137/ 648), 0.748(1163/1554)/0.252(391/1554)] were significantly different between the ethnic populations (chi2 = 7.03, 7.84, 3.94, 4.05, respectively, P <0.05).</p><p><b>CONCLUSION</b>These findings suggested that STK15 Phe31Ile and MMP-2 -735C/T polymorphisms might be the genetic susceptibility factors for esophageal cancer in Mongolian and there should be some differences of genetic susceptibility to esophageal cancer in between Han ethnic Chinese and Mongolian population.</p>

Study on the association between STK15 Phe31Ile polymorphisms and esophageal squamous cell carcinoma / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the relation between single nucleotide polymorphism(SNP) at the 91T-->A(Phe31Ile) position of the STK15 gene and the susceptibility of esophageal squamous cell carcinoma (ESCC) in She county--a ESCC high incidence region in North China.</p><p><b>METHODS</b>Polymerase-chain reaction(PCR)-restriction fragment length polymorphism (RFLP) analysis was used to detect the genotypes of STKl5 Phe31Ile(91T-->A) SNP, and the samples came from 296 ESCC patients and 302 healthy controls.</p><p><b>RESULTS</b>The risk of ESCC significantly increased in the group which had been smoking or having a family history of upper gastrointestinal cancer (UGIC) (the OR = 1.68 and 1.77, 95% CI: 1.34-2.10 and 1.44-2.19, respectively). Rates of the three genotypes (Phe/Phe, Phe/Ile, Ile/Ile) of the STK15 Phe31Ile (91T-->A) SNPs in ESCC patients were 11.5%, 34.8% and 53.7%, respectively, and were not significantly different from that in the healthy group (11.9%, 36.8% and 51.3%) (chi2 = 0.35, P = 0.84). When compared to Phe/Phe genotype, Phe/Ile and Ile/Ile of STK15 91T-->A(Phe31Ile)did not show effect on the risk of ESCC according to the odds ratio results which were 0.98 (95% CI: 0.57-1.69) and 1.09 (0.65-1.82) respectively. STK15 91T-->A (Phe31Ile) SNP also did not significantly influence on the development of ESCC even the samples were stratified by sex, smoking status and family history of upper gastrointestinal cancer.</p><p><b>CONCLUSION</b>The STK15 Phe31Ile(91T-->A) polymorphisms seemed irrelevant with the risk of ESCC in She county.</p>

A study of APin-protein interactions using protein microarray / 대한치과보존학회지

Protein microarray or protein chips is potentially powerful tools for analysis of protein-protein interactions. APin cDNA was previously identified and cloned from a rat odontoblast cDNA library. The purpose of this study was to investigate the APin-protein interactions during ameloblast differentiation. Protein microarray was carried with recombinant APin protein and MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein were selected among 74 interacting proteins. Immortalized ameloblast cells (ALCs) were transfected with pCMV-APin construct and U6-APin siRNA construct. After transfection, the expression of the mRNAs for four proteins selected by protein micoarrays were assessed by RT-PCR. The results were as follows: 1. APin expression was increased and decreased markedly after its over-expression and inactivation, respectively. 2. Over-expression of the APin in the ALCs markedly down-regulated the expression of MEF2 and Aurora kinase A, whereas their expression remained unchanged by its inactivation. 3. Expression of BMPR-IB and EF-hand calcium binding protein were markedly increased by the overexpression of the APin in the ALCs, whereas expression of BMPR-IB remained unchanged and expression of EF-hand calcium binding protein was markedly decreased by its inactivation. These results suggest that APin plays an important role in ameloblast differentiation and mineralization by regulating the expression of MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein.

Mitotic arrest of gastric cancer cells induced by silencing of STK15 gene / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the role of STK15 in regulating mitosis of gastric cancer cells (MKN45) by gene silencing through RNA interference mechanism.</p><p><b>METHODS</b>RNA interference technique was used to inhibit STK15 expression in MKN45 cells. The expression levels of STK15 mRNA and protein were measured by real-time quantitative RT-PCR and Western blot respectively and cell morphological changes were investigated by reverse microscopy. In addition, cell cycle distribution and cellular proliferation were determined by flow-cytometry and MTT assay respectively. Finally, the mitotic phenotype of MKN45 cells was studied by immunofluorescence staining and confocal microscopy.</p><p><b>RESULTS</b>Silencing of STK15 gene by RNA interference was confirmed by marked decrease of STK15 mRNA and protein levels in the treated MKN45 cells. This silencing correlated with rounding of the cells, decreasing of DNA content in G(2) phase (P < 0.05) and a lowered proliferation index (P < 0.05), along with alterations of mitotic phenotype of MKN45 (P < 0.05).</p><p><b>CONCLUSION</b>STK15 gene may play a key role in regulating cellular mitosis and its inhibition by RNA interference leading to mitosis arrest in MKN45 cells.</p>

Association between genetic polymorphism in STK15 and risk of colorectal cancer in a Chinese population / 中华肿瘤杂志

<p><b>OBJECTIVE</b>This case-control study was designed to detect the association between STK15 Phe31Ile polymorphism and colorectal cancer.</p><p><b>METHODS</b>Genotypes were determined in 283 patients with colorectal cancer and 283 controls. The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression model.</p><p><b>RESULTS</b>The frequency of the STK15 Ile/Ile genotype was significantly higher in cancer cases than in controls (50.2% vs. 36.8%; P = 0.02). Subjects with the Ile/Ile genotype had an increased risk for the occurrence of colorectal cancer compared with those with the STK15 Phe/Phe genotype (adjusted OR, 1.92; 95% CI, 1.13 - 3.27). No significant association was observed between this STK15 polymorphism and risk of metastasis of the cancer.</p><p><b>CONCLUSION</b>These findings suggest that STK15 Phe/Ile polymorphism may be a genetic susceptibility factor for colorectal cancer among Chinese.</p>

Analysis of gene expression profiles in gastric cancer cell cycle / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect the gene expression profile in gastric cancer cell cycle and explain the mechanism of gastric cancer cell proliferation by a genomic study.</p><p><b>METHODS</b>Gastric cancer cells MKN45 were synchronized at G2/M and G1/S point by nocodazole-thymidine and double thymidine methods. The synchronizing degree of cells was monitored by flow cytometry. The gene expression profiles at G2/M point, M/G1 transition, G1 early phase, G1 late phase, G1/S point, S early phase, S late phase, G2 early phase and G2 late phase in MKN45 cell cycling were examined using cDNA microarray chips. Hierarchy analysis was conducted with a professional software package and the up-regulated genes at G1 late and G2 phase were analyzed according to gene database. Furthermore, the mRNA level of cyclin E, cyclin B, plk1 and STK15 in above mentioned nine points were measured by quatitative PCR.</p><p><b>RESULTS</b>2001 genes were detected to be available at all 9 points via software processing, out of which 959 appeared up-regulated or down-regulated. 379 genes showed to be up-regulated at late G1 (147) or G2 phases (232), 40 at S and M phases (also up-regulated at G1 late and G2 phases). The 147 up-regulated genes at G1 late phase are involved in DNA metabolism, transcription and translation, protein transportation, ubiquitination and signal transduction, etc. The 232 up-regulated genes in G2 phase are involved in RNA synthesis and processing, intracellular protein transportation, cytoskeleton synthesis, signal transduction, apoptosis and anti-apoptosis, transcription regulation, ubiquitination, mitosis regulation and oncogene expression, etc. The mRNA level of 4 genes detected by quantitative PCR during cell cycle was in agreement with that detected by microarray.</p><p><b>CONCLUSION</b>During MKN45 cell cycling, the preparation for DNA synthesis and chromosome separation are conducted in G1 and G2, which are implicated in multiple genes, may be the main impetus of driving MKN45 cell cycle. Some of these genes may be related to tumor over-proliferation. The cDNA microarray technique has characteristic features such as reliability and can provide a great deal for future research on cell cycle related genes in gastric cancer.</p>

The investigation of STK15 gene amplification and overexpression in laryngeal squamous cell carcinoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the role of STK15 gene amplification and overexpression to genesis and development of laryngeal squamous cell carcinoma (LSCC).</p><p><b>METHODS</b>STK15 gene amplification in 40 cases carcinoma tissues and normal tissues as control was detected by differential PCR approach. STK15 mRNA and protein levels were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry method.</p><p><b>RESULTS</b>In 40 LSCC cases, STK15 gene amplification was found in 14 tumor tissues(35%), mRNA overexpression in 27 tumor tissues(67.5%), and protein upregulated in 29 tumor tissues(72.5%). Statistics analysis showed that STK15 gene amplification and mRNA overexpression were obviously associated to differentiation degree of LSCC, and protein overexpression was closely associated with both differentiation degree and pathological grades of LSCC.</p><p><b>CONCLUSION</b>This research results suggest that STK15 gene amplification contributes to its mRNA and protein overexpression through affecting the exact replication of centrosome and separation of chromosomes. STK15 gene thus plays a role in LSCC oncogenesis and malignant progression.</p>

Inhibition of serine/threonine kinase 15 gene expression induces apoptosis of gastric cancer cells / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To observe the effect of inhibition of serine/threonine kinase15 (STK15) gene expression on apoptosis induction in gastric cancer cell line-MKN45 and discuss the role of STK15 in viability of gastric cancer cells.</p><p><b>METHODS</b>The STK15 expression was inhibited by chemically synthesized siRNA. The STK15 mRNA and protein level were respectively measured by real-time quantitative PCR and western blotting,the change of cell cycle distribution and apoptosis rate were detected by flow-cytometry, cell morphological change was observed by Hoechst staining,and pro-caspase 3 level was also detected by western blot.</p><p><b>RESULTS</b>After treatment by siRNA targeting STK15 after 48 h, STK15 mRNA and protein level decreased obviously. More MKN45 cells accumulated at G(2)/M phase (P< 0.05). The apoptosis rate of STK15 siRNA treated MKN45 cells was higher than that of control cells(P< 0.05) with the pro-caspase 3 level decreased.</p><p><b>CONCLUSIONS</b>Inhibition of STK15 gene expression may induce apoptosis in MKN45 cells through the pathway of caspase3. STK15 gene play a key role in proliferation and viability of MKN45 cells.</p>

Mutation of p53 and overexpression of STK15 in laryngeal squamous-cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the relationship between p53 gene mutations and STK15 abnormal expression in the development of human laryngeal squamous-cell carcinoma (LSCC).</p><p><b>METHODS</b>LSCC tissues and matched normal tissues were taken during operation from 55 patients without previous chemotherapy or radiotherapy. Following polymerase chain reaction amplification direct sequencing single strand conformational polymorphism (PCR-SSCP) combined with silver staining were used to detect mutations of p53 gene in exons 7 and 8 (p53E7 and p53E8) using genomic DNA from 110 specimens including 55 LSCC tissues and 55 matched normal tissues. STK15 expression were evaluated by RT-PCR with beta-actin as internal control.</p><p><b>RESULTS</b>The mutation rate of p53E7 was 30.9% (compared to normal tissues, chi(2) = 8.66, P < 0.01). There was no mutation in p53E8. In 38 of the 55 cases (69.1%), the STK15 mRNA expression level was higher than that of the paired normal tissue. The STK15 to beta-actin ratio of average density value was 1.22 +/- 0.49 in the cancer tissue, and 0.99 +/- 0.54 in the normal tissues (t = 4.539, P < 0.01). In 14 of the 17 cases (82.4%) with p53E7 mutations, the STK15 expression was higher than that of normal tissue. In the 38 cases with STK15 over-expression, p53E7 mutation was found in 14 cases (36.8%). The rate of concurrence of p53E gene mutations and STK15 over-expression (25.5%) was higher than that of only p53E gene mutations (chi(2) = 26.025, P < 0.01).</p><p><b>CONCLUSION</b>There is significant association between p53 gene mutation and STK15 over-expression in laryngeal squamous-cell carcinoma.</p>