miR-218-5p Targeting TPX2 Regulates p53 Pathway and Inhibits Malignant Progression of Lung Adenocarcinoma / 中国肺癌杂志

BACKGROUND@#Lung adenocarcinoma (LUAD) is a major subtype of lung cancer, and its treatment and diagnosis remain a hot research topic. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is highly expressed in a variety of cancer cells and may be associated with the progression of LUAD. This study aimed to investigate the effect of TPX2 on the malignant progression of LUAD cells and the regulatory mechanisms.@*METHODS@#The expression of gene TPX2 in LUAD tissues from The Cancer Genome Atlas (TCGA) database was analyzed by bioinformatics analysis techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of TPX2 and miR-218-5p in human lung normal cell lines and human LUAD cell lines. Western blot was used to detect TPX2 protein expression in cell lines and its effect on the expression of key proteins in the p53 signaling pathway. The relationship between TPX2 and miR-218-5p was predicted using bioinformatics and verified by dual luciferase reporter gene assay. Cell counting kit-8 (CCK-8) assay, cell clone formation, cell scratching, Transwell assay, and flow cytometry were used to detect the effects of miR-218-5p and TPX2 on LUAD cell function.@*RESULTS@#TPX2 was significantly overexpressed in LUAD cells, and knockdown of TPX2 inhibited LUAD cell proliferation, migration, and invasion, promoted apoptosis and induced G2/M phase block, and promoted the expression of key proteins in the p53 signaling pathway. miR-218-5p, an upstream regulator of TPX2, could inhibit its expression. Overexpression of miR-218-5p eliminated the malignant development caused by high expression of TPX2, inhibited the malignant processes of LUAD cells such as proliferation and migration as well as promoted the p53 signaling pathway.@*CONCLUSIONS@#miR-218-5p targets and inhibits TPX2 expression and exerts an inhibitory effect on the malignant progression of LUAD cells via p53.

Expression of TPX2 in kidney renal clear cell carcinoma and its clinical significance / 国际肿瘤学杂志

Objective:To analyze the expression of targeting protein for Xklp2 (TPX2) in kidney renal clear cell carcinoma (KIRC) and its clinical significance.Methods:The postoperative tissue samples of 54 patients with KIRC admitted to the Department of Urology, the First Affiliated Hospital of Bengbu Medical College from July 2017 to June 2019 were collected. Immunohistochemistry was used to detect the protein expression of TPX2 in renal carcinoma and paracancerous tissues. The difference of TPX2 mRNA expression between KIRC tissues and normal tissues was analyzed by using the TIMER database, which verified the immunohistochemical results. The UALCAN database and the Kaplan-Meier plotter database were used to analyze the relationship between TPX2 mRNA expression and clinical stage, molecular subtypes, lymph node metastasis, and prognosis of patients with KIRC. The protein interaction network was constructed by STRING database to obtain TPX2-related proteins, and the genes corresponding to the related proteins were enriched for the KEGG pathway. The relationship between TPX2 expression and immune cell infiltration and the immune checkpoint was studied by using the TIMER database.Results:Immunohistochemical results showed that the positive expression rate of TPX2 protein was 48.15% (26/54) in cancer tissues, which was higher than that in paracancerous tissues (20.37%, 11/54) ( χ2=9.25, P=0.002). The results of bioinformatics analysis showed that TPX2 mRNA expression was significantly up-regulated in KIRC [cancer tissue: 1.89 (1.49, 2.42), normal tissue: 0.35 (0.24, 0.57), U=2 297.00, P<0.001]. The expression of TPX2 mRNA was related to the clinical stage ( χ2=34.36, P<0.001), molecular subtypes ( χ2=30.15, P<0.001), and lymph node metastasis status ( χ2=27.21, P<0.001) of KIRC patients. The 5-year survival rate (53.80%) in patients with high TPX2 expression was lower than that in patients with low TPX2 expression (74.40%, χ2=18.87, P<0.001). STRING database protein interaction network construction obtained 20 TPX2-related proteins, and the genes corresponding to the related proteins were enriched in the cell cycle. The expression of TPX2 was positively correlated with B cells ( r=0.30, P<0.001), CD8 + T cells ( r=0.23, P<0.001), CD4 + T cells ( r=0.18, P<0.001), macrophages ( r=0.20, P<0.001), neutrophils ( r=0.31, P<0.001), dendritic cells ( r=0.39, P<0.001) infiltration and most of its biomarkers (all P<0.05). It was positively correlated with immune checkpoint PD-1 ( r=0.31, P<0.001) and CTLA-4 ( r=0.27, P<0.001), but not correlated with PD-L1 ( r=0.07, P=0.146) . Conclusion:TPX2 is highly expressed in KIRC and is closely associated with poor prognosis. It is expected to be a new therapeutic target for KIRC.

Investigation of the expression of TPX2 in tongue squamous cell carcinoma and its effect on Cal27 cell proliferation and apoptosis / 口腔疾病防治

Objective @#To investigate the expression of targeting protein for xenopus kinesin-like protein2 (TPX2) in tongue squamous cell carcinoma (TSCC) tissues and explore its effect on cell proliferation and apoptosis.@*Methods@#30 cases of TSCC tissues, paired normal tissues were collected in Dongguan People's Hospital during 2013-2016. The mRNA and protein expression level of TPX2 was determined by qRT-PCR and western blot, respectively and analyzed The correlation of TPX2 expression level and clinic opathological parameters. Cal27 cell was transfected with siRNAs to knockdown the expression of TPX2, then cell proliferation, cell apoptosis and related proteins (cleaved caspase 3 and caspase 3) were detected by MTT assay, flow cytometry and western blot. @*Results@#TPX2 was highly expressed in (t=3.254, P=0.002 9) tumor tissues at mRNA level compared to adjacent normal parts. In protein level, TPX2 was highly expressed (66.7%) in tumor tissues, TPX2 expression level of 20 case was higher than the corresponding tissue adjacent to carcinoma (20/30, t=2.862, P=2.862), and high expression of TPX2 was related to T staging, lymph node metastasis of tongue cancer. Knockdown TPX2 effectively reduced cell proliferation, increased apoptosis rate (F=342.9, P < 0.000 1) and upregulated the expression of apoptosis-related proteins cleaved caspase 3 (F=46.98, P=0.001 4) and caspase 3 (F=33.35, P=0.002 7).@*Conclusion@#Overexpression of TPX2 was found in TSCC tissues. Silencing of TPX2 might inhibit cell proliferation and promote cell apoptosis. TPX2 could be a new target for gene therapy of TSCC.

Construction and identification of shRNA lentiviral vector targeting TPX2 gene / 基础医学与临床

Objective To construct the lentiviral RNA interference vector targeting TPX2 and to obtain the human cervical cancer HeLa cell strain stably infected by TPX2-shRNA for studying the relationship between human cervical carcinoma and TPX2 gene.Methods By targeting TPX2 gene,four double-stranded DNA hairpin structures corresponding to shRNA were designed,synthesized and connected with Pglv2-U6-Puro to construct the recombinant plasmids.Then these recombinant plasmids were transformed into DH5α competent cells.The positive clone was extracted and transfected into 293T cells for virus packages after sequenced correctly.Human cervical carcinoma HeLa cell infected by these recombinant lentiviral was screened by Puromycin,then stable cell strain was obtained.The silencing effect of TPX2 in HeLa cell was detected by RT-fluorescent quantitative PCR and Western blot.Cell cycle and cell apoptosis wer detected by Flow cytometry.Results Sequencing results confirmed that 5 lentiviral was packaged successfully.The steady cell strain transfered TPX2-shRNA was screened with 0.4 μg/mL puromycin.HeLa cells infected by recombinant lentivirus all play the gene silencing effect especially in the group of TPX2-shRNA-1.In the group of TPX2-shRNA-1,TPX2mRNA (0.21 ± 0.07) and protein (0.19 ± 0.28) rela tive expression level is lower than those in the control group (1.08±0.07) (P＜0.01) and(0.64±0.03) (P＜ 0.01)respectively;G2 and S-phase cells are higher than those in the control group (P＜0.05)and the apoptosis rate was significantly more than those in the control group (P＜0.05).Conclusions The effective TPX2 genetic interference sequence was obtained,lentiviral vectors carrying TPX2shRNA was successfully constructed,and the HeLa cell strain with TPX2 silenced was successfully screened,which lay the research foundation for the study of the role of TPX2 in cervical cancer.

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

TPX2 promotes migration and invasion of human breast cancer cells

Objective: To investigate the expression of targeting protein for Xenopus kinesin-like protein 2 (TPX2) in breast cancer tissue and to explore its role in proliferation, migration and invasion of breast cancer cells. Methods: The mRNA and protein expressions of TPX2 in breast cancer tissue and cell lines were assessed by quantitative RT-PCR and Western blot. The effect of TPX2 with RNA interference on proliferation, invasion and migration of breast cancer cells was observed by MTT and Transwell assays. Results: Both mRNA and protein expressions of TPX2 were upregulated in breast cancer tissues compared to tumor-adjacent tissue. TPX2 expression was also upregulated in breast cancer cell lines, and the TPX2 interfered by small interfering RNA could inhibit the proliferation, invasion and migration of breast cancer cells by inhibiting matrix metalloproteinase-2 and matrix metalloproteinase-9. Conclusions: Significantly upregulated TPX2 expression is observed in breast cancer tissue and cells, and contributes to promote the proliferation, migration and invasion of breast cancer cells.

Expression of TPX2 in breast cancer tissue and their clinical significances / 临床与实验病理学杂志

Purpose To investigate the expression of TPX2 in breast cancer, analyze the correlation between its expression and clinico-pathologic characteristics and explore the significances of TPX2 in the treatment and prognosis of breast cancer. Methods Expression of TPX2 in breast cancer tissues, adjacent tissue of cancer and normal breast tissue was examined by immunohistochenistry SP method. The mRNA expression of TPX2 in breast cancer tissues, adjacent tissue of cancer and normal breast tissue was detected by reverse-transcription polymerase chain reaction ( RT-PCR) . Results The expression of TPX2 is significantly higher in breast cancer tissues (75. 86%) compared with adjacent tissue of cancer (40. 00%) and normal breast tissue (5. 00%). And the expression of TPX2 cor-related with tumor grade and lymph node metastasis (P0. 05). The expression of TPX2 mRNA is also significantly higher in breast cancer tissues ( 1. 465 7 ± 0. 136 6 ) compared with adjacent tissue of cancer (0. 923 3 ±0. 114 8) and normal breast tissue (0. 326 9 ±0. 097 3) (P<0. 01). Conclusion High expression of TPX2 in breast cancer correlated with tumor grade and lymph node metastasis, so TPX2 might be a risk factor in the tumorigenesis, progression and lymph node metastasis in breast cancer.

TPX2 promotes migration and invasion of human breast cancer cells

OBJECTIVE@#To investigate the expression of targeting protein for Xenopus kinesin-like protein 2 (TPX2) in breast cancer tissue and to explore its role in proliferation, migration and invasion of breast cancer cells.@*METHODS@#The mRNA and protein expressions of TPX2 in breast cancer tissue and cell lines were assessed by quantitative RT-PCR and Western blot. The effect of TPX2 with RNA interference on proliferation, invasion and migration of breast cancer cells was observed by MTT and Transwell assays.@*RESULTS@#Both mRNA and protein expressions of TPX2 were upregulated in breast cancer tissues compared to tumor-adjacent tissue. TPX2 expression was also upregulated in breast cancer cell lines, and the TPX2 interfered by small interfering RNA could inhibit the proliferation, invasion and migration of breast cancer cells by inhibiting matrix metalloproteinase-2 and matrix metalloproteinase-9.@*CONCLUSIONS@#Significantly upregulated TPX2 expression is observed in breast cancer tissue and cells, and contributes to promote the proliferation, migration and invasion of breast cancer cells.

TPX2 gene-targeted short hairpin RNA induces the apoptosis of human lung adenocarcinoma A549 cells and its related mechanism / 肿瘤

Objective: To investigate the effect of short hairpin RNA (shRNA) targeting Xenopus kinesin-like protein 2 (TPX 2) gene on the apoptosis of human lung adenocarcinoma A549 cells and to explore its possible mechanism. Methods: The combination vector of shRNA targeting TPX2 gene was established and then it was transfected into A549 cells. The expression levels of TPX2, Aurora-A, p53 and Bcl-2 mRNAs in A549 cells after transfection with combination vector were detected by RT-PCR, and the expression level of TPX2 protein was determined by Western-blotting. The cell cycle distribution and the apoptosis were analyzed by flow cytometry (FCM). Results: The combination vector of pMagic4.1-shRNA-TPX2 was successfully established. The expression levels of TPX2, Aurora-A and Bcl-2 mRNAs were down-regulated whereas the expression level of p53 mRNA was up-regulated in A549 cells after transfection with pMagic4.1-shRNA-TPX2, and the expression level of TPX2 protein was down-regulated. The apoptosis rate of A549 cells was increased after transfection with pMagic4.1-shRNA-TPX2, and the cell cycle was arrested at S phase. These changes in the pMagic4.1-shRNA-TPX2- transfected group were significantly different from those in the blank control group (without transfection with combination vector) and the negative control group (transfection with pMagic4.1-shRNA-NC) (P<0.05). Conclusion: The shRNA targeting TPX 2 gene can induce the apoptosis of A549 cells, and this effect may be related to up-regulation of p53 expression and down-regulation of Bcl-2 expression. Copyright© 2011 by TUMOR.

Roles of TPX2 in cell mitotic and the relation with tumorigenesis / 肿瘤研究与临床

TPX2(targeting protein for Xklp2)is a microtubule-associated protein in a new family of vertebrate spindle pole components.TPX2 is diffusely distributed all over the nucleus during the cell cycle phases S and G_2 and play important roles in mitotic spindle formation.TPX2 over expression was found in a variety of malignant tumors,that are asssociated with the centrosome amplification,aneuploidy,cell transfor- mation and the biological behavior of tumor.Blocking the expression of TPX2 can inhibit the growth of tumor cells.TPX2 may serve as a new candidate target for tumor therapy.The study progress of its structure and function,expression levels in cell cycle,molecular mechanism etc.was reviewed.

Expression of RHAMM gene and TPX2 gene in esophageal squamous carcinoma and its clinical significance / 重庆医科大学学报

Objective:Toinvestigate the expression of RHAMMgene and TPX2 gene in esophageal squamous cell carcinoma(ESCC)and their relationships with clinicopathological factors.Methods:The expression of RHAMM gene and TPX2 gene were detected by reverse transcriptase polymerase chain reaction(RT-PCR)in ESCC tissues,para-cancer tissues,and matched esophageal normal mucosa tissues of 40 patients.Results:The positive expressions of RHAMM mRNA in ESCC tissues(65.0%)were significantly higher than those in para-cancer tissues(37.5%)and esophageal normal mucosa tissues(22.5%).Significant relationship was observed between positive expression of RHAMM mRNA and lymphnode metastasis(P