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Guanzhang Li,1,* Ting-Wei Chen,2,* Ann-Christin Nickel,2 Sajjad Muhammad,2 Hans-Jakob Steiger,2 Theophilos Tzaridis,3,4 Daniel Hänggi,2 Reinhard Zeidler,5 Wei Zhang,6– 8 Ulf Dietrich Kahlert1,2 1Department of Molecular Neuropathology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, People’s Republic of China; 2Clinic for Neurosurgery, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany; 3Division of Clinical Neurooncology, Department of Neurology and Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, 53127, Germany; 4Tumor Initiation & Maintenance Program, NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, 92037, USA; 5Department for Otorhinolaryngology, Klinikum der Universität München (LMU), Munich, Germany; 6Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, People’s Republic of China; 7China National Clinical Research Center for Neurological Diseases, Beijing, People’s Republic of China; 8Chinese Glioma Genome Atlas Network (CGGA) and Asian Glioma Genome Atlas Network (AGGA), Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ulf Dietrich Kahlert Moorenstrasse 5, Düsseldorf, 40225, GermanyTel +49-211-810 8013Fax +49-211-810 8012Email mail@ulf-kahlert.comBackground: The metabolic enzyme carbonic anhydrase 12 (CA12/CAXII) emerges as a promising cancer therapeutic target with drug development projects underway. Previous reports proposed the relevance of CA12 in the context of glioma but are limited in patient data quantity, ignore ethnic diversity of patients or rely on semi-quantitative, thereby out of date, methodology. Moreover, little is known on the association of CA12 to brain tumor stemness or on the effect of anti-CAXII-directed monotherapies on glioma stem cells (GSCs), in particular their response regarding mesenchymal differentiation status.Methods: We performed in silico analysis on three independent, large-scale patient datasets interrogating state of the art molecular diagnostics alongside clinical outcomes. We analyzed CAXII abundance on a collection of GSCs and functionally tested their response to exposure to CAXII blocking antibody 6A10.Results: CA12 is highly expressed in glial tumors compared with normal tissue and predicts for poor clinical course of tumor patients. CA12 expression in glioblastoma significantly correlates with clinically established, molecular markers of IDH1WT DNA, WHO grade IV or absence of 1p/19q chromosome arm co-deletion. Furthermore, tumors with elevated CA12 cluster into the mesenchymal transcription subclass of the disease. CAXII abundance in different GSCs ranges from almost absent to high levels and does not correlate to stem cell marker CD133/AC133 cell surface expression. Moreover, aiming to pharmacologically block CAXII in our cells with antibody 6A10 caused significant functional response only in one of the tested GSCs models, featuring suppression of cell invasion accompanied by reduction of ZEB1 protein and other stem cell markers.Conclusion: CA12 represents a clinically relevant and molecular brain tumor-subtype specific therapeutic target. Our correlative data from experimental and clinical samples does not support CA12/CAXII to be GSC specific. 6A10 possesses promising potential to impede the invasive capacity of glioma cells and supports the emerging concept that CAXII interacts with cancer EMT programs. However, further mechanistic studies are required to comprehensively assess the therapeutic potential of 6A10 and to identify different resistance mechanisms of GSCs.Keywords: carbonic anhydrase 12, 6A10, glioma, therapeutic antibody, ZEB1, disease modeling

Jianhua Qin,1,2 Tingting Zhu,1,2 Weihua Wu,1,2 Huan Chen,3 Yi He4 1Department of Nephrology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan, People’s Republic of China; 2Sichuan Clinical Research Center for Nephropathy, Luzhou 646000, Sichuan, People’s Republic of China; 3Department of Pathogen Biology, Basic Medical College, Southwest Medical University, Luzhou 646000, Sichuan, People’s Republic of China; 4Department of Urology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan, People’s Republic of ChinaCorrespondence: Yi He Email jingyoufan46@163.comBackground: Long non-coding RNA (lncRNA) has been recognized as the new regulator and biomarker for cancers. However, in clear cell renal cell carcinoma (ccRCC), the functions of lncRNAs are not well characterized. This research aimed to probe the function of lncRNA PCED1B-AS1 in the progression of ccRCC.Materials and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to detect the expression levels of PCED1B-AS1, microRNA-484 (miR-484), and zinc finger E-box binding homeobox 1 (ZEB1) in 40 pairs of human ccRCC tissues and corresponding adjacent kidney tissue samples. Chi-square test was employed to evaluate the association between PCED1B-AS1 expression level and clinicopathological characteristics. The effects of PCED1B-AS1, miR-484 and ZEB1 on the cell proliferation, migration and epithelial-mesenchymal transition (EMT) process of ccRCC cells were studied by CCK-8 assay, EdU cell proliferation assay, wound healing test and Western blotting. The regulatory relationships among PCED1B-AS1, miR-484, ZEB1 were examined by luciferase reporter gene assay and RNA immunoprecipitation assay.Results: PCED1B-AS1 was remarkably up-regulated in ccRCC tissues and cell lines. High expression of PCED1B-AS1 was associated with poor prognosis of the patients. Loss-of-function experiments showed that PCED1B-AS1 could regulate the proliferation, migration and EMT of ccRCC cells. PCED1B-AS1 sponged miR-484 to suppress its expression, and miR-484 targeted the 3ʹ-UTR of ZEB1 to repress the expression of ZEB1. MiR-484 counteracted the functions of PCED1B-AS1 in promoting the proliferation, migration and EMT of ccRCC cells, and PCED1B-AS1 promotes the expression of ZEB1 via repressing miR-484.Conclusion: PCED1B-AS1/miR-484/ZEB1 axis is involved in regulating the progression of ccRCC.Keywords: ccRCC, PCED1B-AS1, miR-484, ZEB1

Lu Ren, Shaoqin Yang, Qinxue Cao, Jun Tian Department of Obstetrics and Gynecology, Huaihe Hospital of Henan University, Kaifeng, 475001 Henan, People’s Republic of ChinaCorrespondence: Jun TianDepartment of Obstetrics and Gynecology, Huaihe Hospital of Henan University, Kaifeng 475001, Henan, People’s Republic of ChinaTel +86 371-23906579Email dfwmex@163.comBackground: Cervical cancer is a lethal gynecologic cancer in women. Long non-coding RNA colorectal neoplasia differentially expressed (LncRNA CRNDE) was recognized as a significant oncogene in multiple cancers. However, the functional role of CRNDE in cervical cancer remains poorly explored.Methods: The expression of CRNDE, microRNA-4262 (miR-4262) and zinc-finger E-box binding homeobox 1 (ZEB1) in cervical cancer tumors and cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Colony formation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were performed to detect cell viability. Flow cytometry and caspase-3 activity assay were conducted to evaluate cell apoptosis. The interaction between miR-4262 and CRNDE or ZEB1 was verified by dual-luciferase reporter system. Transwell assay was employed to evaluate cell migration and invasion. The relative protein expression was assessed by Western blot.Results: CRNDE and ZEB1 were up-regulated, while miR-4262 was down-regulated in cervical cancer tissues and cells. We found that CRNDE sponged miR-4262 and ZEB1 was a target of miR-4262. In addition, miR-4262 inhibitor abolished CRNDE silencing-induced repression on cell proliferation, EMT, migration, invasion and promotion on cell apoptosis. Furthermore, ZEB1 rescued the effects of miR-4262 overexpression or CRNDE deletion on cervical cancer progression. Our data showed that CRNDE targeted miR-4262 to regulate ZEB1 expression in cervical cancer cells. Besides, CRNDE expedited cervical cancer progression through wnt/β-catenin pathway via sponging miR-4262 and altering ZEB1 expression.Conclusion: Our findings demonstrated that CRNDE facilitated the progression of cervical cancer through activation of wnt/β-catenin pathway by regulating miR-4262/ZEB1 axis, representing a prospective targeted therapy for cervical cancer.Keywords: CRNDE, miR-4262, ZEB1, wnt/β-catenin pathway, cervical cancer

Kai Wu,1,* Qiong Feng,2,* Liang Li,1 Yanfei Xiong,3 Shihong Liu,4 Jie Liu,1 Qing Wu1 1Department of Orthopedics, The Second Affiliated Hospital of Nanchang University, Nanchang 300006, People’s Republic of China; 2Nursing School, Nanchang University, Nanchang 300006, People’s Republic of China; 3Department of Orthopedics, Jingan Hospital, Yichun 330600, People’s Republic of China; 4Jingan Hospital of Traditional Chinese Medicine, Yichun 330600, People’s Republic of China*These authors contributed equally to this workCorrespondence: Qing Wu Email wuqing18186@126.comIntroduction: The long-noncoding RNA PCAT6 plays an important regulatory role in the development of several cancers. However, the expression pattern and underlying mechanisms of PCAT6 in osteosarcoma (OS) are yet unknown.Methods: We used real-time PCR to measure PCAT6 expression in 106 tumor pairs and corresponding non-tumor tissues from OS patients. Statistical analyses were applied to evaluate the prognostic value and associations of PCAT6 expression with clinical parameters. Furthermore, the PCAT6 was silenced with siRNA in OS cells. Moreover, phenotype of PCAT6 silenced OS cells was measured using colony formation, CCK-8, cell migration and invasion assay. Finally, the molecular mechanism of PCAT6/miR-143-3p/ZEB1 axis in OS progression was explored.Results: The expression level of PCAT6 in OS tissues was significantly elevated as compared with that in the adjacent normal bone tissues and that high PCAT6 expression closely correlated with the malignant phenotype and poor survival among patients with OS. Multivariate analyses revealed PCAT6 overexpression as an independent prognostic factor for the poor outcome of patients with OS. Functional assay results demonstrated that the knockdown of PCAT6 expression notably suppressed the proliferation, migration, and invasion of OS cells. An elevated PCAT6 level aggravated the malignant phenotype of OS cells via ZEB1 expression upregulation. Mechanistic studies revealed that PCAT6 could sponge endogenous miR-143-3p and inhibit its activity, resulting in an increase in ZEB1 level. Finally, we demonstrated that the tumour-promoting role of PCAT6 in OS was dependent on the regulation of the miR-143-3p/ZEB1 axis.Conclusion: These findings highlight the potential role of PCAT6, which could serve as a valuable prognostic indicator for patients with OS.Keywords: osteosarcoma, PCAT6, ZEB1, miR-143-3p

Lirui He, Yunpeng Lei, Jianing Hou, Jianlong Wu, Guoqing Lv Department of Gastrointestinal Surgery, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518000, People’s Republic of ChinaCorrespondence: Guoqing LvPeking University Shenzhen Hospital, No. 1120 Lianhua Road, Futian District, Shenzhen, Guangdong 518000, People’s Republic of ChinaEmail GuoqingLv123@126.comBackground: Gastric cancer (GC) is an aggressive malignancy with high lethality. Systematic chemotherapy is the main therapeutic strategy for advanced GC patients. The overexpression of Axl is associated with poor prognosis and regulates tumor growth and metastasis in many types of cancer. However, the role of Axl in GC progression remains elusive.Materials and Methods: Western blot and quantitative real-time PCR assay (RT-PCR) assays were used to detect the expression of Gas6, Axl, ZEB1 and epithelial-mesenchymal transition (EMT)-related markers in GC cells. Cell proliferation was determined by EdU cell proliferation assay and CCK-8 assay. Transwell invasion assay was performed to explore the effect of Axl and ZEB1 on cell invasion. Tumor xenografts and lung metastasis models were conducted to examine the effect of Axl on the growth and lung metastasis of GC cells.Results: In our study, we found that high levels of Gas6 and Axl expression were associated with reduced overall survival (OS) in GC patients and the expression of Gas6 and Axl was upregulated in GC cell lines. Ectopic expression of Axl induced EMT and promoted GC cell invasion and proliferation. The knockdown of Axl inhibited EMT and suppressed the proliferation and invasion of GC cell. In vivo study showed that inhibition of Axl impaired tumor growth and lung metastasis of GC cells. Mechanistic investigations revealed that Axl promoted EMT, invasion, and proliferation via upregulating ZEB1 expression in GC cells.Conclusion: Our results demonstrated that the Gas6/Axl/ZEB1 signaling pathway regulated EMT, invasion, and proliferation in GC cells and might represent a potential therapeutic target for GC treatment.Keywords: gastric cancer, Gas6/Axl signaling pathway, ZEB1, EMT, invasion, proliferation

Jingang Shen,1,* Xianbao Lv,1,* Lei Zhang2 1Department of Urology, Chengwu County People’s Hospital, Shandong 274200, People’s Republic of China; 2Department of Urology, Zoucheng People’s Hospital, Shandong 273500, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lei ZhangDepartment of Urology, Zoucheng People’s Hospital, No. 59, Qianquan Road, Zoucheng County, Jining, Shandong 273500, People’s Republic of ChinaTel +86-13506385177Email qifeng2001oy@163.comPurpose: GRHL2 played important roles in different cancers. In this study, we aimed to investigate the roles of GRHL2 in bladder cancer.Methods: The immunohistochemistry assay was performed to detect the expression of GRHL2 in bladder cancer tissues and adjacent noncancerous tissues and the expression levels of GRHL2 and zinc finger E-box binding homeobox (ZEB1) mRNA in tissues were determined by qRT-PCR. In addition, qRT-PCR and Westernblotting were applied to detect the expression levels of GRHL2 and ZEB1 in bladder cancer cell lines (RT4, BIU-87, 5637, T24) and immortalized human bladder epithelial cell line (SV-HUC-1). The cell models with up-regulated and down-regulated expression of GRHL2 were constructed using bladder cancer cell lines T24 and 5637 to investigate the underlying roles of GRHL2 on the proliferation, migration, invasion and EMT process of bladder cancer cells. After that, cell proliferation was evaluated by CCK8 assay, cell cycle assay and colony formation assay. Transwell assay and wound healing assay were performed to determine the invasion and migration ability of the bladder cancer cells. The expressions of epithelial-mesenchymal transition (EMT) related proteins (E-cadherin, Vimentin, Slug and Snail) were assessed by Western blot analysis. Moreover, ZEB1 and GRHL2 were co-transfected into T24 and 5637 cells and their effects on EMT process and invasive capacity of cells were examined.Results: The expression of GRHL2 was down-regulated in bladder cancer tissues and human bladder cancer cell lines compared with the normal bladder tissues and immortalized human bladder epithelial cell line. Besides, down-regulation of GRHL2 improved the proliferation ability of bladder cancer cells and promoted the EMT process through up-regulation of ZEB1. The overexpression of ZEB1 partially reversed the inhibitory effect of GRHL2 on EMT.Conclusion: GRHL2 acts as an anti-oncogene to regulate bladder cancer cell proliferation and inhibit EMT by targeting ZEB1. This study may provide a theoretical basis for further research.Keywords: bladder cancer, GRHL2, EMT, ZEB1

Zhongping Ma,1 Guanghua Gu,1 Weikang Pan,1 Xiaoxiang Chen2 1Department of Obstetrics and Gynecology, Liyang Branch of Jiangsu Provincial People’s Hospital, Changzhou, People’s Republic of China; 2Department of Gynecologic Oncology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Nanjing, People’s Republic of ChinaCorrespondence: Zhongping MaDepartment of Obstetrics and Gynecology, Liyang Branch of Jiangsu Provincial People’s Hospital, No. 70, Construction West Road, Liyang, Changzhou 213300, People’s Republic of ChinaTel +86 519 6809 1065Email gxuqub1972515kmz@yeah.netBackground: Cervical cancer (CC) is a common cancer with a poor prognosis due to the chemoresistance of CC cells to cisplatin. This study aimed to investigate the biological significance of lncRNA prostate cancer-associated transcript 6 (PCAT6) in the carcinogenesis of CC.Materials and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to measure the abundance of PCAT6, miR-543 and zinc finger E-box binding protein 1 (ZEB1) in CC tissues and cells. The combination between miR-543 and lncRNA PCAT6 or ZEB1 was predicted by Starbase and was verified by dual-luciferase reporter assay, RNA-pull down assay and RNA immunoprecipitation (RIP) assay. Cell proliferation and chemoresistance to cisplatin were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis and metastasis were determined by flow cytometry, Western blot and transwell migration and invasion assays.Results: The abundance of ZEB1 protein was measured by Western blot assay. Murine xenograft model was established to confirm the function of lncRNA PCAT6 in vivo. The abundance of lncRNA PCAT6 was enhanced in CC tissues and cells compared with that in corresponding normal tissues and normal cervical epithelial cells Ect1/E6E7. MiR-543 was a target of PCAT6 and was negatively regulated by PCAT6. PCAT6 accelerated the proliferation, metastasis and the chemoresistance of CC cells to cisplatin while suppressed the apoptosis of CC cells. The overexpression of PCAT6 reversed the inhibitory effects of miR-543 accumulation on the proliferation, metastasis and chemoresistance of CC cells to cisplatin and the promoting impact on the apoptosis of CC cells. ZEB1 was a direct target of miR-543, and it functioned as the downstream gene of PCAT6/miR-543 to exert its oncogenic role in CC. PCAT6 promoted the growth of murine xenograft tumor through miR-543/ZEB1 axis in vivo.Conclusion: LncRNA PCAT6 facilitated the proliferation, metastasis and chemoresistance of CC cells to cisplatin while impeded the apoptosis of CC cells via PCAT6/miR-543/ZEB1 axis. PCAT6/miR-543/ZEB1 axis might be a promising target for CC therapy.Keywords: cervical cancer, lncRNA PCAT6, miR-543, ZEB1, chemoresistance

Ying Jiang, Shan Jin, Shisheng Tan, Qi Shen, Yingbo XueDepartment of Oncology, Guizhou Provincial People‘s Hospital, Guiyang, Guizhou, People’s Republic of ChinaObjective: Gastric cancer (GC) is a common tumor malignancy with high incidence and poor prognosis. Radiotherapy is one of the main strategies for GC treatment, while development of radioresistance limits the effectiveness. microRNA-203 (miR-203) has been reported to participate in progression of GC, whereas its interaction with radiosensitivity of GC and the related mechanism remain largely unclear.Methods: The expressions of miR-203 and zinc finger E-box binding homeobox 1 (ZEB1) were measured in GC tissues and cells by quantitative real-time polymerase chain reaction or western blot. Survival fraction, cell viability and apoptosis were measured in GC cells after treatment of radiation by colony formation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay or flow cytometry, respectively. Tumor volume and weight were detected in murine xenograft model after radiation treatment. The interaction between miR-203 and ZEB1 was explored by bioinformatics analysis and luciferase activity assay.Results: miR-203 expression was down-regulated and ZEB1 mRNA level was up-regulated in GC. The expression of miR-203 was associated with radiosensitivity of GC cells. Moreover, overexpression of miR-203 decreased survival fraction, cell viability and tumor growth but promoted cell apoptosis in radiation-treated GC cells. However, knockdown of miR-203 played an opposite effect. ZEB1 was validated as a target of miR-203, and it was involved in miR-203-mediated radiosensitivity of GC cells in vitro and in vivo.Conclusion: miR-203 promoted radiosensitivity of GC cells by targeting ZEB1, indicating miR-203 as a promising radiosensitizer for GC treatment.Keywords: gastric cancer, miR-203, ZEB1, radiosensitizer, radiosensitivity

Ying Jiang, Shan Jin, Shisheng Tan, Qi Shen, Yingbo XueDepartment of Oncology, Guizhou Provincial People‘s Hospital, Guiyang, Guizhou, People’s Republic of ChinaObjective: Gastric cancer (GC) is a common tumor malignancy with high incidence and poor prognosis. Radiotherapy is one of the main strategies for GC treatment, while development of radioresistance limits the effectiveness. microRNA-203 (miR-203) has been reported to participate in progression of GC, whereas its interaction with radiosensitivity of GC and the related mechanism remain largely unclear.Methods: The expressions of miR-203 and zinc finger E-box binding homeobox 1 (ZEB1) were measured in GC tissues and cells by quantitative real-time polymerase chain reaction or western blot. Survival fraction, cell viability and apoptosis were measured in GC cells after treatment of radiation by colony formation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay or flow cytometry, respectively. Tumor volume and weight were detected in murine xenograft model after radiation treatment. The interaction between miR-203 and ZEB1 was explored by bioinformatics analysis and luciferase activity assay.Results: miR-203 expression was down-regulated and ZEB1 mRNA level was up-regulated in GC. The expression of miR-203 was associated with radiosensitivity of GC cells. Moreover, overexpression of miR-203 decreased survival fraction, cell viability and tumor growth but promoted cell apoptosis in radiation-treated GC cells. However, knockdown of miR-203 played an opposite effect. ZEB1 was validated as a target of miR-203, and it was involved in miR-203-mediated radiosensitivity of GC cells in vitro and in vivo.Conclusion: miR-203 promoted radiosensitivity of GC cells by targeting ZEB1, indicating miR-203 as a promising radiosensitizer for GC treatment.Keywords: gastric cancer, miR-203, ZEB1, radiosensitizer, radiosensitivity

Juan Bai,1 Wen-Yu Jiao2 1Department of Oncology, Affiliated Hospital of Chengdu University, Chengdu 610081, People’s Republic of China; 2Department of Respiratory and Critical Care Medicine, Xi’an Daxing Hospital, Xi’an 710016, People’s Republic of ChinaCorrespondence: Wen-Yu Jiao Email xopobs@163.comObjective: MicroRNA-199a-3p (miR-199a-3p or miR-199b-3p) targeting of 3ʹ-UTR of ZEB1 was characterized as an important way to inhibit invasion and metastases in non-small cell lung cancer (NSCLC), one of the most common cancers around the world. Here we aimed to investigate the tumor-suppressive role of miR-199a-3p targeted ZEB1.Materials and Methods: A549 cells were transfected with ZEB1 and/or miR-199a-3p. Then, tumor growth was investigated in xenograft mice. Stem-like property, proliferation and mitochondria injury were further validated in vitro.Results: Overexpression of miR-199a-3p with premiRNAs significantly reduced tumor growth inhibited CD44 and Ki67 and increased Caspase-3 in A549 xenograft mice. Sphere formation and protein expression of stem-like markers showed that miR-199a-3p inhibited stemness of A549 cell. miR-199a-3p reduced proliferation of A549 cells, as showed with EdU staining and reduced expression of Ki67. Transfection of miR-199a-3p also promoted apoptosis, as indicated with increased apoptotic cells with flow cytometry, and increased cleaved Caspase-3/Caspase3 and Bcl-2/Bax. Apoptosis was further validated to be induced with mitochondria dysfunction, which indicated with JC-1 labeled loss of mitochondrial membrane potential, reduced activity of SOD, and increased MDA and LDH. All these effects were inverted with overexpression of ZEB1.Conclusion: Altogether, the findings suggested that the up-regulation of miR-199a-3p significantly inhibited NSCLC growth in vivo, and reduced A549 cell proliferation and promoted mitochondrial-mediated apoptosis, through down-regulation of ZEB1. The findings supported ZEB1 down-expression with miR-199a-3p as a novel therapeutic target for NSCLC treatment.Keywords: lung cancer, miR-199a-3p, miR-199b-3p, ZEB1

Introduction The long-noncoding RNA PCAT6 plays an important regulatory role in the development of several cancers. However, the expression pattern and underlying mechanisms of PCAT6 in osteosarcoma (OS) are yet unknown. Methods We used real-time PCR to measure PCAT6 expression in 106 tumor pairs and corresponding non-tumor tissues from OS patients. Statistical analyses were applied to evaluate the prognostic value and associations of PCAT6 expression with clinical parameters. Furthermore, the PCAT6 was silenced with siRNA in OS cells. Moreover, phenotype of PCAT6 silenced OS cells was measured using colony formation, CCK-8, cell migration and invasion assay. Finally, the molecular mechanism of PCAT6/miR-143-3p/ZEB1 axis in OS progression was explored. Results The expression level of PCAT6 in OS tissues was significantly elevated as compared with that in the adjacent normal bone tissues and that high PCAT6 expression closely correlated with the malignant phenotype and poor survival among patients with OS. Multivariate analyses revealed PCAT6 overexpression as an independent prognostic factor for the poor outcome of patients with OS. Functional assay results demonstrated that the knockdown of PCAT6 expression notably suppressed the proliferation, migration, and invasion of OS cells. An elevated PCAT6 level aggravated the malignant phenotype of OS cells via ZEB1 expression upregulation. Mechanistic studies revealed that PCAT6 could sponge endogenous miR-143-3p and inhibit its activity, resulting in an increase in ZEB1 level. Finally, we demonstrated that the tumour-promoting role of PCAT6 in OS was dependent on the regulation of the miR-143-3p/ZEB1 axis. Conclusion These findings highlight the potential role of PCAT6, which could serve as a valuable prognostic indicator for patients with OS.

Background Gastric cancer (GC) is an aggressive malignancy with high lethality. Systematic chemotherapy is the main therapeutic strategy for advanced GC patients. The overexpression of Axl is associated with poor prognosis and regulates tumor growth and metastasis in many types of cancer. However, the role of Axl in GC progression remains elusive. Materials and Methods Western blot and quantitative real-time PCR assay (RT-PCR) assays were used to detect the expression of Gas6, Axl, ZEB1 and epithelial-mesenchymal transition (EMT)-related markers in GC cells. Cell proliferation was determined by EdU cell proliferation assay and CCK-8 assay. Transwell invasion assay was performed to explore the effect of Axl and ZEB1 on cell invasion. Tumor xenografts and lung metastasis models were conducted to examine the effect of Axl on the growth and lung metastasis of GC cells. Results In our study, we found that high levels of Gas6 and Axl expression were associated with reduced overall survival (OS) in GC patients and the expression of Gas6 and Axl was upregulated in GC cell lines. Ectopic expression of Axl induced EMT and promoted GC cell invasion and proliferation. The knockdown of Axl inhibited EMT and suppressed the proliferation and invasion of GC cell. In vivo study showed that inhibition of Axl impaired tumor growth and lung metastasis of GC cells. Mechanistic investigations revealed that Axl promoted EMT, invasion, and proliferation via upregulating ZEB1 expression in GC cells. Conclusion Our results demonstrated that the Gas6/Axl/ZEB1 signaling pathway regulated EMT, invasion, and proliferation in GC cells and might represent a potential therapeutic target for GC treatment.

Jijun Liu,1 Qi Wu,1 Yonggui Wang,1 Yulong Wei,2 Hong Wu,3 Lijun Duan,1 Qiang Zhang,1 Yonggang Wu1 1Department of Orthopedics, 2Department of Pathology, 3Department of Ultrasound, Bayannaoer City Hospital, Bayannaoer, Inner Mongolia, People’s Republic of China Purpose: Osteosarcoma (OS) is the most common type of primary solid bone tumor. Ovo-like zinc finger 2 (Ovol2), a zinc finger transcription factor, is a mesenchymal–epithelial transition (MET) driver that induces miR-200 expression in prostate cancer, breast cancer, and hepatocellular carcinoma. However, little is known about the expression and function of MET in sarcomas, including OS. This study investigated the expression and clinicopathological significance of Ovol2 and its effect on MET in OS. Patients and methods: The Ovol2 expression in the tumor samples from patients with OS was examined using immunohistochemistry (IHC). We then upregulated the Ovol2 expression in MG-63 and SW1353 cells, detected the expression of MET-associated proteins, and observed the effects of Ovol2 on OS cell proliferation, migration, and cytoskeleton reorganization using Cell Counting Kit-8, transwell invasion, and phalloidin dyeing assays, respectively. The correlation between zinc finger E-box-binding homeobox 1 (ZEB1) and Ovol2 was assessed using the luciferase gene reporter assay in the MG-63 and SW1353 cells and IHC in the human OS tissue samples.Results: The Ovol2 protein overexpression was related to the clinical grade (P=0.02) and the recurrence and metastasis (P=0.02) of OS. Results of the in vitro experiments showed that Ovol2 overexpression can suppress cell migration and invasion and can regulate the expression levels of MET-associated proteins. Ovol2 suppresses ZEB1 expression by binding to the ZEB1 promoter. Ovol2 is concomitant with a reduced IHC expression of ZEB1 in human OS tissues.Conclusion: Ovol2 expression is associated with MET in OS cells and suppresses ZEB1 expression and OS progression. Keywords: Ovol2, mesenchymal–epithelial transition, osteosarcoma, ZEB1

Jijun Liu,1 Qi Wu,1 Yonggui Wang,1 Yulong Wei,2 Hong Wu,3 Lijun Duan,1 Qiang Zhang,1 Yonggang Wu1 1Department of Orthopedics, 2Department of Pathology, 3Department of Ultrasound, Bayannaoer City Hospital, Bayannaoer, Inner Mongolia, People’s Republic of China Purpose: Osteosarcoma (OS) is the most common type of primary solid bone tumor. Ovo-like zinc finger 2 (Ovol2), a zinc finger transcription factor, is a mesenchymal–epithelial transition (MET) driver that induces miR-200 expression in prostate cancer, breast cancer, and hepatocellular carcinoma. However, little is known about the expression and function of MET in sarcomas, including OS. This study investigated the expression and clinicopathological significance of Ovol2 and its effect on MET in OS. Patients and methods: The Ovol2 expression in the tumor samples from patients with OS was examined using immunohistochemistry (IHC). We then upregulated the Ovol2 expression in MG-63 and SW1353 cells, detected the expression of MET-associated proteins, and observed the effects of Ovol2 on OS cell proliferation, migration, and cytoskeleton reorganization using Cell Counting Kit-8, transwell invasion, and phalloidin dyeing assays, respectively. The correlation between zinc finger E-box-binding homeobox 1 (ZEB1) and Ovol2 was assessed using the luciferase gene reporter assay in the MG-63 and SW1353 cells and IHC in the human OS tissue samples.Results: The Ovol2 protein overexpression was related to the clinical grade (P=0.02) and the recurrence and metastasis (P=0.02) of OS. Results of the in vitro experiments showed that Ovol2 overexpression can suppress cell migration and invasion and can regulate the expression levels of MET-associated proteins. Ovol2 suppresses ZEB1 expression by binding to the ZEB1 promoter. Ovol2 is concomitant with a reduced IHC expression of ZEB1 in human OS tissues.Conclusion: Ovol2 expression is associated with MET in OS cells and suppresses ZEB1 expression and OS progression. Keywords: Ovol2, mesenchymal–epithelial transition, osteosarcoma, ZEB1

Jihong An,* Weiling Lv,* Yongzhou Zhang Department of Clinical Pharmacy, Huaihe Hospital of Henan University, Kaifeng, People’s Republic of China *These authors contributed equally to this work Background: Chemoresistance is one of the major obstacles for cancer therapy in the clinic. Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported as an oncogene in most malignancies such as lung cancer, esophageal cancer, and gastric cancer. This study is designed to investigate the function of NEAT1 in paclitaxel (PTX) resistance of ovarian cancer and its potential molecular mechanism. Patients and methods: The expressions of NEAT1 and miR-194 in ovarian cancer tissues and cells were estimated by quantitative real-time polymerase chain reaction (qRT-PCR). MTT, flow cytometry, and Western blot assays were used to assess the effect of NEAT1 on PTX resistance in PTX-resistant ovarian cancer cells. Luciferase reporter assay was applied to examine the association between NEAT1, zinc finger E-box-binding homeobox 1 (ZEB1) and miR-194. Xenograft tumor model was established to confirm the biological role of NEAT1 in PTX resistance of ovarian cancer in vivo. Results: NEAT1 was upregulated, and miR-194 was downregulated in PTX-resistant ovarian cancer tissues and cells. Functionally, NEAT1 knockdown enhanced cell sensitivity to PTX via promoting PTX-induced apoptosis in vitro. NEAT1 was identified as a molecular sponge of miR-194 to upregulate ZEB1 expression. Mechanistically, NEAT1-knockdown-induced PTX sensitivity was mediated by miR-194/ZEB1 axis. Moreover, NEAT1 knockdown improved PTX sensitivity of ovarian cancer in vivo. Conclusion: NEAT1 contributed to PTX resistance of ovarian cancer cells at least partly through upregulating ZEB1 expression by sponging miR-194, elucidating a novel regulatory pathway of chemoresistance in PTX-resistant ovarian cancer cells and providing a possible long noncoding RNA (lncRNA)-targeted therapy for ovarian cancer. Keywords: lncRNA NEAT1, miR-194, ZEB1, PTX, ceRNAs&nbsp

Yuliang Pan,1,2 Jun Zhang,1 Huiqun Fu,1 Liangfang Shen2 1Department of Oncology, The Third Xiangya Hospital of Central South University, Changsha, Hunan, People’s Republic of China; 2Department of Oncology Radiotherapy, Xiangya Hospital of Central South University, Changsha, Hunan, People’s Republic of China Abstract: Breast cancer is the most common cancer in women worldwide. Local invasion, metastasis, and chemotherapy resistance are the obstacles for treatment of breast cancer. In this study, we aim to investigate the role of miR-144 in breast cancer. We demonstrate that the expression of miR-144 is downregulated in breast cancer and cell lines, and lower miR-144 expression is associated with poor differentiation, higher clinical stage, and lymph node metastasis in patients with breast cancer. The rescue of miR-144 expression is able to inhibit the cell proliferation and the ability of cell migration and invasion. In addition, we show that miR-144 can directly target at 3'-untranslation region of zinc finger E-box-binding homeobox 1 and 2, that is, ZEB1 and ZEB2, and regulate their expression at transcriptional and translational levels. Moreover, we also demonstrate that ectopic expression of miR-144 can inhibit the process of epithelial mesenchymal transition in MCF-7 and MDA-MB-231 cells. Thus, we here demonstrate that miR-144 functions as a tumor suppressor in breast cancer at least partly through inhibiting ZEB1/2-mediated epithelial mesenchymal transition process. Our findings indicate that the miR-144-ZEB1/2 signaling could represent a promising therapeutic target for breast cancer treatment. Keywords: breast cancer, miR-144, ZEB1, ZEB2, epithelial mesenchymal transition

Yuliang Pan,1,2 Jun Zhang,1 Huiqun Fu,1 Liangfang Shen2 1Department of Oncology, The Third Xiangya Hospital of Central South University, Changsha, Hunan, People’s Republic of China; 2Department of Oncology Radiotherapy, Xiangya Hospital of Central South University, Changsha, Hunan, People’s Republic of China Abstract: Breast cancer is the most common cancer in women worldwide. Local invasion, metastasis, and chemotherapy resistance are the obstacles for treatment of breast cancer. In this study, we aim to investigate the role of miR-144 in breast cancer. We demonstrate that the expression of miR-144 is downregulated in breast cancer and cell lines, and lower miR-144 expression is associated with poor differentiation, higher clinical stage, and lymph node metastasis in patients with breast cancer. The rescue of miR-144 expression is able to inhibit the cell proliferation and the ability of cell migration and invasion. In addition, we show that miR-144 can directly target at 3'-untranslation region of zinc finger E-box-binding homeobox 1 and 2, that is, ZEB1 and ZEB2, and regulate their expression at transcriptional and translational levels. Moreover, we also demonstrate that ectopic expression of miR-144 can inhibit the process of epithelial mesenchymal transition in MCF-7 and MDA-MB-231 cells. Thus, we here demonstrate that miR-144 functions as a tumor suppressor in breast cancer at least partly through inhibiting ZEB1/2-mediated epithelial mesenchymal transition process. Our findings indicate that the miR-144-ZEB1/2 signaling could represent a promising therapeutic target for breast cancer treatment. Keywords: breast cancer, miR-144, ZEB1, ZEB2, epithelial mesenchymal transition

Rongjie Cheng,1,* Nan Li,2,* Shuyan Yang,1 Lei Liu,1 Shiyu Han1 1Department of Obstetrics and Gynaecology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin City, Heilongjiang Province, People’s Republic of China; 2Department of Pathology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin City, Heilongjiang Province, People’s Republic of China *These authors contributed equally to this work Background: Long non-coding RNAs (lncRNAs) play important roles in cancer initiation and development. The purpose of the present study was to determine the functions and mechanisms of lncRNA ZEB1-AS1 in human cervical cancer (CC).Methods: A total of 106 pairs of CC tissues and adjacent normal epithelial tissues were collected from CC patients who underwent resection. Three human CC cell lines (HeLa, C33A and SiHa) and a normal cervical cell line Crl-2614 and were transfected with human ZEB1-AS1 cDNA, or empty vector as the control. Then, cells were transfected with ZEB1-AS1-specific small interfering RNA (si-ZEB1-AS1), ZEB1-specific siRNA (si-ZEB1) or negative siRNA control (si-NC). The transfection efficiency was confirmed by RT-qPCR analysis. qPCR was applied to determine the qualification of RNA. Cell proliferation was investigated by MTT assay. The apoptosis rate of cells was detected by flow cytometer. Cell invasion was detected by transwell assay. Western blot was applied to determine the expression of proteins. CC xenografts in 12 male BALB/c athymic nude mice were established. And the tumor volumes were measured by vernier caliper.Results: We found that ZEB1-AS1 expression was remarkably increased in human CC tissue samples and cell lines, and its expression levels were closely associated with poor prognosis of CC patients. Moreover, we found that knockdown of ZEB1-AS1 inhibited the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of CC cells in vitro and suppressed CC xenograft tumor growth in vivo. Mechanistically, we found that knockdown of ZEB1-AS1 significantly inhibited ZEB1 expression, and knockdown of ZEB1 could rescue the effects of ZEB1-AS1 overexpression in CC cells.Conclusion: In conclusion, our findings indicated that ZEB1-AS1 serves an oncogenic role in CC, which might become a potential prognostic indicator and therapeutic target in CC. Keywords: cervical cancer, long non-coding RNA, ZEB1-AS1, epithelial–mesenchymal transition, ZEB1&nbsp

Jihong An,* Weiling Lv,* Yongzhou Zhang Department of Clinical Pharmacy, Huaihe Hospital of Henan University, Kaifeng, People’s Republic of China *These authors contributed equally to this work Background: Chemoresistance is one of the major obstacles for cancer therapy in the clinic. Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported as an oncogene in most malignancies such as lung cancer, esophageal cancer, and gastric cancer. This study is designed to investigate the function of NEAT1 in paclitaxel (PTX) resistance of ovarian cancer and its potential molecular mechanism. Patients and methods: The expressions of NEAT1 and miR-194 in ovarian cancer tissues and cells were estimated by quantitative real-time polymerase chain reaction (qRT-PCR). MTT, flow cytometry, and Western blot assays were used to assess the effect of NEAT1 on PTX resistance in PTX-resistant ovarian cancer cells. Luciferase reporter assay was applied to examine the association between NEAT1, zinc finger E-box-binding homeobox 1 (ZEB1) and miR-194. Xenograft tumor model was established to confirm the biological role of NEAT1 in PTX resistance of ovarian cancer in vivo. Results: NEAT1 was upregulated, and miR-194 was downregulated in PTX-resistant ovarian cancer tissues and cells. Functionally, NEAT1 knockdown enhanced cell sensitivity to PTX via promoting PTX-induced apoptosis in vitro. NEAT1 was identified as a molecular sponge of miR-194 to upregulate ZEB1 expression. Mechanistically, NEAT1-knockdown-induced PTX sensitivity was mediated by miR-194/ZEB1 axis. Moreover, NEAT1 knockdown improved PTX sensitivity of ovarian cancer in vivo. Conclusion: NEAT1 contributed to PTX resistance of ovarian cancer cells at least partly through upregulating ZEB1 expression by sponging miR-194, elucidating a novel regulatory pathway of chemoresistance in PTX-resistant ovarian cancer cells and providing a possible long noncoding RNA (lncRNA)-targeted therapy for ovarian cancer. Keywords: lncRNA NEAT1, miR-194, ZEB1, PTX, ceRNAs&nbsp