ZEB2 Promotes Glioma Cell Proliferation, Invasion And Migration By Directly Targeting MiR-637/Akt1/HMGA1 Pathway

BackgroundGlioma is the most common type of all primary brain and central nervous system (CNS) tumors and the current main therapy is maximal safe resection followed by radiotherapy in combination with chemotherapy. Despite the constant progress has been made in the treatment, the therapeutic effect is barely ameliorated. It was reported that the median survival was 14.6 months with radiotherapy plus temozolomide and 12.1 months with radiotherapy alone, and the two-year survival rate was 26.5 percent with radiotherapy plus temozolomide and 10.4 percent with radiotherapy alone. In recent years multiple new targeted drugs have been developed such as Bevacizumab and Cilengitide, however, survival in patients with glioma has not been improved.Due to its high malignancy, infiltrative growth, unclear boundary with surrounding normal brain tissue and easy recurrence, glioma is hard to cure. High malignancy indicates rapid proliferation of glioma cells. Infiltrative growth which manifests invading the normal brain induces incompletely resection and recurrence. Therefore, it is an important direction to study high proliferation and invasion of glioma.Zinc finger E-box Binding homeobox 2 (ZEB2), also known as Smad-Interacting Protein 1 (SIP1), belongs to the E-box binding zinc finger protein family. ZEB2 has two independent separated zinc finger domain that can bind to 5’-CACCT sequence. Thus, ZEB2, as a transcription factor, can bind to 5’-CACCT then inhibit the transcription of downstream genes. Many studies suggested that ZEB2 exert a key role in the genesis and development of cancer, which promotes not only tumor cell proliferation, but also invasion and migration.However, report focuses on ZEB2 in glioma is less compared with that in other cancer, and the mechanism and pathway that ZEB2 involves in are also less reported. Xia etc. found that ZEB2 was upregulated in glioma cells, and knockdown of ZEB2 could inhibit cell invasion and migration. Besides, upregulated ZEB2 inhibited the expression of E-Cadherin and promoted the expression of Fibronectin and Vimentin. Subsequently, Song etc. verified both ZEB2 mRNA and protein level was upregulated in glioma clinical samples compared with normal brain tissues by quantitative RT-PCR, Western blot and immunohistochemistry, and the expression of ZEB2 was positive relate to WHO grading. Moreover, their cell functional assays identified that knockdown of ZEB2 inhibited cell proliferation, invasion and migration, and promoted G1/S phase arrest and cell apoptosis. Further Western blot assay confirmed the above conclusion as well.MicroRNA (miRNA) belongs to endogenous non-coding RNA and its main function is negative regulation of gene transcription. Recently close relationship was confirmed between miRNAs and cancer by many studies, including gliomas. Lots of miRNA mediated proliferation, invasion, migration and resistance of radiotherapy and chemotherapy in cancer through its ability that targeted regulating downstream key genes. Increasing evidence confirmed miRNA has potential value to become candidate marker of cancer and target of drug therapy.Multiple research reported ZEB2 had relationship with miRNAs. And ZEB2, as transcription factor, can binds the promotor region of miRNAs then inhibits or promotes its transcription. Therefore, we performed CombiMatrix miRNAs assay and detected more than 80 miRNAs. Then qRT-PCR confirmed miR-637 was upregulation after knockdown of ZEB2. Further comparison between the promotor sequences of miR-637 and 5’-CACCT in ZEB2 promotor suggested ZEB2 might direct bind to the promotor of miR-637 to inhibit its transcription and expression.In present study, we explored ZEB2 mediated glioma cell proliferation, invasion and migration through miR-637 and the mechanism in this pathway. These findings will provide a theoretical basis for understanding the rapid proliferation and invasion in gliomas, and will be useful in identifying potential candidates for targeted therapeutic intervention of glioma.Content and methodsFurther study on the function and mechanism of ZEB2 in gliomaDesign and construct 4 lentiviral vector that stably knocking down ZEB2 and transfected into glioma cells. Transfection efficiency was detected by qRT-PCR and Western blot. Subsequently, cell proliferation was detected by MTT assay and Edu assay, and clone formation ability was identified by plate clone assay. Scratch assay, Transwell chamber and Boyden chamber were applied to identify ZEB2 knockdown inhibit glioma cell invasion and migration. Tumorigenesis assay in nude mice was utilized to detect ZEB2 promotes cell growth in vivo. Ultimately, Western blot assay was performed to detect the change of protein which associated with cell proliferation, EMT, PI3K/Akt and Wnt/β-catenin pathway.ZEB2 promoted glioma cell growth, invasion and migration through inhibiting the transcription of miR-637CombiMatrix miRNAs assay was performed to detected the change of miRNAs in ZEB2 knockdown U251 cell and further qRT-PCR confirmed miR-637 was upregulation after knockdown of ZEB2. Bioinformatics analysis identified ZEB2 could bind the promotor of miR-637. Subsequently, dual-luciferase reporter assay was performed to confirm ZEB2 bind the promotor of miR-637 and inhibit its transcription. Electrophoretic mobility shift assay and Chromatin immunoprecipitation was utilized to identify ZEB2 bind the promotor of miR-637 in vivo and in vitro, respectively.Design and construct lentiviral vector that stably overexpression miR-637 and transfected into glioma cells. Transfection efficiency was detected by qRT-PCR. Subsequently, MTT assay, Edu assay, plate clone assay and tumorigenesis assay in nude mice was performed to detect the change of cell proliferation, and Transwell chamber and Boyden chamber were applied to identify the change of cell invasion and migration. Furthermore, MTT assay and Edu assay was utilized to confirm ZEB2 promotes cell growth through miR-637, and scratch assay, Transwell chamber and Boyden chamber were applied to identify ZEB2 promotes glioma cell invasion and migration through miR-637.MiR-637 direct targeted Aktl and HMGA1 then inhibited glioma cell function Bioinformatics analysis confirmed Aktl and HMGA1 might be targets of miR-637, and further qRT-PCR and Western blot confirmed Aktl and HMGA1 was downregulation after miR-637 overexpression. Subsequently, dual-luciferase reporter assay was performed to confirm miR-637 can bind the 3’-UTR region of Aktl and HMGA1.Edu assay was utilized to confirm HMGA1 knockdown inhibits cell growth, and Transwell chamber and Boyden chamber were applied to identify HMGA1 knockdown inhibits glioma cell invasion and migration. Subsequently, Edu assay was utilized to confirm miR-637 inhibits cell growth through Aktl and HMGA1, and Transwell chamber and Boyden chamber were applied to identify miR-637 inhibits glioma cell invasion and migration through Aktl and HMGA1.The mechanism of miR-637 mediated glioma cell proliferation, invasion and migration through targeting Aktl and HMGA1Western blot assay was performed to detect the change of protein which associated with cell proliferation, EMT, PI3K/Akt and Wnt/β-catenin pathway after miR-637 overexpression. Western blot assay was performed to detect the change of protein which associated with cell proliferation, EMT, PI3K/Akt and Wnt/β-catenin pathway both in Aktl knockdown glioma cell and miR-637+Aktl overexpression glioma cell.Western blot assay was performed to detect the change of protein which associated with cell proliferation, EMT, PI3K/Akt and Wnt/β-catenin pathway after silencing HMGA1. Western blot assay was performed to detect the change of protein which associated with cell proliferation, EMT, PI3K/Akt and Wnt/p-catenin pathway after miR-637 and HMGA1 co-overexpression.The expression of ZEB2-miR-637-Aktl/HMGAl pathway in clinical samples ZEB2, miR-637, Aktl and HMGA1 mRNA level was detected by qRT-PCR in 45 clinical glioma samples and 15 normal brain tissues. Western blot was performed to verify ZEB2 and HMGA1 protein level in 7 clinical glioma samples and 7 paired normal brain tissues. Immunohistochemistry was performed to detect the expression and localization of ZEB2, Aktl and HMGA1 in 70 clinical glioma samples and 20 normal brain tissues, and the relationship between expression level and clinical pathologic materials was analyzed at the same time. In situ hybrization was performed to detect the expression and localization of miR-637 in 70 clinical glioma samples and 20 normal brain tissues, and the relationship between expression level and clinical pathologic materials was also analyzed. Ultimately, the expressive relationship among ZEB2, miR-637, Aktl and HMGA1 in glioma clinical samples was analyzed by statistical method.ResultsZEB2 promoted glioma cell proliferation, invasion and migration Four lentiviral vector that stably knocking down ZEB2 were transfected into glioma cells and transfection efficiency was detected by qRT-PCR and Western blot. The results showed A segment was the most efficiency in U251 (19.4%) and C segment was the most efficiency in U87 (25.0%).Subsequently, cell proliferation was detected by MTT assay, Edu assay and tumorigenesis assay in nude mice and clone formation ability was identified by plate clone assay. The results of MTT assay identified the decreasing survival ability of shZEB2 group compared with Mock group (P<0.001). Edu assay showed the decreasing percentage of cell in S phase after ZEB2 was silencing(P<0.001). In vivo tumorigenesis assay in nude mice confirmed the volume and weight of tumor in U87 shZEB2 group were small and lighter than that in compared group (P<0.001). Plate clone assay suggested decreasing number of colony formation after ZEB2 knockdown (P<0.001). Those results showed ZEB2 promoted cell growth and colony formation in glioma.Scratch assay, Transwell chamber and Boyden chamber were applied to identify ZEB2 knockdown inhibit glioma cell invasion and migration. Scratch assay showed the rapid decreasing of gap distance in shZEB2 group compared with Mock group (.P<0.001). Transwell chamber and Boyden chamber assay showed less number of transmembrane cells after ZEB2 knockdown (P=0.001 and 0.003).Ultimately, Western blot assay was performed to detect the change of protein which associated with cell proliferation, EMT, PI3K/Akt and Wnt/β-catenin pathway. The results suggested p-PI3K, Akt, p-Akt, CCND1, Vimentin、β-cateni、N-Cadherin were downregulated and p21 was upregulated after knocking down the expression of ZEB2. Other protein such as HMGA1, E2F1, PTEN, Bcl-2, NF-kappa B and Sox2 were upregulated or downregulated.ZEB2 promoted glioma cell growth, invasion and migration via inhibiting the transcription of miR-637CombiMatrix miRNAs assay detected more than 80 changed miRNAs in ZEB2 knockdown U251 cell, and further qRT-PCR confirmed miR-637 was upregulation after knockdown of ZEB2 in U251 and U87 (P=0.02 and P=0.005). Bioinformatics analysis identified ZEB2 could bind the promotor of miR-637. Subsequently, dual-luciferase reporter assay confirmed knockdown of ZEB2 could inhibit the binding between ZEB2 and the promotor of miR-637 (P=0.002), and ZEB2 overexpression could promote this binding process (P=0.004). Meanwhile, electrophoretic mobility shift assay showed ZEB2 overexpression could promote the binding between ZEB2 and the promotor of miR-637, and 50 times dose of untagged competitive probe could inhibit this binding. In addition, chromatin immunoprecipitation identified ZEB2 bind to 0-300bp region of the miR-637 promotor.Lentiviral vector that stably overexpression miR-637 was transfected into glioma cells and qRT-PCR showed miR-637 was overexpressed more than 400 times and 350 times in U251 and U87 cell (both P<0.001). Subsequently, MTT assay, Edu assay, plate clone assay and tumorigenesis assay in nude mice was performed to detect the change of cell proliferation. MTT assay showed the decreasing survival ability in miR-637 overexpression group compared with scramble group (P<0.001). Edu assay showed the decreasing percentage of cell in S phase in miR-637 overexpression group compared with scramble group (P<0.05). In vivo tumorigenesis assay in nude mice confirmed the volume and weight of tumor in U87 miR-637 overexpression group were small and lighter than that in compared group (P<0.05), and the expression of Ki-67 was higher in miR-637 overexpression group than Mock group (P=0.002). Plate clone assay suggested decreasing number of colony formation after miR-637 overexpression (P<0.05). Subsequently, Transwell chamber and Boyden chamber were applied to identify the change of cell invasion and migration. The results of two assays suggested less number of transmembrane cells in miR-637 overexpression group compared with Mock group, and applying miR-637 inhibitor could reverse the decreasing number of transmembrane cells in miR-637 overexpression group (both P<0.05).Furthermore, MTT assay, Edu assay, scratch assay, Transwell chamber and Boyden chamber assay was utilized to confirm ZEB2 promotes cell growth, invasion and migration through miR-637. MTT assay and Edu assay both suggested miR-637 mimics could reverse the cell proliferative promotion induced by ZEB2 overexpression (both P<0.05). And scratch assay, Transwell chamber and Boyden chamber assay also identified that miR-637 mimics could reverse the cell invasive and migrated promotion induced by ZEB2 overexpression (both P<0.05).MiR-637 direct targeted Aktl and HMGA1 then inhibited glioma cell function Bioinformatics analysis confirmed Aktl and HMGA1 might be targets of miR-637, and further qRT-PCR and Western blot confirmed Aktl and HMGA1 was downregulation after miR-637 overexpression (P<0.05). Dual-luciferase reporter assay confirmed miR-637 bound the 3’-UTR region of Aktl and HMGA1 (P<0.05), and miR-637 could not bound the mutative 3’-UTR region of Aktl and HMGA1.Edu assay, Transwell chamber and Boyden chamber assay were utilized to confirm miR-637 inhibits cell growth, invasion and migration through Aktl. Edu assay showed knockdown of Aktl reduced the percentage of cell in S phase which, and applying Akt1 overexpression plasmid could reverse G1/S arrest induced by miR-637 overexpression (P<0.05). Transwell chamber and Boyden chamber assay both suggested knockdown of Akt1 reduced the number of transmembrane cells, and upregulation of Akt1 could reverse the cell invasive and migrated promotion induced by miR-637 overexpression (both P<0.05). Subsequently, Edu assay in HMGA1 knockdown glioma cells confirmed HMGA1 promoted cell growth, and upregulation of HMGA1 could reverse the cell proliferative promotion induced by miR-637 overexpression (P<0.001). Transwell chamber and Boyden chamber assay identified HMGA1 knockdown reduced the number of transmembrane cells, and upregulation of HMGA1 could reverse the cell proliferative promotion induced by miR-637 overexpression (both P<0.05).The mechanism of miR-637 mediated glioma cell proliferation, invasion and migration through targeting Akt1 and HMGA1Western blot assay in miR-637 overexpression cells confirmed Akt1, p-Akt, p-PI3K, β-catenin, p-Foxo1, Cyclin D1, Cyclin E1, p15 and NF-kappa B were downregulated compared with control cell group, and Foxo1, p27/Kip, p21 and hTERT was upregulated as well as N-Cadherin were not changed. Then Western blot assay in miR-637+Akt1 overexpression cells showed p-Akt1, β-catenin, p-Foxo1 and Cyclin D1 were downregulated and Foxo1 was upregulated in Akt1 knockdown cells compared with control group. Moreover, upregulation of Akt1 could rescue the downregulation of p-Akt1, β-catenin, p-Foxo1 and Cyclin D1 and the upregulation of Foxol induced by miR-637 overexpression.Western blot assay in HMGA1 knockdown cells identified Akt, p-Akt, p-PI3K, N-Cadherin, β-catenin, Vimentin, Cyclin D1, Cyclin E1, c-Myc and NF-kappa B were downregulated and p15、p16、CDK6 were upregulated in HMGA1 knockdown cells compared with control group. Moreover, upregulation of HMGA1 could rescue the downregulation of Cyclin D1, β-catenin, NF-kappa B and Vimentin and the upregulation of p15 induced by miR-637 overexpression.The expression of ZEB2-miR-637-Akt1/HMGA1 pathway in clinical samples QRT-PCR assay showed ZEB2, Aktl and HMGA1 mRNA level were both upregulated in clinical glioma samples compared with normal brain tissues (P <0.001), and miR-637 mRNA level was downregulated in clinical glioma samples compared with normal brain tissues (P<0.001). Western blot confirmed ZEB2 and HMGA1 were high expression in glioma samples compared with paired normal brain tissues.Immunohistochemistry showed in normal brain tissues, ZEB2 and HMGA1 mainly expressed in nuclei and few in cytoplasm, but Aktl expressed both in nuclei and in cytoplasm. In glioma tissues, ZEB2, Aktl and HMGA1 both expressed in nuclei and in cytoplasm and their expression level were higher than normal brain tissues. In situ hybrization identified miR-637 mainly expressed in cytoplasm in normal brain tissues, and expressed both in nuclei and in cytoplasm in glioma samples. Besides, miR-637 expression level in glioma samples was lower than that in normal brain tissues. Furthermore, statistical analysis found there is no relationship between these four genes and clinical pathologic materials except WHO grading. At the end, we found that the expressive relationship between ZEB2 and miR-637 (r=-0.358, P=0.002), between miR-637 and Aktl (r=-0.345,P=0.003), as well as between miR-637 and HMGA1 (r=-0.399, P=0.001) were both negative in glioma tissue samples.ConclusionZEB2, as a transcription factor, which could bind to the promotor region of miR-637 then inhibited its transcription, blocked the binding between miR-637 and 3’-UTR region of Aktl and HMGA1. As a result, ZEB2 indirectly promoted cell proliferation through upregulating Cyclin Dl, p-Foxol, and downregulating Foxo1 and p15. Meanwhile, ZEB2 also indirectly enhanced cell invasion and migration via upregulating p-Akt, β-catenin, NF-kappa B and Vimentin. In addition, interaction was observed between HMGA1 and Vimentin in glioma cell. Finally, the expression of ZEB2, Aktl and HMGA1 were both high in glioma tissues compared with normal brain tissues and positive related to WHO grading, as well as miR-637 were low expression in glioma tissues compared with normal brain tissues and its expression was negative related to WHO grading. Furthermore, the expressive relationship between ZEB2 and miR-637, between miR-637 and Aktl, as well as between miR-637 and HMGA1 were both negative in glioma tissue samples.