Circular RNA circFAT1(e2) Facilitates Cell Progression through the miR-30e-5P/MYBL2 Pathway in Glioma

Abstract

Objective: To explore the mechanism of glioma from MYB family genes from the perspective of the circRNA-miRNA-mRNA regulatory network.

Methods: First, the MYB family genes were analyzed by multiple bioinformatics analyses to identify one gene most associated with glioma. Then, the prognostic value and clinical characteristics of the gene were evaluated by bioinformatics analysis and experiments in glioma cells. Next, the target miRNA and circRNA were predicted and verified by dual-luciferase reporter assays. Besides, the functions of target circRNA in glioma were investigated by CCK-8 and Transwell assays. At last, the relation between the screened MYB gene, miRNA, and circRNA in glioma was identified by rescue experiments.

Results: After expression and Cox and survival analysis of six MYB family genes, MYBL2 was identified as the gene most associated with glioma. Then, we found that MYBL2 expression in primary gliomas was higher than those in other histologies, and it had variable expression according to clinical features. Furthermore, MYBL2 knockdown in glioma cells impairs cell growth, invasion, and migration in functional studies. Then, miR-30e-5p and circFAT1(e2) were identified as targets of MYBL2 by bioinformatics prediction and experimental verification. Finally, the relationship between circFAT1(e2), MYBL2, and miR-30e-5p was elucidated by rescue experiments.

Conclusion: circFAT1(e2) could promote glioma development by regulating MYBL2 and miR-30e-5p, and MYBL2 has diagnostic and prognostic values in glioma.

Figure 1

Batch analysis on MYBL2 family genes. (a) Expression analysis of MYB, MYBL2, MYBL1, MYBBP1A, MYPOP, and MYSM1 in three different groups. (b) Forest plot of Cox regression analysis for MYB, MYBL2, MYBL1, MYBBP1A, MYPOP, and MYSM1. (c–g) PFS analysis of genes with significant P values (MYB, MYBL2, MYBL1, MYBBP1A, and MYPOP). ^∗∗P < 0.01 and ^∗∗∗P < 0.001.

Figure 2

Prognostic analysis of MYBL2. (a) Survival analysis on MYBL2 expression in primary glioma. The survival rate of high MYBL2 expression was poor. (b) Survival analysis on MYBL2 expression in recurrent glioma. The survival rate of high MYBL2 expression was poor. (c, d) Cox regression study on MYBL2 and clinical variables (MYBL2, age, gender, race, and grade), both univariate and multivariate. (e) In glioma patients, the nomogram predicts 1-, 3-, and 5-year overall survival. (f) The ideal nomogram is represented by the diagonal dashed line on the calibration curve of the OS nomogram model.

Figure 3

MYBL2 expressions in different glioma histologies and their relation with various clinical features. (a) MYBL2 expression differences in different histologies. The abscissa is the histology, and the ordinate is the gene expression of MYBL2. (b) MYBL2 expression differences in different WHO grades. The expression level of MYBL2 increased with the increase in grade. (c) MYBL2 expression differences in IDH mutation status. The expression level of MYBL2 in the wild type was higher than that in the mutant type. (d) MYBL2 expression differences in 1p/19q codeletion status. The expression level of MYBL2 in noncodel was higher than that in codel. (e) The expression differences of MYBL2 in age status. The expression level of MYBL2 in patients older than 42 years was higher than that in patients younger than 42 years. (f) MYBL2 expression differences in gender. There was no significant difference in the expression level of MYBL2 in patients of different genders.

Figure 4

Downregulation of MYBL2 inhibits the proliferation, invasion, and migration of SW1783 and U373 cells. (a, b) Boxplots, MYBL2 is highly expressed in G1 and tumor tissues. The red ones represent tumor samples, and the blue ones represent normal samples. (c, d) Downregulation of MYBL2 in SW1783 and U373. (e, f) The proliferative capacity of MYBL2 is attenuated in SW1783 and U373 with si-MYBL2. (g, h) The numbers of invading and migrating cells were reduced after the MYBL2 knockdown. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 5

Effects of knocking down circFAT1(e2) on glioma cell proliferation and metastasis. (a) miR-30e-5p is expressed at lower levels in glioma tumors. Brown represents tumor samples, and white represents normal samples. (b) Website prediction of target circRNA of miR-30e-5p. Purple represents ENCORT, yellow represents circbank, and green represents RNAhybrid. (c) circFAT1(e2) knockdown efficiency by si-circ1 and si-circ2 in SW1783. (d) Overcirculation elevated the expression of circFAT1(e2) in U373 cells. (e, f) In comparison to the si-NC group, circ1 silencing decreased the growth of SW1783 and U373 cells. (g, h) The numbers of migrating and invading cells decreased after circFAT1(e2) knockdown. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 6

circFAT1(e2) targets miR-30e-5p and MYBL2 to regulate glioma. (a, b) The activity of luciferase in cells transfected with the WT plasmid tended to rise, but the mutant group's activity appeared to be unchanged. (c) miR-30e-5p had a negative relation with MYBL2. (d) Si-circ1 enhanced miR-30e-5p expression, while miR-30e-5p inhibitors and over-MYBL2 were able to counteract this. (e) Reduced miR-30e-5p expression was caused by over-circ expression, which was reversed by over-miR-30e-5p and si-MYBL2. (f, g) CCK-8 assays were applied to verify the results in (d) and (e). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.