Invasive adenomas are generally pituitary macroadenomas, which often infiltrate the surrounding dura mater, cavernous sinus, brain, and bone tissue . The cavernous sinus invasion rate has been reported to be between 30% and 63% . Studies have shown that invasion of the cavernous sinus and internal carotid artery is an important factor leading to poor prognosis of pituitary adenomas [31, 32]. Invasion of the cavernous sinus and other important tissues poses great challenges to surgical resection and increases the potential risk of cerebrospinal fluid leakage and internal carotid artery injury . The invasiveness of pituitary adenomas is related to the poor prognosis of the patients. Therefore, the study of the progression of invasive pituitary adenomas has important clinical significance to improve the prognosis of patients with pituitary adenomas.
CCNB1 is a member of the cyclin family . CCNB1 can affect the pathogenesis and progression of tumors by affecting the cell cycle [12, 35, 36]. It has been reported that the expression of CCNB1 is upregulated in glioma tissues; overexpression of CCNB1 affects tumor cell invasion, migration, and proliferation, and is associated with poor prognosis . Zhang et al. (2016) reported that CCNB1 was associated with the pathogenesis of pituitary adenomas . In previous research, we demonstrated that CCNB1 affects cavernous sinus invasion in pituitary adenomas through epithelial-mesenchymal transition (EMT) . In this study, we also confirmed that CCNB1 is overexpressed in invasive pituitary adenomas and further affects the ability of invasion and migration of pituitary adenomas.
Previous studies have shown that miR-34a-5p expression exhibits marked changes in various tumors, such as gastric cancer , uterine leiomyosarcoma , lung cancer , hepatocellular carcinoma . Besides, the research also found miR-34a-5p can inhibit EMT in lung cancer, consequently affecting tumor progression . Similarly, in pituitary adenomas, miR-34a-5p can inhibit cell proliferation and promote apoptosis . Furthermore, previous research has revealed that miR-34a-5p affects the biological characteristics of pituitary adenomas through CCNB1, therefore, we speculated that there might be a relationship between miR-34a-5p and CCNB1 . Through the gene prediction websites TargetScan and miRanda, we found that circNFIX (hsa_circ_0005660) had the binding relationship of miR-34a-5p,which was confirmed by the dual-luciferase reporter assays. Therefore, we found a new targeting relationship between miR-34a-5p and circNFIX.
A circRNA is a closed-loop formed by the splicing of precursor RNA, which is mainly formed from exons of protein-coding genes and has a highly conservative ring structure . Recent studies have found that circRNA plays an important role in a variety of biological processes, including cell cycle, apoptosis, vascularization, and invasion [45, 46]. circRNAs can play a wide range of roles including acting as a miRNA sponge to regulate the expression of protein-coding genes . Therefore, circRNA can be used as a potential biomarker for many human diseases . Studies have shown that a circRNA can bind to more than one miRNA . CircNFIX (hsa_circ_0049658) has been reported to affect the progression of gliomas by regulating the miR-378e/RPN2 axis . It has also been reported that circNFIX (hsa_circ_0109102) promotes the proliferation of glioma cells by regulating the expression of miR-34a-5p . However, the function of circNFIX in pituitary adenomas is unknown. Therefore, this study explains the effect of circNFIX (hsa_circ_0005660) on the invasive biological characteristics of pituitary adenomas.
In this study, we explored the role of circNFIX in regulating the expressions of miR-34a-5p and CCNB1 in the development and progression of pituitary adenomas in vivo and in vitro. The roles of circNFIX in pituitary adenomas were summarized in Fig. 6. The results indicated that circNFIX was highly expressed in invasive pituitary adenomas. However, the expression of miR-34a-5p was nearly the opposite. We also revealed that circNFIX silencing or miR-34a-5p overexpression inhibited pituitary adenoma cell invasion, migration, and proliferation. To detect the detailed regulation mechanism between circNFIX and miR-34a-5p, bioinformatics analysis showed there were complementary binding sites between circNFIX and miR-34a-5p. Through dual-luciferase reporter assays and RT-qPCR assays, we confirmed that circNFIX reduced the expression of miR-34a-5p by acting as ceRNA. Further analysis showed that the high expression of circNFIX in pituitary adenomas was related to high Knosp grade and poor prognosis. This evidence suggests that circNFIX is a potential biomarker of pituitary adenomas. Furthermore, our data showed that silencing of circNFIX upregulated miR-34a-5p expression, which in turn downregulated the expression of CCNB1, consequently inhibiting pituitary adenoma cell invasion, migration, and proliferation. All these results demonstrate that circNFIX can sponge miR-34a-5p and competitively suppress its function. Besides, we demonstrated that the inhibitory effect of circNFIX silencing on pituitary adenomas was partially rescued by miR-34a-5p inhibitor. Animal experiments revealed that circNFIX silencing inhibited tumor formation in vivo.