The Molecular Interaction And Regulatory Mechanism Of CD44in The Process Of Malignant Progression Of Gliomas

Background:Glioma, which is easy malignancy, recurrence and characteristics, is one ofthe most common malignant brain tumors. The WHO divided it into four grades:grade Ⅰ and grade Ⅱ are belong to the low malignancy degree, while grade Ⅲand Ⅳ belong to malignant partialhigh. The latest research shows that it is easyfor the low-grade gliomas to transformated to high-grade gliomas.CD44, an unclassified adhesion molecule, which is broaderly participattingin the recurrence and development of tumors. The latest research shows that itplays an important role in malignant progression of glioma, but its exactmechanism remains unclear.MicroRNAs are a class of small non-coding single-stranded RNAmolecules, which will be combined with non-fully complementary pairing withits target mRNA molecules’ the3’untranslated region (3’UTR), and inhibit mRNA translation or degradation of mRNA. Thus, they can inhibit the targetgene’ expression in the post-transcriptional level. Studies have shown thatmicroRNAs not only participat in the basic functions of normal cells fromproliferation to apoptosis regulation [6], but also the tumor occurrence anddevelopment procession.miR34a, a common microRNA, is low expression as a tumor suppressormolecule in a variety of tumors. The latest studies have shown that [8]miR34adirectly targeted-regulated of the CD44gene and inhibited the invasion andmetastasis of prostate cancer stem cells. Therefore, we hypothesized thatmiR34a may also played an important role in the malignant progression ofgliomas.Objective：To validate the relationships between CD44and gliomas. To clarify themechanisms of CD44in malignant progression of glioma with vitro experiments,while looking for the CD44upstream regulation of microRNAs and furtherelucidate the regulatory mechanisms.Methods and Results：In clinical glioma specimens, we found that CD44was positively correlatedwith malignant gliomas in the mRNA and protein levels by using qRT-PCR,immunohistochemistry staining and Western Blot. The glioma cell line U251、A172、U87, which are highly expression of CD44, were detected by indirectimmunofluorescence assay and Western Blot experiment.We design and synthesis of interfering double-stranded siRNA sequence ofCD44, which was transefected to the high experssion of CD44glioma cells. Wefound that with the extension of the time of transfection, the expression of CD44was significantly downregulated in mRNA and protein levels by qRT-PCR andWestern Blot. We found that glioma cells’ proliferation、migration and invasionabilities were significantly reduced, while the number of apoptosis wereincreased after the transfection of CD44siRNA sequence by the MTT assay、cell scratch healing assay、 Transwell chamber invasion experiments and flowcytometry. With the reduction of CD44, Erk1/2、p-p38、p-53and p-21weregradually raised, while the p-Erk1/2was greately reduced in protein levels byWestern Blot. Through the dual luciferase reporter system experiment, weproved that CD44is the target gene of miR34a. In clinical glioma specimens,wefound that miR34a was negatively correlated with gliomas’ grade by qRT-PCR.In glioma cells, the expression of CD44was suppressed after upregulated ofmiR34a by qRT-PCR and Western Blot.Conclusion：CD44can be miR-34a negatively regulate, thereby affecting thedownstream of the multiple molecules involved in malignant progression ofglioma. This provides an important theoretical basis to clear the pathogenesis ofglioma progress.