Screening For MicroRNAs Regulating The Expression Of LRIG1 In Glioma And The Potential Mechanisms

Glioma is the most frequent brain tumor in central nervous system. The prognosis of patients suffered from malignant form, especially glioblastoma, is dismal. Current standard therapies for malignant glioma includes surgical resection followed by adjuvant radiotherapy and chemotherapy. Despite aggressive therapies, survival of patients remains poor. Comprehensive understanding of the molecular mechanisms is the fundamental to develop targets for glioblastoma treatment.Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a pan-negative regulator of receptor tyrosine kinases (RTKs). Upon growth factors binding to their receptors, the genes involving proliferation were activated, as well as LRIG1. Thus, the strength and duration of RTK signals are tightly restrained in normal tissues. It has been reported that the expression of LRIG1 in glioma is frequently down-regulated and the loss is associated with high WHO grade. Previous studies demonstrated that restoring LRIG1 exhibited potent inhibitory effect on glioma in vitro and in vivo. Moreover, it confers sensitivity to radiation and chemotherapeutic agents on glioma cells. Thus, LRJG1 is proposed as a tumor suppressor. However, the mechanisms underlying the impaired expression of LRIG1 in glioma remains largely unknown.microRNAs (miRNAs) are a class of small non-coding RNAs of-22 nucleotides in length that negatively regulate gene expression at the post-transcriptional level by directly binding to the 3’untranslated region (3’UTR) of target mRNAs. Accumulating evidence suggests that deregulated miRNAs play critical roles in the initiation and progression of glioma. In addition, bioinformatic analysis indicates that the 3’UTR of LRIG1 mRNA contains a lot of’seed’sequences matching corresponding miRNAs. In present study, the miRNAs targeting LRIG1 were preliminary screened and their potential values in therapy were also evaluated.In the first part of this study, a subset of miRNAs that were putative to target LRIG1 were retrieved from the database (TargetScan & miRanda). Among them, four miRNAs (miR-19a, miR-93, miR-106b, rmR-23a) have been identified as oncomirs according to the previous studies. Ten glioma samples (WHO grade IV) were subjected to real-time qPCR for detecting the expression of the four miRNAs. Meanwhile, the endogenous levels of LRIG1 protein in corresponding tissues were examined by western blot. Linear regression revealed that there was a strong negative correlation between LRIG1 and miR-19a (correlation coefficient was-0.664, P=0.036), as well as miR-23a (-0.678, P=0.031). However, the correlation between LRIG1 and miR-93 (or miR-106b) was weak (P>0.05). These results suggested that miR-19a and miR-23a may be the post-transcriptional regulators of LRIG1.In the second part, the expression of endogenous miR-19a or miR-23a were down-regulated by using corresponding antisenes oligonucleotides (antagomirs). The level of LRIG1 protein in either U87 or U251 cells was robustly increased following miR-19a knockdown. Although U251 and A172 cells had a relatively high basal level of miR-23a, the level of LRIG1 protein was scarcely affected by antagomir-23a. Furthermore, Luciferase reporter assay demonstrated that LRIG1 was a direct target of miR-19a. Moreover, co-transfection assay revealed that RNA interference-mediated LRIG1 knockout in U87 cells was capable to revert the growth inhibitory effect and increased apoptosis following miR-19a down-regulation. Collectively, these results suggested that LRIG1 contributed to the inhibitory effect of antagomir-19a in glioma cells.In the third part, the U87 cells transfected with antagomir-19a or negative control were implanted in the subcutaneous of nude mice. It was noted that the tumorigenesis in antagomir-19a group was significantly delayed compared with that of control group. Besides, the growth of U87-xenografts in antagomir-19a group was substantially decelerated. The tumors were harvested and weighed at 30d post-injection. The tumors of control group weighed more than that of antagomir-19a group,3.59±0.62g versus 1.44±0.23g (P<0.05). Meanwhile, IHC assay demonstrated that the number of LRIG1 staining cells were significantly increased in the antagomir-19a group compared with control group. The data suggested that down-regulation of miR-19a was able to impaired the growth of U87 cells in vivo and this effect may be mediated by up-regulation of endogenous LRIG1.To conclude, over-expressed miR-19a in glioma is responsible for the impaired LRIG1 expression. Down-regulation of miR-19a could inhibit the growth of glioma cells with restoring LRIG1 protein.