MicroRNA-218-5p accelerates malignant behaviors of breast cancer through LRIG1

Abstract Objective: MicroRNAs play crucial roles in the pathogenesis of cancers. MiRNA-218-5p may act as either an oncogene or a tumor suppressor, but its role in the pathogenesis of Breast Cancer (BC) remains unclear. Methods: Infiltrative breast ductal carcinoma as well as corresponding adjacent normal samples were collected from 30 patients. Mimics and inhibitors of miRNA-218-5p or corresponding negative controls were transfected into BC cells. miRNA-218-5p expression was detected by quantitative PCR. The effects of miRNA-218-5p on the malignant behaviors of BC were assessed. Dual-luciferase reporter assay was employed to evaluate the binding of miRNA-218-5p to LRIG1. Results: BC tissues showed higher miRNA-218-5p expression as compared to the adjacent normal tissues. Ectopic miRNA-218-5p expression accelerated the cell cycle, cell growth and migration of BC, while repressed cell apoptosis. Interestingly, ectopic miRNA-218-5p expression down-regulated LRIG1 expression, and miRNA-218-5p could bind to LRIG1. Also, our study indicated that miRNA-218-5p up-regulated ErbB2 and EGFR expression by targeting LRIG1, suggesting that the LRIG1-mediated signaling pathway contributed to the pro-tumor effects of miRNA-218-5p on BC. Conclusion: MiRNA-218-5p up-regulates ErbB2 and EGFR expression by suppressing LRIG1 expression, thus promoting the malignant behaviors of BC. miRNA-218-5p may exert a pro-tumor effect on BC and serve as a therapeutic target for BC treatment.

LRIG1 expression and its possible suppressor role in oral verrucous carcinoma / 实用口腔医学杂志

Objective:To explore leuncine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) expression in oral verrucous carcinoma(OVC) and its possible mechanism of tumor suppression.Methods:Paraffin specimens of OVC (n =15) and oral squamous cell carcinoma(OSCC,n =30) and corresponding adjacent normal tissues,and 15 normal mucosa tissues(NM) as control from injured patients were collected.The expression levels of LRIG1 and Bcl-2 were examined using immunohistochemical SP method.The correlation between the expression levels of LRIG1 and Bcl-2 was determined using two tailed Pearson's correlation.Results:LRIG1 was significantly lower in OVC tissue than that in NM tissue,but higher than in OSCC tissue.The expression levels of Bcl-2 in OVC were significantly higher than that in NM,but lower than in OSCC.The expression level of LRIG1 was negatively correlated with expression level of Bcl-2 in NM,OVC and OSCC tissue.Conclusion:LRIG1 may inhibit the expression of Bcl-2 and the development of OVC.

Over-expression of LRIG1 suppresses biological function of pituitary adenoma via attenuation of PI3K/AKT and Ras/Raf/ERK pathways in vivo and in vitro / 华中科技大学学报（医学）（英德文版）

Pituitary adenomas (PAs) are well known as a common intracranial benign tumor, and a portion of PAs are refractory to current therapeutic methods. ErbB receptors family signaling pathway regulates the expression of PAs activation associated gene. Inhibition of epidermal growth factor receptor (EGFR) can inhibit proliferation of PAs. Leucine-rich repeats and immunoglobulin-like domains protein 1 ( LRIG1), a negative mediated gene of ErbB receptors family, plays a role in many tumors. However, there are seldom researches about the functional role of LRIG1 in PAs. The aim of this study is to explore the potential effect of LRIG1 and its regulating mechanism in PAs. First, we investigated the role of LRIG1 in cell migration, invasion of PAs with transfected LRIG1 or control. Then, we explored its impact on cell proliferation and apoptosis of PAs in vivo. To study the regulating mechanism of LRIG1, we examined the expression of molecular factor of PI3K/AKT and Ras/Raf/ERK pathway using Western blotting in vitro and RT-PCR in vitro and in vivo. It was found that LRIG1 over-expression inhibited cell migration, invasion and proliferation, and promoted apoptosis of PAs in vivo and in vitro. Furthermore, LRIG1 suppressed the expression of signaling of PI3K/AKT and Ras/Raf/ERK pathways in PAs. LRIG1, as a negative mediated gene of tumor, can inhibit biological function of PAs via inhibiting PI3K/AKT and Ras/Raf/ERK pathways, and it might be a new target for gene therapy of PAs.

LRIG1 Enhances Chemosensitivity by Modulating BCL-2 Expression and Receptor Tyrosine Kinase Signaling in Glioma Cells

PURPOSE: Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) are an inhibitor of receptor tyrosine kinases (RTKs) that was discovered in recent years, and many studies showed that LRIG1 is a tumor suppressor gene and may be related to tumor drug resistance. In this study, we explored whether LRIG1 protein expression can improve the chemosensitivity of glioma cells and what was its mechanism. MATERIALS AND METHODS: We collected 93 cases of glioma tissues and detected the expression of LRIG1 and BCL-2. We constructed a multidrug resistance cell line U251/multidrug resistance (MDR) and examined the change of LRIG1 and BCL-2 at mRNA and protein expression levels. LRIG1 expression was upregulated in U251/MDR cells and we detected the change of multidrug resistance. Meanwhile, we changed the expression of LRIG1 and BCL-2 and explored the relationship between LRIG1 and BCL-2. Finally, we also explored the relationship between LRIG1 and RTKs. RESULTS: LRIG1 was negatively correlated with BCL-2 expression in glioma tissue and U251/MDR cells, and upregulation of LRIG1 can enhance chemosensitivity and inhibit BCL-2 expression. Furthermore, LRIG1 was negatively correlated with RTKs in U251/MDR cells. CONCLUSION: These results demonstrated that LRIG1 can improve chemosensitivity by modulating BCL-2 expression and RTK signaling in glioma cells.

Effects of RNAi-mediated Gene Silencing of LRIG1 on Proliferation and Invasion of Glioma Cells / 华中科技大学学报（医学）（英德文版）

The effects of RNAi-mediated gene silencing of LRIG1 on proliferation and invasion of the human glioma cell line U251-MG and the possible mechanisms were explored in this study.The plasmids pGenesi12-LRIG1-shRNA1 and pGenesi12-LRIGl-shRNA2 were transfected into U251-MG glioma cells respectively by using Lipofectamine 2000 and the transfected cells in which the LRIGI expression was stably suppressed were selected by G418.The cells transfected with negative shRNA served as control.The expression levels of LRIG1 mRNA and protein were measured by qRT-PCR and Western blotting,respectively.The cell cycle was analyzed by flow cytometry.The results showed that LRIG1 mRNA expression was reduced by 70％ and 58％ and LRIG1 protein expression by 58％ and 26％ in U251-MG cells transfected with pGenesil2-LRIGl-shRNAl and pGenesil2-LRIG1-shRNA2 relative to the negative shRNA-transfected U251-MG cells.The proliferative capacity of the LRIG1 specific siRNA-transfected cells was stronger than that of control cells.Cell cycle analysis showed that silencing LRIG 1 significantly increased the percentage of S phase cells and the proliferation index (P＜0.01).Moreover,silencing LRIG1 could promote the invasion of U251-MG cells (P＜0.05).These findings suggested that LRIG1-targeting siRNA can exert a dramatically inhibitory effect on RNA transcription and protein expression of LRIG1,and LRIG1down-regulation could promote the proliferation of U251-MG cells,arrest U251-MG cells in S phase,and enhance the invasion of U251-MG cells.

Association of Expression of Leucine-rich Repeats and Immunoglobulin-like Domains 2 Gene with Invasiveness of Pituitary Adenoma / 华中科技大学学报（医学）（英德文版）

The Leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2) gene expression in pituitary adenoma and its correlation with tumor invasiveness were studied.The expression of LRIG2 mRNA and protein in human pituitary adenoma obtained surgically was detected by RT-PCR (39 cases)and immunohistochemical staining (30 cases).It was found that LRIG2 was mostly localized at the nucleus of the pituitary adenoma cells.Its expression was significantly higher in the invasive cases than in the non-invasive cases.LRIG2 protein was positive in 14 cases out of 21 cases of invasive adenoma,but only 2 cases were positive in 9 cases of non-invasive adenoma.The positive expression rate of LRIG2 mRNA was 91.3％ in invasive cases (total 23 cases) and 62.5％ in non-invasive cases (total 16 cases),respectively.LRIG2 gene is overexpressed in invasive pituitary adenoma.It may play an important role in pituitary adenoma invasiveness and further studies are necessary to elucidate the mechanism under this phenomenon.

Effect of Silencing LRIG3 Gene on the Proliferation and Apoptosis of Bladder Cancer T24 Cells / 华中科技大学学报（医学）（英德文版）

This study examined the effect of silencing LRIG3 expression on the proliferation and apoptosis of bladder cancer T24 cells and explored the role of LRIG3 in the tumorigenesis of bladder cancer.Bladder cancer T24 cells were routinely cultured and pSilencer plasmids were employed to construct LRIG3 eukaryotic expression vector of LRIG3-siRNA,i.e.,pSilencer-LRIG3-siRNA.After confirmation,the vector was transfected into HEK293 cells to make a replication-deficient adenovirus,pAd-LRIG3-siRNA,which was then introduced into bladder cancer T24 cells.RT-PCR,Western-blotting were performed to detect the levels of LRIG3 mRNA and proteins.Cells number was determined by using MTT test.Hoechst33258 staining,transmission microscopy,flow cytometery were conducted to examine the cell apoptosis.Three groups included a blank control group,a negative control group (containing non-interfering plasmids) and a pAd-LRIG3-siRNA group.Our results showed that the recombinant pAd-LRIG3-siRNA was successfully transfected into the bladder cancer T24 cells.The siRNA formed by the transcription of the recombinant plasmids resulted in significantly reduced expressions of LRIG3 gene and protein and significantly decreased cell proliferation and growth in the pAd-LRIG3-siRNA group as compared with the control group (P＜0.01).The siRNA also caused apoptotic changes of some cells,with the apoptosis rate being (17.69±0.75)％,which was significantly different from that of the control group (P＜0.01).It was concluded that recombinant pAd-LRIG3-siRNA plasmids could effectively decrease the expression of LRIG3 mRNA and proteins and,to some extent,inhibit the proliferation and promote the apoptosis of bladder cancer T24 cells.Silencing LRIG3 gene might be a novel alternative for the treatment of bladder cancer.

Effects of RNAi-mediated Gene Silencing of LRIG3 Expression on Cell Cycle and Survival of Glioma Cells / 华中科技大学学报（医学）（英德文版）

The effects of RNAi-mediated gene silencing of LRIG3 expression on cell cycle and survival of human glioma cell line GLI 5 and the possible mechanisms were explored.The plasmids pGenesil2-LRIG3-shRNA l and pGenesil2-LRIG3-shRNA2 were transfected into GL 15 glioma cells respectively by using Metafectine,and the transfected cells that stably suppressed LRIG3 expression were selected by G418.The control cells were transfected with negative control shRNA.The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blot.The apoptosis rate and cell cycle were analyzed by flow cytometry.As compared with the negative shRNA-transfected GL15 cells,LRIG3 mRNA expression in GLI5 cells transfected with pGenesil2-LRIG3-shRNAl and pGenesil2-LRIG3-shRNA2 was silenced by 52.4%,63.8%,and LRIG3 protein expression was re-duced by 50.9% and 67.4% respectively.The LRIG3-specific siRNA transfected cells had higher proliferation rate than control cells.Cell cycle analysis showed that silencing LRIG3 increased the percentage of G2/M phase cells and the proliferation index significantly (P<0.01).Silencing LRIG3 could inhibit the apoptosis of GLl5 cells (P<0.05).These findings suggest that the siRNA targeting LRIG3 gene shows a dramatic inhibitory effect on RNA transcription and protein expression,then promoting the proliferation of GLI5 cells,arresting GLI5 cells in G2/M phase,and suppressing apoptosis ofGL15 cells.

Down-regulation of Leucine-rich Repeats and Immunoglobulin-like Domain Proteins (LRIG1-3) in HP75 Pituitary Adenoma Cell Line / 华中科技大学学报（医学）（英德文版）

Three human leucine-rich repeats and immunoglobulin-like domains (LRIG) genes and proteins, named LRIG1-3, has been previously characterized and it was proposed that they may act as suppressors of tumor growth. The LRIG1 protein can inhibit the growth of tumors of glial cells and the down-regulation of the LRIG1 gene may be involved in the development and progression of the tumor. Real-time reverse transcription-polymerase chain reaction (RT-PCR) is a recently developed technique for quantitative assessment of specific RNA levels. In the current study, it was demonstrated that LRIG1-3 and EGFR mRNA was detected in human pituitary adenoma cell lines and a normal pituitary sample, with differences in the expression levels. Compared to the normal pituitary samples, the expression of LRIG1-3 in HP75 cell line was lower, but the expression of EGFR in HP75 cell line was higher. The results are consistent with LRIG1-3 being tumour suppressor genes, and LRIG genes decreasing the expression of EGFR. The ratio of EGFR/LRIG1 was increased at least 13-fold in HP75 cells compared with the normal pituitary cells, which was also the case for the ratio of EGFR/LRIG2 (14-fold increase in HP75) and EGFR/LRIG3 (11-fold increase in HP75). Further studies were needed to elucidate the explicit role of LRIG genes as negative regulators of oncogenesis in human pituitary adenoma.

Expression of EGFR and LRIG-1 in Human Trigeminal Neurinoma / 华中科技大学学报（医学）（英德文版）

The expression of epidermal growth factor receptor (EGFR) and leucine-rich repeats and immunoglobulinqike domain 1 (LRIG-1) in humantrigeminal neurinoma was investigated and their effect on the origination and development of trigeminal neurinoma, and the relationship between them was studied. By using immunohistochemistry with tissue chip, the expression of EGFR and LRIG-1 was detected in 23 cases of trigeminal neurinoma. It was found that in the 23 cases, the expression rate of EGFR was 21.74 %, while that of the LRIG-1 was 78.26 %. There was a negative correlation between them. It wassuggested that LRIG-1 might inhibit the malignant differentiation and proliferation of the trigeminal neurinoma possibly by the negative feedback loop of EGFR.

Relationship between LRIG1 induced human glioma apoptosis and EGFR gene / 第三军医大学学报

ObjectiveTo explore the molecular mechanism that LRIG1 inhibits signal transduction system of epidermal growth factor receptor(EGFR)by investigating the role of LRIG1 in glioma.MethodsThe plasmid pcDNA3.1-LRIG1 was transfected into primary glioma cells by Lipofectamine.Then,the changes of LRJG1 and EGFR in the transfected glioma cells were measured by RT-PCR and Western blot,and the cell proliferation and apoptosis were analyzed by MTT and flow cytometry.ResultsThe expression levels of LRIG1 mRNA and protein in the glioma cells transfected with pcDNA-LRIG1 were significantly higher than those of control group and pcDNA3.1 transfected glioma cells,while those of EGFR mRNA and protein were significantly lower.The expression of PKC? and Bax was up-regulated,while the expression of bcl-2 was down-regulated.The growth of glioma cells was inhibited and their apoptosis was obviously enhanced.ConclusionBy participating the construction of the negative feedback loop of EGFR,LRIG1 inhibits the occurrence and growth of tumor through several pathways.