Expression Of SOX8 In Non-small Cell Lung Cancer And Therapeutic Potential Of MiRNA-124

PART I Clinicopathologic Study of Sox8 Expression in Non-Small Cell Lung CancerPurposePrimary lung cancer is the most common and lethal malignant tumor worldwide, which ranked the first place as the cause of death by tumor and brought heavy burden to countries on the planet. And non-small cell lung cancer (NSCLC) is the most prevalent type of total malignant primary lung cancers. Accumulating evidence showed that SOX proteins play critical role in the development of tumors and maintenance of tumor stem cells. Despite SOX8 and SOX9 belongs to the E group of SOX protein family and shares high resemblance of functional sequences, however, currently only SOX9 was studied in malignant cancers including NSCLC, while information of SOX8 expression in NSCLC remains unknown. Considering the fact that both SOX8 and SOX9 proteins were found to be up-regulated and involved in the tumorogenesis of hepatocellular carcinomas, we decided to study SOX8 expression in NSCLC. We provide descriptive evidence of SOX8 expression in 80 NSCLC specimens and 7 normal lung tissues at the transcriptional and translational levels in this section. Moreover, we studied the correlation between SOX8 expression and various clinical factors. In addition,77 NSCLC patients received follow-up for survival study of the predictive value of SOX8 in NSCLC.Materials and Methods1. Immunohistochemistry was used to examine SOX8 protein expression in 80 NSCLC tissues and 7 normal bronchial lung tissues (5 cm away from the gross border of tumor lesions). The correlation between the expression of SOX8 with a variety of clinical factors including gender, age, cell differentiation, lymph node metastasis, and clinical stage was analyzed.2. The mRNA expression of SOX8 in NSCLC and normal lung tissues at transcriptional level were studied by quantitative real-time chain reaction (qRT-PCR).3. Seventy-seven NSCLC patients available for follow-up was included for survival analysis. Univariate survival analysis and Log-rank test was used to determine the predictive significance of SOX8 over-expression in NSCLC patients.Results1. SOX8 protein expression in NSCLC and normal lung tissuesAt translational level, immunohistochemistry results revealed that SOX8 was found to be little or no expression in normal lung tissues, while overexpressed in NSCLC tissues (P<0.01). SOX8 protein was generally to be nuclear immunopositive. Total immunopositive SOX8 cases accounted for 86.25% of total NSCLC cases (69/80), and intense expression accounted for 62.5% (50/80).2. Correlation between SOX8 expression and clinicopathologic factorsTo further analyze the significance of SOX8 expression in NSCLC, we studied the correlation between SOX8 expression and clinicopathologic factors of NSCLC patients. Correlation was revealed between SOX8 expression and tumor size (P< 0.001), cell differentiation (P=0.015) and clinical stage (P=0.013). But no difference between SOX8 expression and other factors such as pathological classification, gender, age and tobacco/alcohol history was found.3. SOX8 mRNA expression in NSCLCqRT-PCR results with ANOVA analysis and Tukey’s post hoc test revealed that SOX8 Mrna was upregulated in NSCLC tissues of low differentiation group (6.52-fold, P= 0.047) and high differentiation (11.71-fold, P<0.001) group than in normal lung tissues. Moreover, SOX8 mRNA expression in low cell differentiation group was significantly higher than in high cell differentiation group (1.80-fold, P<0.001).4. Survival analysis of SOX8 in NSCLCLog-rank test showed that SOX8 protein over-expression was related to significantly shortened overall survival (48.7 months, P=0.006).Conclusion1. No SOX8 expression was found in normal lung tissues.2. SOX8 was overexpressed in most NSCLC tissues, and its expression was found to correlate with tumor size, cell differentiation, lymph node metastasis, and clinical stage, which suggests SOX8 might be involved in the cell proliferation and migration in NSLCL tumor cells.3. NSCLC with SOX8 overexpression manifested unfavorable overall survival which indicate SOX8 protein expression might play a predictive role in clinical practice.Part Ⅱ In Vitro Study of Therapeutic Efficacy of miRNA-124 in Non-Small Cell Lung CancerPurposeMicroRNA(miRNA) is one type of small molecular RNA with 19-25 nucleotides which prevalently exists in enkaryocytes. Despite of a short sequence, miRNA could bind to target mRNA and down-regulate its expression at transcriptional level and execute regulatory function in cells. In tumors, miRNA could either be protector or oncogenes. Utilize the regulatory impact of miRNA on functional mRNAs, miRNA is becoming a promising therapeutic option in medical research. However, little was kown about the regulatory miRNA on SOX8 in NSCLC. In this section, by using TargetScan and 2 other softwares, we predicted the potential miRNA as miRNA-124 and verified the interaction between miRNA-124 and SOX8. Next we study the impact of miRNA-124 overexpression and low expression on the proliferation and migration in NSCLC cell lines in vitro.Material and methods1. Prediction of regulatory miRNA on SOX8TargetScan, MicroCosm Targets version 5, and microRNA.org data base were used to predict the regulatory miRNA on SOX8 and the biding site on 3’UTR of SOX8.2. MiRNA expression in 9 NSCLC samplesqRT-PCR was used to examine the paired expression of miRNA-124 and SOX8 mRNA in 9 NSCLC samples.3. Verification of interaction between miRNA-124 and SOX8 Luciferase assay was used to compare the interaction between SOX8 mRNA and miRNA-124 wildtype and mutant types.4. Impact of MiRNA-124 on the viability and migration on NSCLC cell lines Human NSCLC cell lines A549 and H1299 was transfected with miRNA-124 mimic and miRNA-124 inhibitor, and then CCK-8 was used to test the viability of cells and Transwell was used to examine the migration of cells.Results1. Decreased expression of miRNA-124 in NSCLC PatientsIt is established that miRNA-124 is down-regulated in various kinds of tumors. Real time PCR was performed to test whether miRNA-124 also decreased in NSCLC patients. Significantly reduced expression of miRNA-124 was observed in 9 NSCLC samples comparing with normal adjacent tissues.2. Sox8 as a target gene of miRNA-124Due to the cardinal role of SOX8 in NSCLC and the advantages of microRNA in cancer development and metastasis, we further analyzed the sequences of 3’UTR in Sox8 mRNA and some predicted miRNAs through the website service Targetscan. Given the number of publications of miRNA-124 in cancer, we chose miRNA-124 for further validation. In our dual luciferase reporter assay, we found miRNA-124 decreased the relative luciferase activity of firefly carrying the wild type, but not mutant,3’UTR of SOX8 mRNA (P= 0.002). Additionally, SOX8 protein was decreased in the H1299 cells with miRNA-124 mimics compared to vector control, supporting our validation of SOX8 as a target gene of miRNA-124.3. Cell proliferation is regulated by miRNA-124 in NSCLC cell linesWith information about the potential role of SOX8 and the relationship between SOX8 and miRNA-124 in patient samples, we further analyzed whether miRNA-124 truly related to some functions of NSCLC in cell-based assays. miRNA-124 mimics and inhibitors in two types of NSCLC cells provided more evidence to indicate that miRNA-124 can regulate NSCLC cell growth.4. Cell migration is regulated by miRNA-124 in NSCLC cell linesBy trans-well assay, upregulated miRNA-124 significantly inhibited tumor cell migration capability in both A549 and H1299 cell lines.Conclusion1. Decreased expression of miRNA-124 was found in NSCLC Patients.2. Sox8 acts as a target gene of miRNA-124, and over-expression decreased expression of SOX8 expression at both translational and transcriptional levels.3. Over-expression of miRNA-124 significantly inhibited viability and migration in NSCLC cell line A549 and H1299 in vitroPart Ⅲ In vivo study of Therapeutic Efficacy of miRNA-124 in Non-Small Cell Lung CancerPurposeDue to the great difference of physiological circumstance between in vitro and in vivo conditions, thus we decided to study the therapeutic efficacy of miRNA agomir-124 on xenograft subcutaneous tumor models of human NSCLC cell line A549 on nude mice.Materials and methodsNSCLC xenograft nude mice model was established using cell line A549, and subsequently treated with miRNA-124 agomir-124 (1 nmol/50μL) every 4 days since day 8 after the establishment of animal model. Tumor size was measured every 4 days during the treatment of miRNA agomir-124 and the mice were sacrificed on day 36.ResultsEfficacy of miRNA agomir-124 in the NSCLC xenograft modelGiven the result that miRNA-124 mimic could inhibit tumor cell viability and migration ability in vitro, in our present study we evaluated the anticancer efficacy of miRNA agomir-124 in A549 subcutaneous xenograft models using doses of 1 nmol/L of miRNA agomir-124 and an equal amount of PBS as a control. As shown in Figure 5A, from day 16 after the first treatment, miRNA agomir-124 considerably inhibited subcutaneous tumor growth. At the end of the observation, we also compared mean final tumor weights between treatment and control groups. The data confirmed that difference in the mean final tumor weights between the two groups was statistically significant(P< 0.05).Conclusion1. Nude mice NSCLC xenograft model was successfully established.2. MiRNA agomir-124 is an efficient tool in the functional study of miRNA-124 in vivo.3. MiRNA agomir-124 inhibited the tumor growth in nude mice tumor model which suggested that miRNA agomir-124 is potentially an efficient therapeutic option in NSCLC.