MicroRNA-96 Promotes Cell Migration And Invasion In A549 Lung Cancer Cells Through Directly Targeting SARI

Objective Lung cancer is one of most harmful cancers in the world. Lung cancer is the leading cause of cancer mortality and morbidity among men and women in China,and non-small cell lung cancer(NSCLC) accounts for more than 80% of such death.Surgical resection remains the main treatment of choice for NSCLC, but <50% of patients can undergo curative resection and even for early-stage NSCLC, 5-y survival after curative resection has been reported to range from only 30–60%. Invasion and metastasis of lung cancer also affect patients’ effects and survival time. The clear pathogenesis and metastasis mechanism is still unclear, and the clinical work lacks effective therapy. Therefore, novel therapeutic strategies are urgently needed to improve its poor prognosis. SARI(Suppressor of AP-l, regulated by IFN), which also named BATF2, is a novel type I IFN-inducible early response gene. SARI is a novel tumor suppressor gene found in the past few years. The expression of SARI was constitutively detected in multiple lineage-specific normal cells, but not detected in their tumorigenic counterparts. Our previous research show that SARI inhibits the invasion and metastasis of lung adenocarcinoma by modulating EMT(epithelial-mesenchymal transition). However, the suppression mechanisms of SARI expression in NSCLC are still obscure. micro RNAs(mi RNAs) are a class of endogenous, small non-coding single RNA molecules that regulate gene expression at the posttranscriptional level. Several studies demonstrated that mi RNA-96 is abnormally expressed in various tumors and directly involved in human cancer processes. In NSCLC, the role of mi RNA-96 remains largely unknown. Recent results identified the expression of mi RNA-96 in lung cancer tumor were significantly higher than that of their normal counterparts. The high expression of mi RNA-96 in tumors was associated with overall poor survival in patients with lung cancer. The aim of this study was to investigate the expressions of SARI and mi RNA-96 in NSCLC, and to clarify the regulation of mi RNA-96 through directly targeting SARI; to observe the effect of mi RNA-96 and SARI on biological behavior in A549 lung cancer cells. This study can provide new experimental evidences of carcinogenesis and progress mechanism, and laying a foundation for formulating novel therapeutic target for treatment of NSCLC.Methods 1. The expression of SARI was evaluated in NSCLC tissues of 120 patients whounderwent curative surgery by immunohistochemistry. Further, SARI m RNA levels were also detected in cancer tissues and adjacent non-tumor tissues of 30 patients by RT-PCR. In addition, survival analysis was performed between clinicopathologic variables and prognosis of NSCLC patients. 2.Taqman stem-loop real-time q RT-PCR method was used to assess the expression of mi RNA-96 in the NSCLC. The correlation of mi RNA-96 expression and clinicopathological features of NSCLC was analyzed, and the correlation of mi RNA-96 expression and SARI m RNA expression was also analyzed. 3.By using Target Scan, mi Randa and Microcosm Targets, we speculated that SARI might be one of the target genes of mi RNA-96. Luciferase reporter assay was used to verify mi RNA-96 directly regulated SARI expression. RT-PCR and western blotting were used to examine the m RNA and protein expression of SARI respectively in A549 cells transfected with mi RNA-96 inhibitors. 4. A549 cells were transfected with mi RNA-96 inhibitors to down-regulate mi RNA-96 expression, then MTT assay were used for evaluating cell proliferation, wound-healing and transwell invasion assays were employed for investigating the metastatic properties. RNAi technique was used to down-regulate SARI expression in A549 cells, then its effect on migration and invasion were examined.Results 1. In 120 NSCLC samples, 41.7 % of cases(50/120) showed positive expression of SARI. The relative m RNA expression value of SARI in NSCLC tissues were significantly much lower than those in non-tumor adjacent tissues(0.37±0.14 VS 1.02±0.27,fold change 0.36, P=0.025). SARI protein expression was significantly associated with several clinicopathologic variables in NSCLC tissues as follows: tumor differentiation, extent of metastatic lymph nodes, T stage and TNM stage. However, only extent of metastatic lymph nodes and TNM stage were identified as the independent indicators of SARI protein expression in NSCLC tissues with the binary logistic regression multivariate analysis(P<0.05). In survival analysis, patients with positive SARI expression had better overall survival compared to the negative SARI expression group(67.5% vs 41.6%,χ2=8.993,P=0.003). Multivariate Cox regression analysis showed that SARI expression status was the independent prognostic factors for NSCLC.2.The expression level of mi RNA-96 was significantly higher in cancer tissues than that in non-tumor adjacent tissues(fold change 2.16, P<0.05).The expression level of mi RNA-96 had correlation with tumor differentiation, extent of metastatic lymph nodes and T stage(P<0.05), and no correlation with sex, age, smoking status, histology and tumor location(P>0.05). And overexpression of mi RNA-96 is associated with reduced expression of SARI in NSCLC tumor tissues. 3. Bioinformatics predicted that SARI might be a target of mi RNA-96. Luciferase reporter assay showed that mi RNA-96 remarkably repressed the expression of luciferase from a vector also containing the wild-type mi RNA-96 binding site, but had no effect on mutant vector. Inhibition of mi RNA-96 could upregulate the m RNA and protein level of SARI in A549 cells. 4. After t ransfection of mi RNA-96 inhibitor, the speed of Cell A549 growth is obviously slower than that of the controls, and decreased potential of migration and invasion in vitro.Silencing of SARI with si RNA in A549 cells led to increased cell migration and invasion.Conclusions 1. The SARI m RNA and protein expression in gastric tumor tissues were significantly lower than those in adjacent non-tumor tissues. The expression of SARI was associated with the lymph node metastases and TNM stage. The survival analysis demonstrated that SARI expression status was the independent prognostic indicators for NSCLC. It suggested that SARI may be a tumor suppressor gene. 2. The higher expression level of mi RNA-96 in NSCLC tumor tissues and correlation with clinicopathological features indicate that the mi RNA-96 play as oncogene roles in NSCLC. The expression level of mi RNA-96 tended to significant negative correlate with SARI expression. 3. mi RNA-96 post-transcriptionally down-regulates the expression of tumor suppressor SARI.mi RNA-96 inhibitor transfected in A549 cells exhibited markedly increased SARI expression. 4. mi RNA-96 promotes cell migration and invasion in A549 cells, at least in part, by directly repressing SARI expression. Both of them are likely to be new potential therapeutic target for treatment of NSCLC.