| Background and Objective: Lung cancer was the leading cause of death among the malignant tumors worldwide.It was estimated that China endured 0.6 million lung cancer deaths and 0.7 million new lung cancer cases during 2015 in China. Non-small cell lung cancer (NSCLC) accounts for 80% of all lung carcinomas. It is still urgent and critical to explore the molecules that drive lung cancer growth, survival, and metastasis and identify novel biomarkers during the tumor progression as the potential therapeutic targets. G-protein coupled receptors (GPCRs) are the main extracellular surface receptors involved in the signal transduction. Growing literatures suggest that different expression levels and activation of GPCRs are associated with different types of tumors.Furthermore, evidences showedthat there was excessive activation of GPCRs in NSCLC,while there have been few drugs inhibiting GPCRs directly. The regulator of G-protein signaling (RGS) family of proteins is the regulatory molecule of GPCR-mediated responses in cells. RGS could serve as GTPase activation protein (GAP) to accelerate GTP hydrolysis. Many data indicated that genetic alterations of RGS members were involved inseveral disease states including schizophrenia and tumorigenesis.Theup-regulation of RGS4 has been reported in colorectal cancers, and was associated with increased cell viability, invasion, and/or motility of thyroid cancer, glioma, ovarian ascites, and triple negative breast cancer cells. However, the role of RGS4 has been poorly understood in lung cancers up to date. The purpose of this study was to determine whether RGS4 plays roles and what kinds of roles it plays in regulating NSCLC development.Methods: Immunohistochemistry (IHC), Cell Culture and Transfection, RNA isolation and quantitative RT-PCR, Cell Counting Kit-8 Assay, Colony Formation Assay,Assessment of apoptosis, Immunofluorescence Assay, Wound Healing Assay, Transwell Assay, Protein Extraction and Western Blotting, Statistical analysis.Results:1. RGS4 is over-expressed in certain NSCLC tissuesWe performed immunohistochemistry assays to detect RGS4 expression in tissue microarrays (TMAs) containing 41 NSCLC tissues and 7normal tissues. The RGS4 protein displayed high expression in 20 NSCLC cases, and showed mediumor low staining in normal tissues. Statistical analysis revealed that RGS4 protein was significantly overexpressed in NSCLC tissues than in non-tumor tissues. Overexpression of RGS4 was found more in adenocarcinoma than squamous cell carcinoma accounting for 62.5% and18.2% (P□=□ 0.0228). The subcellular expression pattern of RGS4 in NSCLC sample was investigated by immunofluorescence analysis further. RGS4 showed the major cytoplasmic localization in tumor tissues; while in lung tissues, the staining is mainly localized at stroma. Further western blotting analysis also indicated that the protein level of RGS4 is higher in certain NSCLC samples.All above results suggested that RGS4 is significant overexpressed in some NSCLC samples compared to that of normal lung tissues.2. RGS4 knockdown decreased cell proliferation, but not migration and invasion in H1299 cellsWe examined whether the deficiency of RGS4 affects cell functions. RGS4 was knocked down by siRNA and CCK-8 assay was performed to evaluate the effect of RGS4 knockdown on the proliferation activity of H1299 cells. CCK-8 assay results indicated that the OD450 values for the RGS4-siRNA group on 48h and 72h weresignificantly inhibited compared with NC groups suggesting that RGS4 knockdown greatly inhibited cell proliferation.In clonogenic assays, the shRGS4group showed decreased colony formation ability by 47% compared with shNC control indicating the inhibition of cell colony formation by RGS4 knockdown. The wound healing and transwell assays were performed to determine the migratory and invasive abilities of cells after transfection with shRNA. The wound healing assay revealed that the migratory ability of H1299 cells in the shRNA transfection group were not significantly affected at both 24h and 48h post-wounding compared with both NC groups. Meanwhile, the cell invasion assay by transwell showedthat the number of invading cells was not significantly affected either in the shRGS4 group compared to NC control group in H1299 cells.3. RGS4 knockdown or inhibition promotes apoptosis in H1299 cellsWe detected the apoptosis of H1299 cells upon serum starvation by TUNEL assay. The percentage of TUNEL-positive cells had significantly increased after 40h of RGS4 siRNA transfection suggesting the inhibition of RGS4 promotes cell apoptosis. Compared with the H1299 cells in the shNC group, the activation of proapoptotic protein caspase-3 were significantly increased in H1299 cells after 48 h of RGS4 shRNA group transfection.The expression of proapoptotic protein Bax was also remarkably increased in the RGS4 shRNA group compared with the NC groups (P<0.05). In contrast, there was no significant difference in the expression of anti-apoptotic protein Bcl-2 between the RGS4 shRNA group and the NC groups.4. RGS4 knockdownincreased microRNA-16 expression and inhibited BDNF-TrkB signaling in H1299 cellsWe next detected the connection between RGS4 and miR-16. It was found that when the RGS4 was knockdown by shRNA, the miR-16 expression was elevated. To investigate whether RGS4 is functional through miR-16, we co-transfected miR-16 inhibitor and RGS4 siRNA in H1299 cells, and then detected the activation of Caspase-3. MiR-16 inhibitor could abolish the RGS4 shRNA effects on Caspase-3 activation and pro-apoptotic protein Bax expression, suggesting RGS4 knockdown activated apoptosis through the induction of tumor suppressor miR-16. We found that miR16 inhibitor significantly increase the mRNA of RGS4 in H1299 cells. That is to say, the increased RGS4 mRNA levels in NSCLC may result in and from decreased expression of miR16.Our results showed that RSG4 knockdown indeed decreased BDNF expression and the phosphorylation of TrkB, the receptor of BDNF, suggesting that BDNF-TrkB pathway could be the downstream signaling of RGS4. Moreover, BDNF expression was significantly elevated by the inhibition of miR16. Furthermore, we found that the specific inhibitor of Trk, K252a, blocked the function of miR-16 inhibitor on cell proliferation. In summary,RGS4 and miR16 could regulate each other’s expression as a feedback loop and control the downstream BDNF-TrkB signaling in NSCLC cell lines.Conclusion: Our study showed that the expression level of RGS4 was up-regulated in certain NSCLC samples, and the distribution of RGS4 in normal and NSCLC tissues were different.RGS4 was mainly shown the cytoplasmic localization in tumor cells but stroma localization in normal cells, which suggested that it may refer the cancer cells some stroma character. We also found that decreasing the protein level of RGS4 or decreasing its activity by inhibitor caused corresponding proliferation decrease andapoptosis, without affecting migration and invasion. Moreover, when the RGS4 was knockdown by siRNA, the tumor-suppressor miR-16 expression was elevated.Furthermore, when blocking miR-16 by the inhibitor, the mRNA level of RGS4 was elevated in turn and the role of RGS4 silencing on proliferation and apoptosis was abolished. |
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