Department of Biochemistry, AIIMS, New Delhi, India.
Department of Urology, AIIMS, New Delhi, India.
Mol Cell Biochem. 2020 Jan;464(1-2):51-63. doi: 10.1007/s11010-019-03648-y. Epub 2019 Nov 21.
RASSF1A is a tumor suppressor gene, and its hypermethylation has been observed in cancers. RASSF1A acts as an upstream regulator of Hippo pathway and modulates its function. The aim of this study was to analyze expression of RASSF1A, Hippo pathway molecules (YAP, MST) and downstream targets (CTGF, Cyr61 and AREG) in bladder cancer patients. Later, the link between RASSF1A and Hippo pathway and a potential therapeutic scope of this link in UBC were also studied. MSPCR was performed to study methylation of RASSF1A promoter. Expression of molecules was studied using qPCR, Western blot and IHC. The link between RASSF1A and Hippo pathway was studied using Spearman's correlation in patients and validated by overexpressing RASSF1A in HT1376 cells and its effect on Hippo pathway was observed using qPCR and Western blot. Further therapeutic potential of this link was studied using MTT and PI assays. The expression of RASSF1A was lower, whereas the expression of YAP, CTGF and CYR61 was higher. The expression of RASSF1A protein gradually decreased, while the expression of YAP, CTGF and CYR61 increased with severity of disease. Based on Spearman's correlation, RASSF1A showed a negative correlation with YAP, CTGF and CYR61. YAP showed a positive correlation with CTGF and CYR61. To validate this link, RASSF1A was overexpressed in HT1376 cells. Overexpressed RASSF1A activated Hippo pathway, followed by a decrease in CTGF and CYR61 at mRNA, and enhanced cytotoxicity to chemotherapeutic drugs. This study finds a previously unrecognized role of RASSF1A in the regulation of CTGF and CYR61 through mediation of Hippo pathway in UBC and supports the significance of this link as a potential therapeutic target for UBC.
RASSF1A 是一种肿瘤抑制基因，其在癌症中存在高甲基化。RASSF1A 作为 Hippo 通路的上游调节剂，调节其功能。本研究旨在分析膀胱癌患者中 RASSF1A、Hippo 通路分子（YAP、MST）和下游靶点（CTGF、 Cyr61 和 AREG）的表达。之后，还研究了 RASSF1A 与 Hippo 通路的联系及其在 UBC 中的潜在治疗范围。MSPCR 用于研究 RASSF1A 启动子的甲基化。使用 qPCR、Western blot 和 IHC 研究分子的表达。通过 Spearman 相关性在患者中研究 RASSF1A 与 Hippo 通路之间的联系，并通过在 HT1376 细胞中转染 RASSF1A 来验证其对 Hippo 通路的影响，并使用 qPCR 和 Western blot 观察其影响。进一步通过 MTT 和 PI 测定研究该联系的潜在治疗作用。RASSF1A 的表达降低，而 YAP、CTGF 和 CYR61 的表达升高。RASSF1A 蛋白的表达逐渐降低，而 YAP、CTGF 和 CYR61 的表达随着疾病的严重程度而增加。根据 Spearman 相关性，RASSF1A 与 YAP、CTGF 和 CYR61 呈负相关。YAP 与 CTGF 和 CYR61 呈正相关。为了验证这一联系，在 HT1376 细胞中转染 RASSF1A。过表达的 RASSF1A 激活 Hippo 通路，随后 CTGF 和 CYR61 的 mRNA 水平下降，并增强了对化疗药物的细胞毒性。本研究发现 RASSF1A 在调节 UBC 中的 CTGF 和 CYR61 方面发挥了以前未被认识的作用，通过 Hippo 通路介导，并支持该联系作为 UBC 潜在治疗靶点的重要性。
