Hypoxic Regulation of the KLK4 Gene in two Different Prostate Cancer Cells Treated with TGF- β.

IF 1.8 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Cell Biochemistry and Biophysics Pub Date : 2024-07-19 DOI:10.1007/s12013-024-01396-5

Fatma Poyrazlı, Derya Okuyan, Feray Köçkar, Sümeyye Aydoğan Türkoğlu

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Abstract

The human kallikrein-related peptidase (KLK) family which consists of 15 members is associated with prostate cancer and other cancers. It has been reported that overexpression of KLK4 in prostate cancer correlates with bone metastasis or advanced stage. Hypoxia occurs in the early stages of prostate cancer due to the accumulation of acidic metabolites or reactive oxygen species (ROS). In our study, KLK4 gene expression in hypoxic conditions in PC-3 and LNCaP cells which are treated with TGF-β was evaluated with mRNA, protein, and promoter activity levels. A chemical hypoxia model was created and confirmed at mRNA and protein level. No statistically significant cytotoxic effect of CoCl₂ and TGF-β was observed in PC-3 and LNCaP cells with the MTT test. Four different truncated KLK4 gene promoter constructs were cloned in pmetLuc expression vector and basal activities of all promoter fragments were analyzed. The activities of P1 (-447/ + 657), P2 (-103/ + 657), and P3 (-267/ + 657) promoter fragments increased in hypoxic conditions except P4 (+555/ + 657), which does not contain the SMAD and HRE region. KLK4 mRNA levels in both PC-3 and LNCaP cells increased in the hypoxia and hypoxia/TGF groups compared to the non-treated groups. The stimulating effect of TGF-β is correlated with the increase in SMAD2/3 mRNA levels. KLK4 expression is up-regulated by TGF-β, especially under hypoxic conditions, and its interaction with the SMAD pathway is determined with different inhibitor experiments. HIF-1α and SMAD transcription factors bind to the KLK4 promoter showing the direct interaction of HIF-1α (-80/-52) and SMAD (+163/+194) regions with EMSA.

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用 TGF- β 处理的两种不同前列腺癌细胞中 KLK4 基因的缺氧调控。

由 15 个成员组成的人类 Kallikrein 相关肽酶（KLK）家族与前列腺癌和其他癌症有关。据报道，KLK4 在前列腺癌中的过表达与骨转移或晚期相关。前列腺癌早期由于酸性代谢产物或活性氧（ROS）的积累而发生缺氧。我们的研究通过mRNA、蛋白质和启动子活性水平评估了PC-3和LNCaP细胞在缺氧条件下的KLK4基因表达。建立了化学缺氧模型，并在 mRNA 和蛋白质水平上进行了确认。通过 MTT 试验，在 PC-3 和 LNCaP 细胞中没有观察到 CoCl2 和 TGF-β 有统计学意义的细胞毒性作用。在 pmetLuc 表达载体中克隆了四种不同的截短 KLK4 基因启动子构建体，并分析了所有启动子片段的基础活性。在缺氧条件下，P1（-447/ + 657）、P2（-103/ + 657）和P3（-267/ + 657）启动子片段的活性增加，但不含SMAD和HRE区域的P4（+555/ + 657）除外。与未处理组相比，缺氧组和缺氧/TGF 组 PC-3 和 LNCaP 细胞中的 KLK4 mRNA 水平均有所增加。TGF-β的刺激作用与SMAD2/3 mRNA水平的增加相关。KLK4的表达受TGF-β上调，特别是在缺氧条件下，其与SMAD通路的相互作用通过不同的抑制剂实验来确定。HIF-1α和SMAD转录因子与KLK4启动子结合，通过EMSA显示了HIF-1α（-80/-52）和SMAD（+163/+194）区域的直接相互作用。

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来源期刊

Cell Biochemistry and Biophysics 生物-生化与分子生物学

CiteScore

4.40

自引率

0.00%

发文量

审稿时长

7.5 months

期刊介绍： Cell Biochemistry and Biophysics (CBB) aims to publish papers on the nature of the biochemical and biophysical mechanisms underlying the structure, control and function of cellular systems The reports should be within the framework of modern biochemistry and chemistry, biophysics and cell physiology, physics and engineering, molecular and structural biology. The relationship between molecular structure and function under investigation is emphasized. Examples of subject areas that CBB publishes are: · biochemical and biophysical aspects of cell structure and function; · interactions of cells and their molecular/macromolecular constituents; · innovative developments in genetic and biomolecular engineering; · computer-based analysis of tissues, cells, cell networks, organelles, and molecular/macromolecular assemblies; · photometric, spectroscopic, microscopic, mechanical, and electrical methodologies/techniques in analytical cytology, cytometry and innovative instrument design For articles that focus on computational aspects, authors should be clear about which docking and molecular dynamics algorithms or software packages are being used as well as details on the system parameterization, simulations conditions etc. In addition, docking calculations (virtual screening, QSAR, etc.) should be validated either by experimental studies or one or more reliable theoretical cross-validation methods.

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