INPP4B促进结直肠癌转移及其作用机制的初步探索

赖萌; 毛志刚; 唐邓; 兰思琪; 晏蕊婷; 向琦; 赵暹暹; 苏宓; 王玉芳

doi:10.12182/20240960205

INPP4B促进结直肠癌转移及其作用机制的初步探索

Preliminary Study of the Role of INPP4B in Promoting Colorectal Cancer Metastasis and the Mechanisms Involved

摘要

摘要:
目的明确肌醇多磷酸4-磷酸酶Ⅱ型（inositol polyphosphate-4-phosphatase type Ⅱ B, INPP4B）在结直肠癌（colorectal cancer, CRC）中的表达及临床意义，明确CRC细胞中INPP4B与基质金属蛋白酶7（matrix metallopeptidase 7, MMP7）的关系，并初步探究INPP4B对CRC细胞的增殖、迁移的影响及机制。
方法使用TIMER2.0和GEPIA2数据库分析INPP4B在癌（瘤）和癌旁（瘤旁）组织中的表达差异和对CRC预后的影响；通过免疫组化检测临床手术切除的102例CRC肿瘤中INPP4B的表达，并分析INPP4B与临床病理指标的相关性；在过表达/敲减INPP4B的CRC细胞中，实时荧光定量PCR检测INPP4B和MMP7的基因表达，Western blot检测INPP4B蛋白表达，利用CellTiter 96® AQueous One检测细胞增殖，划痕实验、实时无标记动态细胞分析技术（RTCA）检测细胞迁移和侵袭；结合LinkedOmics数据库分析与INPP4B功能相关信号通路，并在细胞水平验证潜在的关键分子。
结果数据库分析结果显示，与正常组织相比（结肠癌：1.91，直肠癌：1.89），在CRC中INPP4B表达升高（结肠癌：2.30，直肠癌：2.33）。免疫组化检测临床肿瘤组织和肿瘤旁组织也证实INPP4B在CRC中表达增高（P<0.001）。Cox回归模型分析显示INPP4B〔风险比（HR）=1.457，95%置信区间（CI）：1.003～2.115〕影响CRC预后，Kaplan–Meier曲线显示INPP4B高表达患者生存期短（P<0.05）；χ²检验分析INPP4B表达与临床病理指标，发现INPP4B的高表达与淋巴结转移（χ²=3.997，P=0.046）和神经浸润（χ²=8.511，P=0.004）相关。体外实验中，与对照组细胞相比，过表达INPP4B的CRC细胞增殖和迁移能力增加（P<0.05）。通过LinkedOmics数据库分析显示INPP4B与细胞外基质重塑和细胞转移相关；Pearson相关性分析显示MMP7与INPP4B正相关（r=0.3782，P<0.001）；体外过表达或敲减INPP4B后MMP7表达水平也随之升高和下降。
结论 INPP4B在结直肠肿瘤组织中呈高表达并与淋巴结转移、神经浸润、患者预后相关。MMP7可能介导了INPP4B促进CRC细胞迁移和侵袭的作用。

Abstract:
Objective To investigate the expression of inositol polyphosphate 4-phosphatase type Ⅱ B (INPP4B) in colorectal cancer (CRC) and the relevant clinical significance, to determine the relationship between INPP4B and matrix metallopeptidase 7 (MMP7) in CRC cells, and to make preliminary exploration of the effects of INPP4B on the proliferation and migration of CRC cells and mechanisms involved.
Methods The TIMER2.0 and GEPIA2 databases were used to analyze the differences in INPP4B expression between cancer and para-cancerous tissues and the effects of such differences on the prognosis of CRC. The expression of INPP4B in 102 surgically resected CRC tumors was determined by immunohistochemistry (IHC), and the correlation between INPP4B and clinical pathological indicators was analyzed. In CRC cells with overexpressed/knocked-down INPP4B, the expression of INPP4B and MMP7 were examined by real time fluorogenic quantitative PCR, the protein expression of INPP4B was assessed by Western blot, cell proliferation was determined using the CellTiter 96® AQueous One assay, and cell migration and invasion were assessed using wound healing assay and real-time label-free dynamic cell analysis (RTCA). The LinkedOmics database was used to analyze signaling pathways related to INPP4B function, and the role of potential key molecules was validated at the cellular level.
Results Analysis with the TIMER2.0 database and GEPIA2 database showed elevated INPP4B expression (colon adenocarcinoma COAD: 2.30, rectal adenocarcinoma READ: 2.33) in CRC compared to normal tissue (COAD: 1.91, READ: 1.89). IHC testing confirmed that INPP4B was upregulated in clinical CRC tissues and paracancerous tissues (P<0.001). Cox regression model analysis showed that INPP4B (hazards ratio HR=1.457, 95% confidence interval CI: 1.003-2.115) affected the prognosis of CRC, and the Kaplan-Meier curve showed that patients with high INPP4B expression had shorter overall survival (P<0.05). χ² test was performed to analyze the relationship between INPP4B expression and clinicopathological indexes, and it was found that high expression of INPP4B was correlated with lymph node metastasis (χ²=3.997, P=0.046) and neural invasion(χ²=8.511, P=0.004). In in vitro experiments, CRC cells overexpressing INPP4B showed a significantly increased cell proliferation and migration compared to the cells in the control group (P<0.05). Analysis using the LinkedOmics database showed that INPP4B was correlated with extracellular matrix remodeling and cell migration. Pearson's correlation analysis showed that MMP7 was positively correlated with INPP4B (r=0.3782, P<0.001). INPP4B overexpression or knockdown in vitro also led to the upregulation or the downregulation of MMP7 expression in CRC cells.
Conclusion INPP4B is highly expressed in CRC tissues and significantly correlated with lymph node metastasis, neural invasion, and patient prognosis. MMP7 may mediate the role of INPP4B in promoting CRC cell migration and invasion.

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