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

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.