Abstract:

Objective To discuss the expression and clinical significance of inositol polyphosphate-4-phosphatase type Ⅱ （INPP4B） gene in hepatocellular carcinoma （HCC） based on The Cancer Genome Atlas （TCGA） database and experimental verification with clinical samples. Methods Based on data from 424 clinical samples in the TCGA database （including 374 HCC tissues and 50 paracarcinoma tissues）， Kaplan-Meier method and Cox regression analysis were used to evaluate the relationship between INPP4B gene and the clinical characteristics and survival prognosis of the HCC patients. The correlations between INPP4B gene and the number of 24 types of immune cells， matrix， immune cell infiltration and tumor purity in tumor tissue， and the expression level of the high-frequency mutant gene tumor protein 53（TP53） in HCC were analyzed. The clinicopathological data and paraffin-embedded tissue sections of 60 HCC patients treated with surgical resection from December 2022 to December 2023 were collected. According to clinical diagnosis， they were divided into poorly differentiated group （HCC-L group）， moderately differentiated group （HCC-M group） and well-differentiated group （HCC-H group）， with 20 cases in each group； 20 patients during the same period who underwent biopsy and were pathologically diagnosed as non-tumor were selected as normal group， and their clinicopathologic data and liver tissue paraffin sections were collected. HE staining was used to observe the pathomorphology of HCC tissue and normal liver tissue of the subjects in various groups； immunohistochemistry method was used to detect the expressions of Ki-67 and INPP4B proteins in the HCC tissue and normal liver tissue of the subjects in various groups. Results The TCGA database analysis results showed that compared with normal tissue， the expression level of INPP4B mRNA in HCC tissue was significantly increased （P<0.01）. Compared with INPP4B low expression group， the overall survival（OS） of the patients in INPP4B high expression group was significantly prolonged （P<0.05）. The univariate Cox regression analysis results showed that tumor stage， pathological stage， tumor status and residual tumor had impacts on OS of the HCC patients （P<0.05）. The univariate regression analysis results showed that the INPP4B prognostic risk model score ratio was HR=0.781， 95% confidence interval（CI）： 0.552-1.105， P=0.168. The AUC value for the impact of INPP4B on OS of the HCC patients was 0.558， indicating that the INPP4B gene prognostic risk model had certain predictive value in survival prognosis. The INPP4B mRNA expression level was not correlated with TNM stage， stage， patient gender， age， race or body mass index （BMI） （P>0.05）. In tumor tissue with high and low INPP4B expression， 22 types of immune cells showed statistically significant differences （P<0.05）； the INPP4B mRNA expression level was positively correlated with the number of 23 types of immune cells except T helper （Th） 17 cells （r>0）， among which all Th cells except natural killer （NK） CD56⁺ cells were statistically significant （P<0.01）； INPP4B was significantly correlated with matrix （r=0.475）， immune cell infiltration （r=0.641） and tumor purity （r=0.599） in tumor tissue （P<0.01）. INPP4B was correlated with TP53 （r=0.287， P<0.01）. The HE staining results showed that clear and complete lobular structure， neatly arranged cells and slight inflammatory cell infiltration were observed in liver tissue of the subjects in normal group； completely destroyed lobular structure， significant hepatocellular steatosis， massive inflammatory cell infiltration， and lesions such as ballooning degeneration and small cell hyperplasia in some cells were observed in HCC tissue of the patients in HCC-L， HCC-M and HCC-H groups， and the lower the HCC differentiation degree， the more severe the tissue destruction； The immunohistochemistry results showed that compared with normal group， the expression levels of Ki-67 protein in HCC tissue of the patients in HCC-L， HCC-M and HCC-H groups were significantly increased （P<0.01）， and the lower the differentiation degree of the HCC patients， the higher the Ki-67 positive rate. Brownish-yellow granules evenly distributed in the cells and INPP4B protein was highly expressed in liver tissue of the subjects in normal group； compared with normal group， the expression levels of INPP4B protein in HCC tissue of the patients in HCC-L， HCC-M and HCC-H groups were significantly decreased （P<0.01）， and the lower the differentiation degree of the HCC tissue， the lower the INPP4B positive rate. Conclusion INPP4B is a protective factor for the prognosis of HCC patients； as a new tumor suppressor gene， INPP4B may become a potential target for new drug screening in HCC treatment.

Key words: Hepatocellular carcinoma, Inositol polyphosphate-4-phosphatase type Ⅱ gene, The Cancer Genome Atlas database, Phosphatidylinositol signaling system, Immune cells