The Expression Of ACP5 In Colorectal Cancer And Its Regulatory Mechanism Of Malignant Biological Behavior

OBJECTIVES: Colorectal cancer is one of the most common malignant tumors,and the morbidity and mortality are increasing year by year.To analyze the molecular mechanism of the development of colorectal cancer,to find biomarkers suggesting prognosis,and to find new therapeutic targets is a hot spot in the field of colorectal cancer research.Recent studies have found that tartrate-resistant acid phosphatase 5plays a crucial role in the development of malignant tumors,but its effects on the development of colorectal cancer have not been reported.This study will detect the relationship between ACP5 expression and clinicopathological features and prognosis of colorectal cancer patients;explore the effect of ACP5 on proliferation and invasion of colorectal cancer cells at the cellular level and the molecular mechanism of ACP5 may be involved in the regulation of colorectal cancer cell development;The effect of ACP5 levels in tumor cells on the development of colorectal cancer cells was described in a nude mouse tumor model.METHODS:(1)The expression of ACP5 in the colorectal cancer database was analyzed using biological information technology.In clinical tissue samples,we used RT-q PCR and Western blot to detect the difference of ACP5 m RNA and protein expression levels in normal colorectal epithelial cells and colorectal cancer cells;then we collected 285 cases of CRC patients undergoing radical surgery from January2008 to December 2017.And 32 cases of CRC fresh tissue specimens from January20 to June 2018 was also collected to use RT-q PCR,Western blot and immunohisctochemistry indicated that ACP5 m RNA and protein were differentially expressed in colorectal cancer tissues and corresponding adjacent tissues.Chi-square test and Fisher test were used to analyze the correlation between ACP5 expression level and clinicopathological features of CRC patients.Kaplan-Meier method and log-rank test were used to compare the difference in overall survival rate and disease-free survival between ACP5 high expression group and low expression group.univariate and multivariate analyses was used to predict the difference of ACP5 expression level in clinical outcome of CRC patients.(2)We overexpressed or knocked down the expression level of ACP5 gene in colorectal cancer cell and verified the transfection by RT-q PCR and Western blot.The effect of ACP5 on the proliferation and invasion of colorectal cancer cells was detected by CCK8 assay and transwell assay;Western blot was used to detect the effect of ACP5 overexpression or knockdown on FAK/PI3K/AKT signaling pathway.We added si RNA-FAK in colorectal cancer cell lines with overexpressing ACP5 or pc DNA3.1-FAK in a colorectal cancer cell line with knock-down ACP5 or AKT inhibition in colorectal cancer cell lines with overexpressing ACP5 to demonstrate that ACP5 promotes proliferation and invasion of colorectal cancer cells through the FAK/PI3K/AKT signaling pathway.(3)In the animal model,sh RNA-ACP5 in HCT116 cell line was constructed,and the effect of knockdown of ACP5 on tumor volume and size was observed by subcutaneous transplantation tumor experiment.Western blot analysis was used to detect FAK/PI3K/AKT signaling pathway.RESULTS:(1)We first analyzed two independent microarray datasets from GEO databases(GSE9689,GSE21510).The results showed that ACP5 expression was significantly increased in tumor tissues compared with normal colorectal tissue.(2)The RT-q PCR results showed that ACP5 m RNA was increased in CRC tissues(n=32)compared with matched cancer-adjacent tissues(n=32).Furthermore,Western blotting data showed that ACP5 protein was obviously increased in CRC tissues(n=12)compared with matched cancer-adjacent tissues(n=12).Finally,we also found that the immunoreactivity of ACP5 was low or high expressed in 113(39.65%)or 172(60.35%)of the 285 colorectal cancer samples,respectively.ACP5 expression was prominently correlated with tumor size(P=0.031),tumor classification(P=0.027),lymphatic metastasis(P=0.001),distant metastasis(P=0.003)and TNM stage(P=0.005);whereas no significant relevance was found with age,gender,tumor location and CEA level.We measured the correlation of ACP5 expression with overall survival(OS)and disease-free survival(DFS)using Kaplan-Meier analysis and log-rank test,and high ACP5 expression was remarkably associated with decreased OS(P=0.001)and DFS(P=0.001),which indicates that the OS and DFS are better in CRC patients with low ACP5 expression than in those with high ACP5 expression.The multivariate analysis using the Cox proportional hazards model revealed that ACP5 expression was a significant independent prognostic factor and recurrent factor for CRC patients.(3)Using CCK-8 assays,we observed that the growth rate of pc DNA3.1-ACP5 treated cells was promoted compared with pc DNA3.1-vector cells,whereas cells with si RNAs-ACP5 had a prominently lower proliferative capacity than negative control in CRC cells.To determine whether ACP5 can affect CRC cell invasive capacity,the transwell assay results showed that pc DNA3.1-ACP5 significantly enhanced the invasive potential of HT-29 and SW480 cells.While the invasion of HCT116 and SW1116 cells with si RNAs-ACP5 were significantly decreased.We attempted to determine the effect of ACP5 on FAK signaling in CRC cells.As a result,we revealed that phospho-FAK,phospho-PI3 K and phospho-Akt are marked increased in cells by pc DNA3.1-ACP5,while total-FAK,total-PI3 K and total-Akt protein level were unaffected,compared with controls.Conversely,the phospho-FAK,phospho-PI3 K and phospho-Akt protein are marked decreased in cells by si RNA-ACP5.Then we silenced FAK to illustrate the proliferative and invasive potential of CRC cells.Indeed,silencing of FAK suppressed the proliferative and invasive ability of CRC cells and also compromised the role of pc DNA3.1-ACP5.And overexpression of FAK completely recovered the proliferative and invasive potential of si RNAs-ACP5 cells.Furthermore,the inhibition of activity by MK2206(Akt phosphorylation inhibitors)could partially decrease the positive effects of ACP5 on CRC cell proliferation and invasion.These results suggest that ACP5 promoted CRC progression through the activation of the FAK/PI3K/AKT signaling pathway.(4)Sh RNA-ACP5-HCT116 cells were implanted subcutaneously into the flanks of nude mice.The growth of tumor cells in mice injected with sh RNA-ACP5 cells slower than control animals at different time points.And the average tumor weight in sh RNA-ACP5 group was 0.4-fold smaller than in control animals.Furthermore,the expression of downstream targets of ACP5,and FAK/PI3K/AKT signaling pathway were markedly decreased in sh RNA-ACP5 group compared with that in control mice.COMCLUSION: 1)The expression level of ACP5 m RNA and protein in cancer tissues is significantly increased in clinical colorectal cancer patients.The overall survival rate and disease-free survival rate of patients with ACP5 low expression group were significantly higher than those of ACP5 high expression group.ACP5 can be used as one of the prognostic indicators of CRC patients.2)At the level of cellular function,ACP5 can promote the proliferation and invasion of colorectal cancer cells;and promote the development of colorectal cancer cells through FAK/PI3K/AKT signaling pathway.3)In the animal model of transplanted tumor,down-regulation of ACP5 gene expression can inhibit the growth of colorectal cancer xenografts in nude mice and inhibit FAK/PI3K/AKT signaling pathway.4)The present results elucidate a potential mechanism underlying the tumor-oncogenic role of ACP5 in colorectal cancer,and indicate that ACP5 could be a useful marker and potential therapeutic target in colorectal cancer.