| PURPOSE: Since the 20 th century,the incidence of cancer has been increasing year by year,and tumor has become the number one disease that seriously threatens human health.As one of the most common gastrointestinal tumors,colorectal cancer is a malignant tumor with high incidence,high mortality and high invasiveness.At present,the common clinical treatments for colorectal cancer mainly include surgical resection,radiotherapy,chemotherapy,immunotherapy,molecular targeted therapy and so on.However,because the initial symptoms of colorectal cancers are not obvious,early examination is not common and difficult to detect,most of the colorectal cancer patients in China have progressed to the middle and late stages when they are first diagnosed,and the 5-year survival rate of many patients is less than 20%.In addition,local recurrence and metastasis of tumor are also very common,which all add many difficulties to the treatment of colorectal cancer,resulting in high mortality and poor prognosis of colorectal cancer.Therefore,it is important to find new effective treatment options and prognosis determination for colorectal cancer.Ferroptosis is a newly discovered form of regulated cell death,mainly caused by the accumulation of iron-dependent lipid peroxides above a certain level,which is significantly different from the traditional cell death modalities such as necrosis,apoptosis,autophagy and scorch death.An increasing number of studies have confirmed that ferroptosis also has a regulatory role in colorectal cancer,which is closely related to tumor growth and progression,and that induction of ferroptosis in tumor cells is likely to be a novel and effective targeted therapeutic strategy,but its specific mechanism is still unclear.Therefore,this study aims to find ferroptosis-related proteins in colorectal cancer and explore their regulatory roles and mechanisms in colorectal cancer cells,so as to provide new ideas and directions for clinical targeted treatment of other neoplastic diseases such as colorectal cancer and improvement of patients’ prognosis.METHODS: Sequencing data of colorectal cancer tissues were first downloaded from the Tumor Genome Atlas TCGA database,and the expression of WIPI2 in colorectal cancer and normal tissues was analyzed by limma package,and the relationship between clinical traits and WIPI2 expression and prognosis was assessed by univariate and multifactorial cox analysis.The accuracy of the risk model constructed with WIPI2 was also tested by ROC curves as well as survival analysis,and the relationship between WIPI2 expression and clinicopathological features was examined using cox analysis.At the human tissue level,immunohistochemical experiments were performed using colorectal cancer tissues and corresponding paracancerous normal tissues collected from Jiangda Diagnostic Pathology Center Center of CRC patients to verify the expression of WIPI2,ACSL4,and GPX4 in colorectal cancer and normal paraneoplastic tissues.In vitro,transient transfection of si-RNA was used to knock down the expression level of WIPI2 in CRC cells,and transfection of WIPI2-Flag overexpression plasmid was used to elevate its expression level.The effects of WIPI2 on CRC cell growth and proliferation were verified by CCK-8 and cell clone formation assays.We further examined the changes in the expression levels of WIPI2 and ferroptosis-related proteins in colorectal cancer cells treated with knockdown and overexpression of WIPI2 and ferroptosis inducer Erastin to investigate whether WIPI2 can affect the growth activity of colorectal cancer cells through the ferroptosis pathway.RESULTS: Public data from the TCGA platform and immunohistochemical results from this study showed that the expression of WIPI2,ACSL4 and GPX4 were significantly higher in colorectal cancer tissues compared with paracancerous normal tissues,and the high expression of WIPI2 predicted poor prognosis of CRC patients.Meanwhile,the results of both bioinformatics database analysis and immunohistochemical semi-quantitative analysis suggested that the expression level of WIPI2 correlated with several clinicopathological features and could jointly predict the prognosis of colorectal cancer patients with other factors.In vitro experiments,we demonstrated that WIPI2 could promote the growth and proliferation of HCT116 and HT29 cells by both knocking down and overexpressing WIPI2.Moreover,WIPI2 was able to induce an increase expression level of ferroptosis protein ACSL4 and a decrease in the expression of GPX4,suggesting that WIPI2 can potentially positively regulate CRC cells ferroptosis.Meanwhile,both NC and si groups were able to further inhibit cell growth activity,as well as increase WIPI2 and decrease GPX4 expression when treated with Erastin,but the rate of cell viability inhibition and the trend of protein changes were more significantly in the NC group than si group,which suggested that WIPI2 could enhance the sensitivity of colorectal cancer cells to Erastin.In summary,our study suggests that WIPI2 promotes colorectal cancer cell growth and also plays an important role in the development of CRC through the ferroptosis pathway.CONCLUSIONS: WIPI2 is differentially highly expressed in colorectal cancer tissues and correlates with poor patient prognosis and several clinicopathological features(e.g.,degree of differentiation and age,etc.).In vitro,WIPI2 can promote the growth and proliferation ability of colorectal cancer cells,and it can also induce changes in the expression of ferroptosis protein and enhance the sensitivity of colorectal cancer cells to ferroptosis inducers.Therefore,WIPI2 is expected to be a new prognostic biomarker for colorectal cancer and a potential target for molecular therapy. |
