COE Inhibits Migration And Invasion In Colorectal Cancer By CHIP-mediated Ubiquitination Of Galectin-1

Colorectal cancer(CRC)is one of the digestive malignant tumors.The incidence of CRC varies around the world,with North America,Northern Europe and other countries ranking first.However,the incidence rate is extremely low in Asia,Africa.With the continuous development of social economy and changes in people’s lifestyles,the incidence of CRC in our country is increasing,and there is a trend of younger age at onset.At present,the treatment of CRC is still based on surgery,chemotherapy,radiotherapy and molecular targeted agents.Although great progress has been made in various clinical treatments,the mortality of CRC is still gradually increasing.The reason for this was mainly due to local recurrence or distant metastasis.Therefore,we need to find molecular markers that can predict the therapeutic effect and prognosis of CRC,so as to develop more effective treatment programs to reduce the mortality of CRC.CHIP is an E3 ubiquitin ligase which degrades many oncogenic and tumor suppressor proteins by ubiquitination.Gal-1 is a cellular autocrine protein which exert an enormous function on the development of tumors.GAL-1 has been shown to be specifically expressed in many tumor tissues and regulates a variety of biological activities of cancer cells,such as breast cancer and gastric cancer.However,its role in CRC is rarely studied.Chinese herbal medicine is the treasure of traditional medicine in China.In recent years,more and more researchers are committed to looking for anticancer drugs from Chinese herbal medicine.Celastrus orbiculatus Thunb belongs to Celastraceae,which has the effects of dispelling wind and activating blood,detumescent and analgesic.Our previous research results show that Celastrus orbiculatus extracts(COE)can inhibit the proliferation,invasion and metastasis of tumor cells.Further drug sensitivity test showed that digestive system tumors were most sensitive to COE.The extraction technology and application of Celastrus orbiculatus extract have been authorized by the national invention patent(patent number:ZL200710025343.3).The purpose of this study was to investigate the effects of COE on the proliferation,migration and invasion of HCT 116 and SW 480 cells,and further explore its specific mechanism.Through the experimental study,we preliminarily expounded the role and mechanism of CHIP and Gal-1 in CRC,and explored the mechanism of COE regulating the migration and invasion of CRC.The results showed that COE could inhibit the migration and invasion of colorectal cancer through CHIP-mediated ubiquitination of Galectin-1.This study was divided into four parts.Part Ⅰ Human colon cancer HCT 116 and SW 480 stably expressed cell lines with high expression of CHIP,low expression of CHIP,high expression of GAL-1,low expression of GAL-1,and their control groups were constructedObjective:To construct HCT 116 and SW480 cells with CHIP and Gal-1 high expression and low expression.Methods:Human colon cancer HCT 116 and SW 480 cells were cultured in RPMI-1640 culture medium containing 10%fetal bovine serum.High expression of CHIP(LV-CHIP),high expression of CHIP(LV-CHIP-Ctrl),low expression of CHIP(LV-CHIP-shRNA),lentivirus and high expression of Gal-1(LV-CHIP-shRNA),high expression of Gal-1(LV-Gal-1-ctrl),low expression of Gal-1(LV-Gal-1-shRNA),and low expression of Gal-1(LV-Gal-1-shRNA-CTRL)lentivirus infects human colon cancer HCT 116,SW 480 cells.Lentivirus-infected cells were screened by RPMI-1640 medium containing 2ug/ml puromycin and 10%fetal bovine serum until HCT 116/LV-CHIP,HCT 116/LV-CHIP-ctrl,HCT 116/LV-CHIP-shRNA and HCT 116/LV-CHEP-shRNA were stably expressed.HCT 116/LV-Gal-1,HCT 116/LV-Gal-1-ctrl,HCT 116/LV-Gal-1-shRNA,HCT 116/LV-Gal-1-shRNA,HCT 116/LV-Gal-1-shRNA,HCT 116/LV-CHIP,SW480/LV-CHIP,SW480/LV-CHIP-shRNA,SW480/LV-CHIP-shRNA-ctrl,And SW 480/LV-Gal-1,SW 480/LV-Gal-1-ctrl,SW 480/LV-Gal-1-shRNA,SW 480/LV-Gal-1-shRNA-ctrl.Then Western blot was used to detect the relative expression of CHIP and Gal-1 protein in cells.Results:Stable expression of HCT 116/LV-CHIP、HCT 116/LV-CHIP-shRNA、SW480/LV-CHIP、SW480/LV-CHIP-shRNA、HCT 116/LV-Gal-1、HCT 116/LV-Gal-1-shRNA、SW480/LV-Gal-1、SW480/LV-Gal-1-shRNA colorectal cancer cell lines were successfully constructed.Part Ⅱ Effects of CHIP and Gal-1 on proliferation,migration and invasion of human colorectal cancer HCT-116 and SW-480 cell lines and their role in predicting prognosis of colorectal cancer patientsObjective:To verify the effect of CHIP and Gal-1 on the proliferation,migration and invasion of CRC,and whether they can effectively predict the prognosis of colon cancer patientsMethods:Expression levels of CHIP and GAL-1 in 8 pairs of fresh human colorectal cancer tissues and their adjacent tissues were determined by RT-PCR,Western blot and immunohistochemistry,respectively.The expression levels of CHIP and GAL-1 in 470 human colon cancer specimens were detected by TMA,and the correlation between their expression levels and clinicopathological characteristics was statistically analyzed.Cox univariate and multivariate analyses of the correlation between CHIP and GAL-1 and prognosis of human colon cancer patients.The role of CHIP and Gal-1 in predicting patient prognosis was analyzed in combination with the expression of CHIP and Gal-1 and clinical risk score.Then CCK-8 and EDU experiments were used to detect the change of proliferation ability of cells infected with lentivirus,and Transwell experiments were used to detect the change of invasion and metastasis ability of cells.To further explore the interaction between CHIP and Gal-1,HCT 116/LV-CHIP and HCT 116/LV-CHIP-shRNA cells were re-infected with lentivirus with high expression of Gal-1(LV-Gal-1)and low expression of Gal-1(LV-Gal-1-shRNA),respectively.Changes in cell proliferation ability were detected by CCK8 and EDU methods,and changes in cell invasion and metastasis ability were detected by Transwell method.HCT 116/LV-CHIP,HCT 116/LV-CHIP-shRNA and their control cells were selected.After 0.9%normal saline was suspended to 1×107/ml,200uL of each was inoculated into the right subcutaneous and abdominal cavity of nude mice,and animal models of subcutaneous xenograft and abdominal metastatic tumor were constructed.Nude mice were randomly divided into 8 groups with 6 mice in each group.4 groups were subcutaneous transplantation tumor group,and 4 groups were abdominal metastasis tumor group.The groups were HCT 116/LV-CHIP group,HCT 116/LV-CHIP-ctrl group,HCT 116/LV-CHIP-shRNA group,and HCT 116/LV-CHIP-shRNA-ctrl group.The length and diameter of tumors in nude mice were measured every three days and the tumor volume was calculated.The body weight of nude mice was measured every two days.After 28 days,all nude mice were sacrificed by cervical dislocation.The subcutaneous transplantation tumor and tumor lumps in the abdominal mesentery of nude mice were isolated,weighed,and photographed for recording.Results:The expression of CHIP in CRC tissues was lower than that in the corresponding adjacent normal tissues,while the expression of Gal-1 was higher than that in the corresponding adjacent normal tissues.Kaplan-Meier survival curve analysis showed that high expression of CHIP and low expression of GAL-1 could significantly prolong the 5-year survival of patients.COX univariate and multivariate analyses indicated that CHIP and GAL-1 were independent markers in predicting the prognosis of colon cancer.CHIP is an independent positive prognostic factor for CRC patients,Gal-1 is an independent negative prognostic factor for CRC patients,and high CHIP and low Gal-1 expression are the most effective independent prognostic factors in each group.RT-PCR results showed that compared with HCT 116/LV-CHIP-ctrl,the expression level of CHIP mRNA in HCT 116/LV-CHIP cells was significantly increased(P<0.05).Compared with HCT 116/LV-CHIP-shRNA-ctrl,the expression level of CHIP mRNA in HCT 116/LV-CHIP-shRNA cells was significantly reduced(P<0.05),and CHIP could not regulate GAL-1 at the protein level.CCK-8 and EDU results showed that CHIP could inhibit the proliferation of intestinal cancer cells,while GAL-1 could promote the proliferation of intestinal cancer cells.Transwell showed that CHIP could inhibit the invasion and metastasis of colorectal cancer cells,while GAL-1 could promote the invasion and metastasis of colorectal cancer cells.Meanwhile,LV-GAL-1 can improve the proliferation,invasion and metastasis of HCT 116/LV-CHIP cells,while LV-Gal-1-shRNA can reduce the proliferation,invasion and metastasis of HCT 116/LV-CHIP-shRNA cells.Part Ⅲ The molecular mechanism of CHIP regulating Gal-1Objective:To investigate how CHIP regulates GAL-1 to inhibit the proliferation,invasion and metastasis of colorectal cancer cellsMethods:HCT 116/LV-CHIP were treated with MG132,and the changes of Gal-1 protein level were detected by Western blot.Gal-1 related proteins were obtained by IP assay for ubiquitination.LV-chip,LV-U-box and LV-TPR lentivirus infected HCT116 cells,and the ubiquitination functional domain required by CHIP degradation of Gal-1 was determined by IP and WB methods.Results:CHIP significantly promoted the ubiquitination of Gal-1 protein.As an E3 ligase,CHIP mediated the ubiquitination of Gal-1 through its U-box domain.Part Ⅳ Effects of COE on proliferation and migration of human colon cancer HCT-116 cells and its mechanismObjective:To investigate the effect of COE inhibition on proliferation and migration of colorectal cancer by chip-mediated gal-1 ubiquitination.Methods:Human colorectal cancer HCT 116、HCT 116/LV-CHIP-shRNA and HCT 116/LV-CHIP-shRNA-ctrl cells were treated with 1/2 48h IC50 concentration of COE.Western blot assay was used to detect the protein expression levels of CHIP and Gal-1 in each group after COE treatment.MTT was used to detect the changes of cell proliferation in each group after COE treatment.High content cell migration assay was used to detect the effect of COE on the metastasis ability of colorectal cancer cells.Results:Western blot results showed that after COE treatment,the expression level of CHIP in HCT 116 cells was significantly increased,and the expression level of Gal-1 was decreased,while the expression of CHIP in HCT 116/LV-CHIP-shRNA cells was recovered,and the expression of Gal-1 was decreased.After 12,24,36 and 48h,compared with HCT 116(negative control group),the proliferation ability of cells treated with COE was significantly reduced.Compared with HCT 116/LV-CHIP-shRNA(negative control group),the proliferation ability of cells treated with COE was also reduced,but still stronger than that in the HCT 116 cells.High-content cell migration assay results showed that compared with the corresponding control group,after 48 hours of COE treatment,the migration ability of HCT 116 cells was significantly decreased,and the cell migration ability of HCT 116/LV-CHIP-shRNA cells was also reduced,but still stronger than that in the HCT 116 cells.