Expression Of ALAS1 In Colorectal Cancer And Its Effect On Cell Behavior

Colorectal cancer(CRC)is the third most common malignant disease in the world and the fourth most common cause of cancer death.Although CRC has made research progress in diagnosis and treatment,because the early symptoms of the disease are not obvious.The incidence of primary diagnostic metastases is high,and the prognosis of patients is often poor.As a result,CRC is a serious threat to human health and has become the focus of global attention.With the rapid development of China’s economy in recent years,the quality of life of residents has significantly improved,but due to the faster pace of life and worse living habits,the incidence and mortality of CRC cancer in China have increased year by year.Therefore,It is of great clinical significance to further study the mechanism of colorectal cancer.5-aminolevulinic acid synthase(ALAS)is encoded by two genes,ALAS1 and ALAS2.ALAS1 belongs to the housekeeping gene and is a rate-limiting enzyme for heme biosynthesis,widely expressed throughout the body and involved in many cellular functions.Recent studies have found that the synergistic effect of histone deacetylase inhibitors(HDACi)with artemisinin and its derivatives(ARTs)increases the expression of ALAS1 the resulting heme leads to enhanced cytotoxicity in ARTs,which induces cell death.Zhang li et al.found that the expression level of ALAS1 protein was significantly up-regulated in non-small cell lung cancer(NSCLC),and inhibition of ALAS1 activity reduced the ability of NSCLC cells to proliferate,colony form and migrate.However,relevant research about ALAS1 in the occurrence and development of CRC is rare in the present.This experimental study mainly carried out the following three aspects: First,to detect the expression of ALAS1 in colorectal cancer and adjacent normal tissues,to analyze the relationship between ALAS1 protein expression and clinicopathological parameters of patients,and the relationship between abnormal expression of ALAS1 and prognosis of patients.Second,The activity of ALAS1 in HCT116 cells was inhibited by si RNA or succinylacetone(SA),and the effects of ALAS1 on cell proliferation,migration and invasion were detected by cck-8,flow cytometry,plate cloning,scratch assay,Transwell chamber migration and matrix gel assay.Third,the effect of ALAS1 on apoptosis was detected by flow Annexin-FITC/PI double staining after si RNA or SA inhibiting the activity in HCT116 cells.This study provides a new idea to explore the relevant role of ALAS1 in the development of colorectal cancer,and also provides a theoretical basis for further clinical application of ALAS1.Part Ⅰ: Expression of ALAS1 in colorectal cancer tissues and its clinical significanceObjective: The aim of this part is to analyze the relationship between ALAS1 protein expression and clinicopathological parameters and the relationship between ALAS1 abnormal expression and prognosis of CRC patients by comparing the expression of ALAS1 in tumor tissues and adjacent paired normal tissues.Methods: 67 patients with CRC were selected.All the selected cases were confirmed as adenocarcinoma by pathology.After the specimen was removed from the body,the tumor tissue and the normal tissue near the heart of the tumor were rapidly removed(the tumor margin was greater than 10 cm).The expression of ALAS1 in tumor tissue and adjacent paired normal tissue was detected by q RT-PCR,Western blot and Immunofluorescence.The expression of ALAS1 in human colonic mucosal epithelial cell lines FHC cells and human colorectal cancer cell lines HCT116 was detected by Western blot,immunofluorescence assay.Results:1.The expression level of ALAS1 in colorectal cancer tissues was significantly increasedThe expression of ALAS1 in colorectal cancer tissues was significantly higher than that in adjacent normal tissues by q RT-PCR,Western blot,immunostaining experiments(P<0.05).There was no correlation between the expression level of ALAS1 protein in colorectal cancer tissues and patients’ age,gender and tumor differentiation(P>0.05),which was closely related to the invasion depth,N stage and tumor size(P<0.05).Kaplan-Meier survival curves were used to analyze the relationship between ALAS1 expression levels and postoperative survival in colorectal cancer patients.We found that the overall survival rate of patients with ALAS1 high expression was significantly lower than that of patients with ALAS1 low expression(P =0.04).2.ALAS1 expression in colorectal cancer cell line HCT116 was significantly increasedThe expression of ALAS1 in FHC cells and HCT116 cells was detected by Western blot and immunofluorescence experiments.The results showed that the expression of ALAS1 protein in the HCT116 cells was significantly higher than that in the FHC cells,and the difference was statistically significant(P<0.05).Conclusion: ALAS1 expression in colorectal cancer tissues is higher than that in adjacent tissues,and its expression is positively correlated with invasion depth,N stage and tumor size,and negatively correlated with a prognosis.The expression level of ALAS1 protein can be used as an independent factor to evaluate the prognosis of colon cancer patients.Part Ⅱ: Effects of inhibition of ALAS1 activity on proliferation,migration and invasion of HCT116 cellsObjective: By observing the effect of inhibiting ALAS1 activity on the proliferation,migration and invasion of HCT116 cells,the mechanism of action was preliminarily explored.Methods: HCT116 cells were used to inhibit ALAS1 activity by si RNA transfection or SA,and the effects of ALAS1 on cell proliferation,migration and invasion were detected by flow cytometry,plate cloning,scratch test,Transwell chamber migration and matrix gel invasion experiment.The changes of cell proliferation-related genes C-myc,PCNA and transfer-related genes MMP-2 and MMP-9 were detected by Western blot.Results:1.Validation of ALAS1-si RNA transfection of HCT116 cellsThe transfection effect of HCT116 cells transfected with si RNA was verified by Western blot,and the silencing efficiency of cells in group alas1-sirna #2 was the highest.2.The effect of inhibition of ALAS1 activity on proliferation activity of HCT116 cellsThe effect of ALAS1 on HCT116 proliferation activity was detected by CCK-8 experiments,After knockdown ALAS1 expression,cell proliferation activity was significantly inhibited.The results of plate cloning showed that the number of cell colony formations in the experimental group was significantly decreased.To further detect the effect of ALAS1 on the proliferation status of HCT116,the cell cycle was detected by flow cytometry.The results showed that cell cycle was inhibited in G0/G1 phase after inhibition of ALAS1 expression,and the distribution of the S stage was relatively reduced.The experimental results were consistent with knockdown ALAS1 expression after treated with SA.These results suggest that ALAS1 plays an important role in colorectal cancer cell proliferation.3.The effect of inhibition ALAS1 activity on migration and invasion activity of HCT116 cellsThe effects of ALAS1 on HCT116 cell invasion and migration were investigated by Transwell chamber migration and matrix gel invasion experiments.Compared with the control group,the migration and invasion ability of the experimental group was decreased(P<0.01).The cell scratch test showed that the cell growth in the experimental group was slow and the cell migration area was obviously reduced.The migration and invasion ability of cells in the experimental group was also significantly inhibited after the cells were treated with SA.(P<0.01).These results suggest that ALAS1 plays an important role in the migration and invasion of colorectal cancer cells.4.The effect of inhibition ALAS1 activity on proliferation and metastasis related genes in HCT116 cellsThe western blot was used to detect the effects of inhibition of ALAS1 activity on proliferation related genes C-myc,PCNA and transfer-related genes MMP-2 and MMP-9 in HCT116 cells.Compared with the control group,after inhibiting the activity of ALAS1,the protein expression levels of C-myc and PCNA were significantly down-regulated,and the protein expression levels of MMP-2 and MMP-9 were also down-regulated.Conclusion:1.The ALAS1-si RNA could be transfected into HCT116 cells by RNA interference technique to inhibit ALAS1 expression,and the transfection effect of ALAS1-si RNA#2 group of cells is better.2.After inhibiting the activity of ALAS1,the proliferation capacity of CRC cells was significantly inhibited,possibly by down-regulating the expression level of PCNA c-myc,thus playing an important role in the proliferation of colorectal cancer cells.3.After inhibiting the activity of ALAS1,the ability of colorectal cancer cell migration and invasion are significantly inhibited.It may be through down-regulating the expression levels of MMP-2 and MMP-9,thus playing an important role in the migration and invasion of colorectal cancer cells.Part Ⅲ: Effects of inhibition of ALAS1 activity on apoptosis of HCT116 cellsObjective:By observing the effect of inhibiting the activity of ALAS1 on the apoptosis of HCT116 cells,the mechanism of action was preliminarily discussed.Methods: HCT116 cells were used as research objects,the activity of ALAS1 was inhibited by si RNA transfection or SA,the effect of ALAS1 on apoptosis was detected by flow annexin-fitc /PI double staining,and the effect of ALAS1 on apoptosis-related genes such as P53,Bax,bcl-2 and Caspase3 was detected by Western blot.Results:1.Inhibition of ALAS1 activity had a significant effect on HCT116 apoptosisFlow annexin-fitc /PI double staining method was used to detect the effect of ALAS1 on the apoptotic ability of HCT116.The results showed that the apoptotic rate of the experimental group was significantly increased,and the difference was statistically significant compared with the control group.These results suggest that ALAS1 plays an important role in inhibiting the apoptosis of colorectal cancer cells.2.Regulation of apoptosis-related genes in colorectal cancer cells by inhibiting the activity of ALAS1The effect of inhibiting the activity of ALAS1 enzyme on apoptosis-related genes such as P53,Bax bcl-2 and Caspase3 in HCT116 cells was detected by Western blot.Compared with the control group,protein expression levels of apoptosis-related genes such as P53,Bax and Caspase3 protein were significantly increased after inhibition of ALAS1 enzyme activity,while the expression levels of bcl-2 protein were down-regulated,with statistically significant differences(P<0.05).These results suggest that the effect of inhibiting ALAS1 activity on the apoptosis of colorectal cancer cells may be related to the regulation of the above genes.Conclusion:The inhibition of ALAS1 activity attenuated the inhibition of apoptosis in HCT116 cells,possibly by down-regulating the expression level of Bcl-2 in cells and simultaneously up-regulating the expression level of P53,Bax,Caspase-3,thereby playing a role in the apoptosis process of colorectal cancer cells.