The Effect Of ARL2 On Proliferation,migration And Invasion Of Colorectal Cancer Cells And Related Mechanism

Part I The expression level of ARL2 in colorectal cancer and its correlation with clinicopathological factors and prognosisBackground and objective:As a member of the RAS superfamily,ADP ribosylation factor-like protein 2(ARL2)plays key roles in regulating the dynamics of microtubule and mitochondrial functions.Recent studies have shown that ARL2 plays an important role in the occurrence,progression and prognosis evaluation of many malignant tumors.ARL2 can affect the proliferation and invasion of tumor cells by regulating the receptor tyrosine kinase AXL.However,the role of ARL2 in colorectal cancer(CRC)has not been reported.In this study,the ALR2 mRNA and protein expression were first explored in the cancer tissue of CRC patients,so as to analyze the correlation of ARL2 expression and clinical pathological features,the prognosis of CRC patients.Methods:1.Real-time fluorescence quantitative PCR(qPCR)and Western blot were used to detect the expression of ARL2 mRNA and protein in fresh tissues of 20 patients with CRC after operation.2.Immunohistochemistry(IHC)method was used to detect the expression of ARL2 and AXL protein in paraffin section samples of 182 patients with CRC after surgery,to analyze the relationship between ARL2 and clinicopathological features.Patients’ survival data were followed up to analyze the effect of ARL2 on the survival of patients,and the correlation between ARL2 and AXL was analyzed.Results:1.The qPCR results showed that the adjacent tissues of CRC patients were used as controls,the relative expression level of ARL2 mRNA in CRC cancer tissues was 2.52±1.74,which was higher than that in paracancerous tissues,and the difference was statistically significant(P<0.05).Western blot analysis showed that the protein expression level of ARL2 in cancer tissues(0.59±0.20)of CRC patients was significantly higher than that in paracancerous tissues(0.26±0.12),and the difference was statistically significant(P<0.05).2.IHC detection indicated that the positive rate of ARL2 in cancer tissues of CRC patients was 79.67%(145/182),and its expression was mainly distributed in the cytoplasm.Immunohistochemical score of ARL2 in cancer tissues(3.87±3.20)was higher than that in paracancerous tissues(0.14±0.21),and the difference was statistically significant(P<0.001).In CRC,ARL2 positive rate is associated with TNM stage,patients with TNM stage Ⅰ+ⅡARL2 of cancerous tissue were 90.82%(89/98)higher than that of patients with TNM stageⅢ+Ⅳ ARL2 were 66.67%(56/84),the difference was statistically significant(P<0.001),but with the patient’s age,sex,tumor size,lymph node metastasis,distant metastasis and diameter has no associated with the degree of differentiation(P>0.05).3.Kaplan-Meier survival curve indicated that the 5-year survival rate of CRC patients with ARL2 positive was significantly higher than that of CRC patients with negative expression,and the difference was statistically significant(P<0.001).Univariate Cox regression model analysis showed that gender,lymph node metastasis,distant metastasis,TNM stage,and ARL2 expression were important prognostic factors for 5-year survival of CRC patients(P<0.05);Multivariate Cox regression model analysis showed that ARL2 expression and distant metastatic status were independent prognostic factors affecting 5-year survival of CRC patients(P<0.05).4.In order to further elucidate the role of ARL2 positive expression in 5-year survival in CRC patients,we performed a stratified analysis,the results showed ARL2 positive expression in men and women,colon and rectum,TNM stage Ⅰ+Ⅱ and TNM stage Ⅲ+Ⅳ,with lymph node metastasis and without lymph node metastasis,age 65 or higher,without distant metastasis,the 5-year survival rate in patients of CRC were higher than the negative expression of CRC patients(P<0.05),and there was no difference in 5-year survival among CRC patients younger than 65 years or with distant metastases,whether positive or negative for ARL2 expression(P>0.05).5.IHC results showed that the positive rate of AXL in cancer tissues of CRC patients was 42.86%(78/182),and its expression was mainly distributed in the cytoplasm.Semiquantitative analysis of IHC staining showed that the AXL immunohistochemical score in cancer tissues(1.25±1.83)was higher than that in paracancerous tissues(0.02±0.13),showing a significant difference(P<0.001).In CRC TNM stage Ⅲ+Ⅳ phase AXL positive expression rate of as follows:47.62%(40/84),significantly higher than TNM stage I+II phase AXL positive expression rate of 38.78%(38/98),with significant difference(P<0.05),however,AXL protein expression and CRC patients’ age,sex,tumor size,lymph node metastasis,distant metastasis and diameter not differentiation degree(P>0.05).Spearman correlation analysis showed that the expression of ARL2 and AXL in CRC cancer tissues was negatively correlated(r=-0.375,P<0.001).Conclusion:1.The changes of ARL2 expression in cancer tissues of CRC patients were associated with TNM stage,and the expression of ARL2 and distant metastatic status were independent prognostic factors affecting the 5-year survival of CRC patients.2.The positive expression of ARL2 in cancer tissue of CRC patients indicates good prognosis,but there was no difference in 5-year survival between ARL2 positive and ARL2 negative patients at age less than 65 years or with distant metastasis.Part II ARL2 affects the proliferation,migration and invasion ability of colorectal cancer cells by regulating AXL negativelyObjective:To observe the effects of ARL2 expression on AXL expression in colorectal cancer cell lines HCT8 and HCT116,and cell proliferation,migration and invasion by up-regulating and down-regulating the expression level of ARL2.Methods:1.HCT8 and HCT116 cell lines with ARL2 overexpression were constructed by transfection with eukaryotic plasmid pcDNA3.1(pARL2 group:ARL2 overexpression group;vector group:empty plasmid control group);2.HCT8 and HCT116 cell lines with ARL2 knockdown were constructed by shRNA lentivirus infection(shARL2 group:ARL2 interference group;shCtrl group:empty virus control);3.Western blot and Immunofluorescence(IF)experiments were used to identify whether the constructed cell lines were successful;4.CCK8 assay was used to identify cell proliferation;Clone formation assay was used to identify the ability of cell clone formation;Cell migration ability was determined by scratch test.Transwell assay was used to determine cell invasion ability.5.AXL protein expression in cells was detected by Western blot,AXL mRNA expression in cells was detected by qPCR.Results:1.Identification of HCT8 and HCT116 cell lines with overexpression of ARL2:Western blot analysis showed that the expression level of ARL2 protein in HCT8 and HCT116 cells after transfection of ARL2 overexpressed plasmid(0.96±0.19 and 0.81±0.07)was significantly higher than that in vector group(0.48±0.07 and 0.53±0.15),and the difference was statistically significant(P<0.001);The results of IF identification showed that the expression level of ARL2 protein in HCT8 and HCT116 cells after transfection with ARL2 overexpressed plasmid(24.17±0,92 and 25.18±0.97)was significantly higher than that in vector group(12.56±0.48 and 12.68±0.53),and the difference was statistically significant(P<0.001);All the above results indicated that ARL2 overexpressed in HCT8 and HCT116 cell lines was successfully constructed.The results of CCK8 method showed that the proliferation ability of HCT8 and HCT116 cells in pARL2 group was significantly decreased compared with vector group(P<0.05);The results of cloning-formation experiment showed that the cloning-formation ability of HCT8 and HCT116 cells in pARL2 group was significantly decreased compared with vector group(0.84±0.18 Vs 0.43±0.58,1.41±0.48 Vs 0.66±0.51,all P<0.05);In the scratch experiment,compared with the vector group,the scratch repair rate of HCT8 and HCT116 cells in pARL2 group was decreased,and the cell migration was inhibited,with statistical significance(1.19±0.33 Vs 0.48±0.25,1.17±0.32 Vs 0.49±0.18,all P<0.05);Transwell invasion experiment showed that compared with vector group,the number of transmembrane cells and the invasion ability of HCT8 and HCT116 cells in pARL2 group were decreased,and the difference was statistically significant(2.94±0.38 Vs 1.37±0.32,2.87±0.51 Vs 1.13±0.25,all P<0.05);Western blot results showed that compared with the vector group,the expression level of AXL in HCT8 and HCT116 cells after ARL2 overexpression was significantly decreased(0.52±0.35 Vs 0.24±0.18,0.59±0.38 Vs 0.19±0.14,all P<0.05);The qPCR results showed that the vector group was used as the control group.After ARL2 knockdown expression,the relative expression levels of AXL mRNA in HCT8 and HCT116 cells were 0.46 ± 0.53 and 0.48 ± 0.47,respectively,which were significantly lower than those in Vector group,with statistical significance(P<0.05).The above results suggested that both the protein and mRNA levels of AXL were decreased after ARL2 overexpression.2.Identification of HCT8 and HCT116 cell lines with ARL2 knockdown:Western blot analysis showed that the expression level of ARL2 protein in HCT8 and HCT116 cells after transfection with lentivirus shARL2(0.16±0.13 and 0.17±0.12)was significantly lower than that in shCtrl group(0.39±0.27 and 0.43±0.17),and the difference was statistically significant(P<0.05);The results of IF identification showed that the expression level of ARL2 protein in HCT8 and HCT116 cells after transfection with lentivirus shARL2(4.98±0.93 and 4.76±0.91)was significantly lower than that in shCtrl group(13.26±0.18 and 13.24±0.73),and the difference was statistically significant(P<0,001);All the above results suggested that the knockdown expression of ARL2 was successfully constructed in HCT8 and HCT116 cell lines.CCK8 assay showed that compared with shCtrl group,the proliferation ability of HCT8 and HCT116 cells in shARL2 group was significantly increased(P<0.05);The results of clone formation assay showed that compared with shCtrl group,the clone formation ability of HCT8 and HCT116 cells in shARL2 group was significantly increased(0.91±0.05 Vs 2.67±0.48,1.21±0.08 Vs 2.94±0.78,P<0.05);In the scratch test,compared with the shCtrl group,the scratch repair rate of HCT8 and HCT116 cells in the shARL2 group was increased,and the cell migration ability was enhanced,with statistical significance(1.13±0.25 Vs 2.02±0.51,1.06±0.22 Vs 2.32±0.41,P<0.05);Transwell invasion assay showed that compared with shCtrl group,the number of transmembrane cells in HCT8 and HCT116 cells in shARL2 group was increased,and the invasion ability was enhanced,with statistical significance(2.93±0.40 5.37±0.71,2.900±0.53 Vs 4.93±0.76,all P<0.05);Western blot analysis showed that compared with shCtrl group,the expression level of AXL protein in HCT8 and HCT116 cells after ARL2 knockdown was significantly increased(0.54±0.26 Vs 1.21±0.33,0.56±0.16 Vs 0.98±0.19,P<0.05);The qPCR results showed that the relative expression levels of AXL mRNA in HCT8 and HCT116 cells after ARL2 knockdown were 4.73 ± 3.83 and 2.57 ± 0.95,respectively,which were higher than those in the shCtrl group(P<0.05).These results suggested that both the protein and mRNA levels of AXL were increased after ARL2 knockdown expression.Conclusion:ARL2 can regulate the proliferation,clonal formation,migration and invasion of HCT8 and HCT116 cells in vitro,and the mechanism may be related to the negative regulation of AXL expression.