| Objective: The incidence of colorectal cancer(CRC)in the developed countries in Europe and the United States has begun to decline significantly,but the incidence of colorectal cancer in our country is still slowly increasing year by year,and the longterm efficacy of advanced colorectal cancer patients still cannot make people satisfaction.Therefore,it is extremely urgent to explore the pathogenesis of colorectal cancer,looking for the molecules of carcinogenesis and development of colorectal cancer and malignant biological behavior as a new marker for early diagnosis and targeted therapy to improve the rate of early diagnosis and prolong survival time for clinicians.Uncontrolled cell abnormal proliferation is one of the important preconditions leading to tumorigenesis.Its essence is the activation of the protooncogenes and the inactivation of the tumor suppressor gene.B-cell translocation gene 3(BTG3),as a member of the family of antiproliferative genes,plays an important role in participating in DNA damage repair,maintaining genome stability and inducing abnormal cellular senescence.BTG3 is highly expressed in normal human tissues.However,BTG3 is over-expressed in many human malignancies,and silencing BTG3 gene can increase the proliferation,migration and invasion of cancer cells,and attenuate the apoptosis.This suggests that BTG3 may be a potential tumor suppressor gene.However,the role of BTG3 in the development of colorectal cancer,malignant phenotype and prognostic value is unclear.This research intends to explore the function of BTG3 in the carcinogenesis and development of colorectal cancer and the molecular mechanism of regulating malignant biological behavior of CRC.Methods: 1,Collected 140 cases of colorectal cancer patients with cancer and adjacent normal colorectal tissue(pathologically confirmed that no cancer cells).Immunohistochemistry(IHC)staining was used to compare the expression of BTG3 in colorectal cancer tissues and paired normal tissues.The relationship between abnormal BTG3 expression and the clinicopathological features of patients with colorectal cancer was analyzed by t-test or ANOVA.The Kaplan-Meier method was used to analyze the relationship between the expression of BTG3 and the overall survival(OS)and disease-free survival(DFS).Cox regression model as multivariate analysis was used to evaluate the overall survival and disease-free survival.Construction of ROC curve to evaluate the diagnostic value of BTG3 in colorectal cancer.2,The colorectal cancer cell lines with relatively higher BTG3 protein expression were screened.The expression of BTG3 gene was down-regulated by lentivirus and the stable cell line was constructed.The changes of BTG3 m RNA and protein level were detected by Real-time PCR and Western blot to evaluate the gene knockdown efficiency.CCK-8 assay was used to detect the effect of down-regulation of BTG3 on cell proliferation;Propidium Iodide(PI)staining was used to detect the effect of down-regulation of BTG3 on cell cycle;Annexin V-PE / 7-AAD double staining was used to detect the effect of cell apoptosis in early stage;Transwell assay was used to detect the effect of BTG3 down-regulation on cell migration and invasion.3,Affymetrix Gene Chip was used to detect the changes of total gene expression profiles after down-regulation of BTG3 expression in colorectal cancer cells.Ingenuity Pathway Analysis(IPA)was used to analyze the differential gene expression,disease and function classification and enrichment of classical signaling pathways analysis.Western blot was used to detect the expression of differentially expressed proteins of some downstream candidate pathways in order to verify the reliability of the IPA bioinformatics analysis and to reveal the possible molecular mechanisms by which BTG3 exerts its biological function in colorectal cancer.Results: 1,IHC results showed that the average value of MOD of BTG3 positive expression in cancer tissues was 0.267,which was significantly lower than that in adjacent normal tissues(0.295,P <0.001).The abnormal expression of BTG3 correlated with tumor differentiation(P = 0.037),tumor invasion depth(P = 0.016),distant metastasis of tumor(P = 0.024)and TNM stage(P = 0.007).There was no significant correlation between age,tumor location,lymph node metastasis,vascular or nerve invasion(P> 0.05).Patients with colorectal cancer with relatively high expression of BTG3 had significantly higher OS and DFS than patients with relatively low BTG3 expression(OS P = 0.0045;DFS P 0.0011).BTG3 expression was an independent prognostic factor affecting OS and DFS in patients with colorectal cancer(OS P = 0.016;DFS P = 0.005).BTG3 has a certain reference value for the diagnosis of colorectal cancer.The area under the ROC curve is 0.638(P <0.0001).2,BTG3 protein is highly expressed in HCT116 and Lo Vo cell lines.The lentivirus could significantly down-regulate the m RNA and protein expression of BTG3 after both cells were infected successfully(all P <0.05).The effect of downregulation of BTG3 gene on cell proliferation by CCK8 assay: The proliferation of HCT116 and Lo Vo cells of knockdown group(sh RNA1 and sh RNA2)was significantly higher than that of NC group.Proliferation of HCT116 cells in sh RNA1 and sh RNA2 group at 24 h and 48 h were higher than that of NC group(4.022 ± 0.419 vs 2.279 ± 0.054 P <0.0001;3.323 ± 0.207 vs 2.279 ± 0.054 P = 0.0008)(8.993 ± 1.043 vs 4.649 ± 0.078,P <0.0001;7.667 ± 0.246 vs 4.649 ± 0.078,P <0.0001).Compared with NC group,sh RNA1 group and sh RNA2 group at 96 h were significantly higher than NC group(12.71 ± 1.732 vs 6.736 ± 1.601 P <0.0001;12.04 ± 0.530 vs 6.736 ± 1.601 P <0.0001).The proliferation of Lo Vo cells at 48 h was significantly higher than that of NC group(3.002 ± 0.425 vs 2.588 ± 0.505 P = 0.2949;2.837 ± 0.036 vs 2.588 ± 0.505 P = 0.6167,respectively).The proliferation rates of sh RNA1 and sh RNA2 groups at 72 h were significantly higher than those in NC group(5.984 ± 0.220 vs 5.232 ± 0.109 P <0.0001,5.612 ± 0.036 vs 5.232 ± 0.109 P = 0.0091),96 h cell proliferation ratio sh RNA1 group and sh RNA2 group were higher than NC group(9.287 ± 0.381 vs 7.527 ± 0.139 P <0.0001;8.374 ± 0.274 vs 7.527 ± 0.139 P = 0.0044).Detection of BTG3 down-regulation of cell cycle: HCT116 cells,G1 phase cells sh RNA1 group than NC cells increased(75.105 ± 2.586 vs 61.357 ± 2.525 P = 0.0002),sh RNA2 group than in NC group cells but no significant difference(64.777 ± 3.867 vs 61.357 ± 2.525 P = 0.431).The expression of S-phase cells in sh RNA1 and sh RNA2 groups was not significantly different from NC group(14.910 ± 3.929 vs 17.383 ± 4.381,21.010 ± 4.259 vs 17.383 ± 4.381,P <0.05)The number of cells in sh RNA1 group was significantly lower than that in NC group(9.985 ± 3.617 vs 21.257 ± 2.020 P = 0.0016),the number of cells in sh RNA2 group was lower than that in NC group(14.180 ± 0.625 vs 21.257 ± 2.020 P = 0.0435)(54.767 ± 2.778 vs 63.153 ± 3.199 P = 0.0139,P <0.05).The number of cells in sh RNA2 group was significantly lower than that in NC group(59.300 ± 4.859 vs 63.153 ± 3.199 P = 0.3342)Group cells increased(32.457 ± 2.45 7 vs 14.877 ± 4.351 P <0.0001).The number of cells in sh RNA2 group was significantly higher than that in NC group(26.717 ± 5.061 vs 14.877 ± 4.351 P = 0.0008)= 0.0072).The number of cells in sh RNA2 group was lower than that in NC group(13.983 ± 0.466 vs 21.970 ± 1.304 P = 0.0192).Down-regulation of BTG3 can promote the proliferation of HCT116 cells by decreasing G2 arrest,and down-regulating BTG3 can promote the proliferation of Lo Vo cells by increasing S phase DNA synthesis and reducing G2 arrest.Annexin VPE / 7-AAD double staining method detected the effect of BTG3 gene downregulation on cell apoptosis: HCT116 cells,sh RNA1 group apoptosis rate was lower than NC group(8.627 ± 4.307 vs 23.117 ± 4.737 P = 0.0065),sh RNA2 group The apoptosis rate of Lo Vo cells and sh RNA1 group was lower than that of NC group(0.713 ± 0.268 vs 4.783 ± 1.590 P = 0.0034),and the ratio of sh RNA2 group was lower than that of NC group(13.890 ± 1.154 vs 23.117 ± 4.737 P = 0.0469)The apoptosis rate of NC group decreased(2.283 ± 0.284 vs 4.783 ± 1.590 P = 0.0311),indicating that silenced BTG3 gene can inhibit the apoptosis of colorectal cancer cells.Transwell assay detected the effect of down-regulation of BTG3 gene on cell migration.HCT116 cells,sh RNA1 group than NC group(186 ± 8 vs 55 ± 7 P <0.0001),sh RNA2 group than NC group(147 ± 8 vs 55 ± P <0.0001).In Lo Vo cells,the number of cells in sh RNA1 group increased more than that in NC group(124 ± 12 vs 74 ± 6 P <0.0001),and the number of cells in sh RNA2 group increased more than those in NC group(98 ± 7 vs 74 ± 6 P = 0.0156).The invasion ability of HCT116 cells was significantly higher in sh RNA1 group than in NC group(130 ± 5 vs 36 ± 6 P <0.0001),and in sh RNA2 group was higher than that in NC group(92 ± 8 vs 36 ± 6 P < 0.0001).In Lo Vo cells,the number of cells in sh RNA1 group was significantly higher than that in NC group(106 ± 7 vs 47 ± 8 P <0.0001),and the number of cells in sh RNA2 group was more than that of NC group(85 ± 8 vs 47 ± 8 P <0.0001).BTG3 gene may enhances cell migration and metastasis.3,After down-regulation of BTG3 gene,554 differentially expressed genes were screened by gene expression microarray,including 281 up-regulated genes and 273 down-regulated genes.Differential genes mainly affect cell morphology,cytology,cell cycle,apoptosis and tumor formation,tumor cell death,proliferation and so on.Differential genes mainly involve multiple classical signaling pathways including the ERK / MAPK pathway.The protein expression of nine differential genes involved in ERK / MAPK was detected by Western blot.The up-regulated expressions of PAK2,YWHAB,RPS6KA5 and STAT3 were detected.The down-regulated expressions of RAP1 A,DUSP6 and STAT1 were detected.CFOS and ATF4 were unchanged.The overall trend is in line with the result of the gene chip analysis.Conclusions: 1,The expression of BTG3 in colorectal cancer was significantly lower than that in adjacent normal tissues and closely correlated with the degree of tumor differentiation,invasion depth,distant metastasis and TNM stage.BTG3 exerted tumor inhibition in the carcinogenesis and development of colorectal cancer.BTG3 can be used as an independent prognostic indicator of colorectal cancer;BTG3 has some reference value for the diagnosis of colorectal cancer.2,Down-regulation of BTG3 gene can significantly increase the proliferation,migration and invasion ability of colorectal cancer cells,meanwhile,it can inhibit apoptosis and cell cycle arrest.BTG3 plays an important role in promoting the malignant phenotype of colorectal cancer.3,That BTG3 gene regulated several molecules involved in ERK / MAPK pathway may be the possible molecular mechanisms on aggressive colorectal cancer behavior. |
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