LASS2/TMSG1 Gene Participates In The Proliferation And Apoptosis Of Human Lung Cancer A549 Cells Through The Ceramide Pathway And Its Mechanism

Objective To discuss the effect of LASS2 / TMSG1 on the proliferation and apoptosis of human lung cancer A549 cells in vitro,and to observe the effect of LASS2 / TMSG1 on the growth process of subcutaneously transplanted tumors in nude mice.Furthermore,to investigate the specific molecular mechanism of LASS2 / TMSG1 inducing apoptosis of human lung cancer A549 cells.Methods 1.Flow cytometry combined with Annexin V-FITC / PI double staining method was used to detect the effect of LASS2 / TMSG1 gene on human lung cancer A549 cells.2.Plate clone formation test was used to detect the effect of LASS2 / TMSG1 gene on the proliferation of human lung cancer A549 cells in vitro.3.Construct a subcutaneous xenograft model of human lung cancer in nude mice and observe the effect of the LASS2 / TMSG1 gene on the growth of subcutaneous xenografts in nude mice.4.Hematoxylin-eosin staining was used to observe the microscopic morphology,colony formation,apoptotic necrosis and tumor metastasis of subcutaneous tumor tissue in nude mice.5.Immunohistochemical staining to observe the effect of LASS2 / TMSG1 gene on the expression of Ki67 in subcutaneous tumor tissue of nude mice.6.ELISA method to detect the effect of LASS2 / TMSG1 gene on the expression of ceramide and P38 MAPK protein in human lung cancer A549 cell supernatants and nude mice tumor tissue homogenates.7.Data analysis was performed using SPSS22.0 software,P <0.05(as the analysis standard),which was considered statistically significant.Results(1)The results of previous experiments showed that the expression level of LASS2 / TMSG1 protein in human lung cancer A549 cells of the over-expression group was increased by Western Blot detection,indicating that the LASS2 / TMSG1 gene over-expression group was successfully constructed.(2)The results of flow cytometry showed that the early apoptosis rate(%)of the LASS2 / TMSG1 gene overexpression group was significantly higher than that of the negative control group,respectively: 31.51 ± 1.41,11.72 ± 1.07,(t = 1.10,P < 0.05).(3)The results of plate clone formation experiments showed that the percentage of cell clone formation(%)in the LASS2 / TMSG1 gene overexpression group was significantly lower than that in the negative control group,respectively: 12.17 ± 4.51,31.83 ± 10.13,(t = 3.07,P <0.05).(4)The results of nude mice xenograft formation experiments showed that compared with the negative control group,the tumor volume of the nude mice in the LASS2 / TMSG1 gene overexpression group increased slowly(P> 0.05).The final mass and volume of tumor tissue in two groups of nude mice were measured.The average mass of tumor tissue in the LASS2 / TMSG1 gene overexpression group was slightly lower than that in the negative control group,which were 0.23 ± 0.10,0.28 ± 0.13,(t = 0.75,P> 0.05).);The average volume of tumor tissue in the LASS2 / TMSG1 gene overexpression group was lower than that in the negative control group,which were 90.60 ± 45.26,143.90 ± 89.40,respectively(t = 1.41,P> 0.05),and the difference was not significant.(5)Hematoxylin-eosin staining revealed that the tumor cells in both groups showed poorly differentiated adenocarcinoma morphology;the average number of tumor cell clones(1.29 ± 0.49)in the LASS2 / TMSG1 gene overexpression group was significantly lower than that in the negative control group.(2.29 ± 0.95),(t = 2.48,P <0.05);compared with the negative control group,the necrosis of the tumor tissue in the LASS2 / TMSG1 gene overexpression group was larger and more thorough.No liver,spleen,kidney and lymph node metastasis were found in the two groups of nude mice.(6)Immunohistochemical staining revealed that Ki67 positive rate(%)(27.15 ± 9.36)in tumor tissue of nude mice in the LASS2 / TMSG1 gene overexpression group was significantly lower than that in the negative control group(44.80 ± 6.05),(t = 4.48,P <0.05).(7)ELISA test results showed that the ceramide content in the LASS2 / TMSG1 gene overexpression group of human lung cancer A549 cell supernatant was significantly higher than that of the negative control group,which were 155.00 ± 3.83,128.50 ± 13.71,(t = 3.22,P <0.05);The ceramide content of the LASS2 / TMSG1 gene over-expression group in nude mice tumor homogenates was significantly higher than that in the negative control group,which were 265.00 ± 19.34 and 226.20 ± 7.99,respectively(t = 3.21,P <0.05);The content of P38 MAPK protein in the LASS2 / TMSG1 gene overexpression group of human lung cancer A549 cell supernatant was significantly higher than that of the negative control group,which were 86.47 ± 1.04 and 78.38 ± 1.38,respectively(t = 8.14,P <0.05);tumor tissue of nude mice The content of P38 MAPK protein in the LASS2 / TMSG1 gene overexpression group in the homogenate was significantly higher than that in the negative control group,which were 89.93 ± 3.92,62.32 ± 3.54,respectively,and the difference was statistically significant(t = 9.06,P <0.05).Conclusion As a tumor metastasis suppressor gene,LASS2 / TMSG1 gene can not only affect the proliferation of tumor cells,but also regulate the apoptosis of tumor cells.The LASS2 / TMSG1 gene may activate cascade signaling pathways by inducing the synthesis of ceramide,further activating its downstream effector molecule,P38 MAPK protein,etc.,and thus play a role in promoting human lung cancer cell apoptosis and inhibiting human lung cancer cell proliferation.