Study On The Mechanism Of LASS2/TMSG1 Inducing Autophagy In Human Lung Cancer Cells Via Synthesizing Ceramide

Objective:LASS2/TMSG1 is a newly discovered tumor metastasis suppressor gene,which can inhibit tumor proliferation,invasion and metastasis.It has two functional domains,HOX and TLC,in which TLC domain may be involved in the synthesis of ceramide.Ceramide is not only a lipid in eukaryotic cells,but also a bioactive substance that can regulate cell growth,differentiation and autophagy.The ceramides synthesized by LASS2/TMSG1 gene in human lung cancer cells were qualitatively and quantitatively analyzed based on ultra high performance liquid chromatography-tandem mass spectrometry to determine the types of ceramides induced by LASS2/TMSG1 gene and whether there are differences in the content of different types of ceramides,and to explore whether ceramide is involved in autophagy in human lung cancer cells by activating JNK signal pathway and its related molecular mechanism.Methods LASS2/TMSG1 gene silencing cell line was constructed by lentivirus infection experiment;LASS2/TMSG1 gene overexpression cell line was constructed by liposome transfection method;the type and content of ceramide induced by LASS2/TMSG1 gene were detected by ultra high performance liquid chromatography-tandem mass spectrometry;and the effects of LASS2/TMSG1 gene silencing and overexpression on autophagy vesicles of human lung cancer cells were detected by autophagy MDC assay.AO staining was used to detect the effects of LASS2/TMSG1 gene silencing and overexpression on autophagosomes in human lung cancer cells;and Western blot method was used to detect the protein expression levels of JNK,p-JNK,c-Jun,p-c-Jun,Beclin1 and LC3 in human lung cancer cells before and after LASS2/TMSG1 gene silencing and overexpression.Using SPSS22.0 statistical software to analyze and process the data,Three groups of samples were analyzed by one-way ANOVA whether there is any difference in this mean,and then compare the samples in pairs.P<0.05 means that the difference is significant and statistically significant.Results（1）The levels of LASS2/TMSG1 m RNA and protein in LASS2/TMSG1 gene silencing group were significantly lower than those in blank control group and negative control group（real-time fluorescence quantitative PCR method:F=17.457,P=0.001;P silence group-blank control group=0.002;P silence group-negative control group=0.003.Western blot method:F=13.631,p=0.000;P blank control group-silence group=0.001;P negative control group-silence group=0.000）,the expression levels of LASS2/TMSG1 m RNA and protein in LASS2/TMSG1 overexpression group were on the contrary(real-time fluorescence quantitative PCR method:F=43.520,p=0.000;P overexpression group-blank control group=0.000;P overexpression group-negative control group=0.000.Western blot method:F=5.145,P=0.023;P overexpression group-blank control group=0.009;P overexpression group-negative control _group=0.029).The results of MDC staining showed that the number of autophagy vesicles in the LASS2/TMSG1 gene silencing group was significantly lower than that in the blank control group and negative control group(F=19.696,P=0.000;P _{silence group-blank control}group=0.000;P silence group-negative control group=0.001).The number of autophagic vesicles in the overexpression group of LASS2/TMSG1 gene was significantly more than that in the blank control group and negative control group(F=24.422,P=0.000;P _{overexpression}group-blank control group=0.000;P overexpression group-negative control group=0.000).The results of AO staining showed that the number of autophagosomes in the LASS2/TMSG1 gene silencing group was significantly lower than that in the blank control group and the negative control group(F=13.088,P=0.006;P silence group-blank control group=0.004;P silence _{group-negative control group}=0.005).The number of autophagosomes in the overexpression group of LASS2/TMSG1 gene was significantly more than that in the blank control group and negative control group（F=18.781,P=0.003;P overexpression group-blank control group=0.002;P overexpression group-negative control group=0.001）.20 kinds of ceramides in cells were analyzed by liquid chromatography-tandem mass spectrometry（LC-MS）.The results showed that the total contents of ceramides and the contents of d18:1/20:0、d18:1/22:0、d18:1/24:0、d18:1/24:1and d18:1/26:0 in the LASS2/TMSG1 gene silencing group were significantly lower than those in the blank control group and negative control group（F total ceramide=32.154,P=0.001;P silence group-blank control group=0.000;P silence group-negative control group=0.001.Fd18:1/20:0=5.669,P=0.041;P silence group-blank control group=0.034;P silence group-negative control group=0.022.F d18:1/22:0=12.626,P=0.007;P silence group-blank control group=0.003;P silence group-negative control group=0.009.Fd18:1/24:0=52.056,P=0.000;P silence group-blank control group=0.000;P silence group-negative control group=0.000.F d18:1/24:1=6.392,P=0.033;P silence group-blank control group=0.014;P silence group-negative control group=0.042.F d18:1/26:0=16.263,P=0.004;P silence group-blank control group=0.003;P silence group-negative control group=0.002）;The total ceramide content and the contents of d18:1/20:0、d18:1/22:0、d18:1/24:0、d18:1/24:1、d18:1/26:0in LASS2/TMSG1 gene overexpression group were opposite to those in LASS2/TMSG1 silencing group.(F total ceramide=23.893,P=0.001;P overexpression group-blank control group=0.001;P overexpression group-negative control group=0.001.Fd18:1/20:0=36.385,P=0.001;P overexpressiongroup-blankcontrolgroup=0.000;P overexpression group-negative control group=0.000.Fd18:1/22:0=41.524,P=0.000;P overexpression group-blank control group=0.000;P overexpression group-negative control group=0.000.Fd18:1/24:0=30.987,P=0.001;P overexpression group-blank control group=0.001;P overexpression group-negative control group=0.000.Fd18:1/24:1=71.125,P=0.000;P overexpressiongroup-blankcontrolgroup=0.000;P overexpression group-negative control group=0.000.Fd18:1/26:0=78.546,P=0.000;P overexpression group-blank control group=0.000;P overexpression _{group-negative control group}=0.000).Western blot method was used to detect the expression of proteins related to the autophagy pathway.There was no significant difference in JNK（SAPK）protein between the LASS2/TMSG1 gene silence group and the blank control group and the negative control group(F=1.078,P=0.398;P _{silence group-blank control}group=0.919;P silence group-negative control group=0.235;P blank control group-negative control group=0.270);p-JNK,c-Jun,p-c-Jun,LC3 and Beclin1 in the LASS2/TMSG1 gene silencing group were significantly lower than those in the blank control group and the negative control group（Fp-JNK=16.015,P=0.004;P silence group-blank control group=0.001;P silence group-negative control group=0.009.Fc-Jun=9.416,P=0.014;P silence group-blank control group=0.006;P silence group-negative control group=0.017.Fp-c-Jun=21.259,P=0.002;P silence group-blank control group=0.002;P silence group-negative control group=0.001.FBeclin1=7.554,P=0.023;P silence group-blank control group=0.011;P silence group-negative control group=0.022.FLC3Ⅱ/LC3Ⅰ=12.285,P=0.008;P silence group-blank control group=0.045;P silence group-negative control group=0.003）;There was no significant difference in JNK（SAPK）between the LASS2/TMSG1 gene overexpression group and the blank control group and the negative control group（F=0.105,P=0.902;P overexpression group-blank control group=0.671,P overexpression group-negative control group=0.899,P blank control group-negative control group= 0.764）;p-JNK,c-Jun,p-c-Jun,LC3 and Beclin1 were significantly higher than those of the blank control group and the negative control group,and the difference was statistically significant.(F=p-JNK=5.796,P=0.040;P overexpression group-blank control group=0.025;P overexpression group-negative control group=0.026.Fc-Jun=17.952,P=0.003;P overexpression group-blank control group=0.007;P overexpression group-negative control group=0.001.Fp-c-Jun=7.782,P=0.021;P overexpression group-blank control group=0.010;P overexpression group-negative control group=0.024.FBeclin1=11.834,P=0.008; P overexpression group-blank control group=0.006;P overexpression group-negative control group=0.005.FLC3Ⅱ/LC3Ⅰ=11.645,P=0.009;P overexpression group-blank control group=0.014;P overexpression group-negative control _group=0.003).Conclusion After the silencing of LASS2/TMSG1 gene,autophagy in human lung cancer cells decreased and autophagy level decreased.After the overexpression of LASS2/TMSG1 gene,autophagy in human lung cancer cells increased and autophagy level increased.It is speculated that LASS2/TMSG1 may be involved in inducing autophagy in human lung cancer cells.The mechanism may be that the synthesis of ceramide,especially long-chain ceramides d18:1/20:0,d18:1/22:0,d18:1/24:0,d18:1/24:1 and d18:1/26:0,etc.activate the JNK-c-Jun signaling pathway to promote the expression of autophagy-related proteins Beclin1 and LC3 proteins,thereby induce autophagy in tumor cells.