Expression Of SPTLC1 In Xuanwei Non-Smoking Female Lung Adenocarcinoma And Its Effect On Cell Biological Function

Objectives:The purpose of this study was to investigate the expression of SPTLC1 in Xuanwei non-smoking female lung adenocarcinoma tissues,verify its effect on the biological function of lung adenocarcinoma cells,and predict the possible mechanism of its biological function in lung adenocarcinoma.Methods:1.Expression of SPTLC1 protein in lung adenocarcinoma of Xuanwei non-smoking womenIn this study,105 pairs of tumor tissues and paired paracancer tissues were collected from surgically removed Xuanwei non-smoking female lung adenocarcinoma patients,and examined the expression level of SPTLC1 protein by immunohistochemistry to compare the protein expression differences between the two groups.2.Regulation of SPTLC1 gene on biological function of lung adenocarcinoma cells2.1 Screening of tool cell linesWestern Blot was used to detect and compare the expression levels of SPTLC1 in human lung adenocarcinoma cell lines A549,H838,H1734,H1299,H1975,XWLC-05 and HCC827,using immortalized human bronchial epithelial cell line Beas-2b as control.According to the relative expression level of SPTLC1 protein,the cell lines with the highest relative expression level and the cells with the lowest relative expression level were selected as tool cells.2.2 Construction of tool cell linesSPTLC1 knockout cell lines were constructed using CRISPR/Cas9 technique,and SPTLC1 stable overexpression cell lines were constructed by lentvirus transfection.2.3 Effect of SPTLC1 gene knockout and overexpression on biological function of lung adenocarcinoma cellsCCK-8 assay,Transwell invasion assay,scratch assay and flow cytometry were used to investigate the effects of SPTLC1 on the proliferative ability,invasion ability,migration ability,cell growth cycle and apoptosis of lung adenocarcinoma cells at the cell level.3.To predict the possible molecular mechanism of SPTLC1 gene regulating biological function of lung adenocarcinomaTranscriptome sequencing of SPTLC1 overexpressing cell lines,and differentially expressed genes were screened for GO enrichment analysis and KEGG signaling pathway enrichment analysis to predict the possible mechanism of SPTLC1 exerting biological function.Results:1.Expression of SPTLC1 protein in lung adenocarcinoma of Xuanwei non-smoking womenThe positive expression rate of SPTLC1 in Xuanwei non-smoking female lung adenocarcinoma tissues was 94.29%(99/105),and the positive expression rate in paired adjacent tissues was 24.76%(26/105),the positive expression rate of SPTLC1 gene in Xuanwei non-smoking female lung adenocarcinoma tissue was significantly higher than that in paired adjacent tissue,and the difference was significant(P<0.01).2.Regulation of SPTLC1 gene on biological function of lung adenocarcinoma cells2.1 Screening of tool cell linesThe expression level of SPTLC1 protein in lung adenocarcinoma cell lines A549,H838,H1734,H1299,H1975 and HCC827 was higher than that of Beas-2b.The relative expression level of A549 was the highest,and the expression level of SPTLC1 protein in XWLC-05 was lower than that of Beas-2b.Therefore,A549 cells and XWLC-05 cells were selected as tool cells for follow-up experiments.2.2 SPTLC1 gene knockout and overexpression cell lines were constructed successfullyThree SPTLC1 knockout plasmids were successfully constructed.The CRISPR-Cas9 knockout vector and STPLC1 overexpression vector were successfully transfected into A549 cells and XWLC-05 cells using lentvirus.Astern the SPTLC1 protein expression was detected by Weblot.The protein expression level of SPTLC1 in A549 cell knockout group was lower than that in control group,while the protein expression level of SPTLC1 in XWLC-05 cell overexpression group was higher than that in control group.SPTLC1 gene knockout and overexpression cell line were successfully constructed.2.3 Effects of SPTLC1 gene knockout and overexpression on biological function of lung adenocarcinoma cells(1)The proliferation ability of SPTLC1 knockout group was enhanced compared with control group;The proliferative ability of SPTLC1 overexpression group was not significantly changed compared with control group.The proliferation ability of SPTLC1 knockout group was enhanced compared with control group,and the difference was statistically significant(P<0.05);The proliferation ability of SPTLC1 overexpression group was not significantly different from that of control group,and the difference was not statistically significant(P>0.05).(2)There was no significant change in the invasion ability of SPTLC1 knockout group compared with control group,and the difference was statistically significant(P<0.05);The invasion ability of SPTLC1 overexpression group was lower than that of control group,and the difference was statistically significant(P<0.05).(3)The migration ability of SPTLC1 knockout group was enhanced compared with control groupand the difference was statistically significant(P<0.05);The migration ability of SPTLC1 overexpression group was not significantly changed compared with control group,and the difference was not statistically significant(P>0.05).(4)Compared with the control group,the proportion of G0/G1 phase in SPTLC1 knockout group decreased significantly,and the difference was statistically significant(P<0.05),the proportion of S stage was significantly increased,and the difference was statistically significant(P<0.05),G2/M phase ratio did not change significantly,the difference was not statistically significan(P<0.05).Combined with the results of CCK-8 experiment,SPTLC1 gene knockout can promote A549 cells to enter the S phase,thus promoting cell proliferation.Compared with the control group,the G0/G1 phase ratio of SPTLC1 overexpression group had no significant change,and the difference was not statistically significant(P>0.05),there was no significant change in S stage,the difference was not statistically significant(P>0.05),G2/M phase ratio did not change significantly,the difference was not statistically significant(P>0.05).Combined with the results of CCK-8 experiment,SPTLC1 gene overexpression had no significant effect on the growth cycle of XWLC-05 cells(5)Compared with the control group,the proportion of apoptosis in SPTLC1 knockout group was not significantly changed(P>0.05).;Compared with the control group,the proportion of apoptosis in SPTLC1 overexpression group was not significantly changed(P>0.05).3.Transcriptome sequencing results and analysisA total of 27 differentially expressed genes(DEGs)were screened by transcriptome sequencing and analysis.GO functional enrichment analysis showed that,SPTLC1 may be associated with mitochondrial respiratory chain complex I assembly,chromatin remodeling,Wnt signaling pathway,NADH dehydrogenase(ubiquitin-one)activity,protein depalmitylation,proton-driven mitochondrial ATP synthesis,aerobic respiration and other potential factors regulating the tumor microenvironment.KEGG signal pathway enrichment analysis showed that SPTLC1 may be involved in oxidative phosphorylation pathway and sphingolipid signal pathway.Conclusions:SPTLC1 protein was highly expressed in Xuanwei non-smoking female lung adenocarcinoma tissues,which may be the different-expressed protein of Xuanwei non-smoking female lung adenocarcinoma.Knockout of SPTLC1 can enhance the migration and invasion of lung adenocarcinoma cells,and promote the proliferation of lung adenocarcinoma cells and their entry into the stage of division.SPTLC1 may regulate the tumor microenvironment by participating in oxidative phosphorylation pathway and sphingolipid signaling pathway,and may become a potential target to inhibit the malignant biological behavior of lung adenocarcinoma cells.