Abstract:

Objective To investigate the expression of chondroitin sulfate proteoglycan 4 pseudogene 12 （CSPG4P12） in the small cell lung cancer （SCLC） tissue， its relationship with immune infiltration， and its effect on cell biological functions， and to clarify its effect in the occurrence and development of SCLC. Methods The E-GEOD-60052 cohort was obtained by searching the ArrayExpress database for SCLC. The R language Bioconductor package was used to complete data filtering standardization， and 63 samples of SCLC tumor tissues and 7 samples of normal tissues were obtained. The Mann-Whitney U test was used to analyze the difference in CSPG4P12 expression levels between two groups. Pearson correlation analysis was used to evaluate the associations between CSPG4P12 expression levels and 47 immune checkpoint genes. The ESTIMATE algorithm and CIBERSORT algorithm were used to evaluate the correlations between CSPG4P12 expression and tumor immune cell infiltration. A case-control study was used to analyze the clinical data. A total of 230 patients with SCLC were selected as case group， and 230 healthy subjects were selected as control group. The genotyping of CSPG4P12 rs2880765， rs6496932 and rs8040855 was performed using TaqMan-MGB fluorescent probe labeling method. Odds ratio （OR） and 95% confidence interval （CI） were calculated by unconditional Logistic regression model to analyze the association between polymorphic genetic variation of CSPG4P12 gene and the risk of SCLC. The SCLC DMS114 cells were transfected with pUC-57 plasmid （control group） and CSPG4P12 over-expression plasmid （OV-CSPG4P12 group）， respectively. The efficiencies of CSPG4P12 over-expression in two groups were verified by real-time fluorescence quantitative PCR（RT-qPCR） method. Cell counting kit-8 （CCK-8） method was used to detect the cell proliferation activities of cells in two groups. Transwell chamber assay was used to detect the numbers of migration and invasion cells in two groups， respectively. Hoechst 33342 fluorescence staining was used to observe the cell apoptosis in two groups. Results The ArrayExpress database E-GEOD-60052 cohort analysis showed that the expression level of CSPG4P12 mRNA in SCLC tissue was decreased compared with normal tissue （P<0.001）. The expression of CSPG4P12 had positive correlations with the immune checkpoint genes including leukocyte associated immunoglobulin like receptor 1 （LAIR1） （r=0.47， P<0.001）， tumor necrosis factor（TNF） receptor superfamily member 9 （TNFRSF9） （r=0.38， P<0.01）， and TNF superfamily member 9 （TNFSF9） （r=0.44， P<0.001）. The ESTIMATE algorithm results showed that the matrix score， immune score and ESTIMATE composite score of the patients in CSPG4P12 low expression group were lower than those in CSPG4P12 high expression group （P<0.01）. The CIBERSORT algorithm results showed that compared with CSPG4P12 high expression group， the infiltration of M0 macrophages in CSPG4P12 low expression group was increased （P<0.05） and the infiltration of mast cells resting was decreased （P<0.05）. The CSPG4P12 expression level had positive correlations with infiltration of mast cells resting （r=0.35， P=0.03） and mononuclear cell infiltration （r=0.34， P=0.034）. In case-control studies， compared with AA genotype， CSPG4P12 rs2880765 AT and TT genotype carriers had a higher risk of SCLC （OR=1.68， 95%CI=1.15-2.45， P<0.01）. The stratified analysis showed that genetic variation of rs2880765 A>T increased the risk of SCLC in the male， younger age group（≤60 years） and smoking subgroups （males： OR=1.86， 95%CI=1.18-2.93， P<0.01； ≤60 years： OR=1.73， 95%CI=1.11-2.68， P<0.01； smoking： OR=2.76， 95%CI=1.49-5.13， P=0.001）. The cell biology experiment showed that compared with control group， the proliferation abilities of the cells in OV-CSPG4P12 group were significantly decreased at 48 and 72 h （P<0.01）， while the number of migration cells at 24 h was significantly decreased （P<0.01）， the number of apoptotic cells at 24 h was increased （P<0.05） and the number of invasion cells at 48 h was significantly decreased （P<0.01）. Conclusion CSPG4P12 is lowly expressed in SCLC tumor tissue， which is associated with immune infiltration. The genetic variation of CSPG4P12 rs2880765 A>T can increase the risk of SCLC， and its over-expression can inhibit cell proliferation， migration and invasion， and promote apoptosis.

Key words: Chondroitin sulfate proteoglycan 4 pseudogene 12, Immune infiltration, Genetic variation, Cell biological function, Small cell lung cancer