Function And Mechanism Of Pseudogene CSPG4P12 In Non-small Cell Lung Cancer

Objectives This study aimed to investigate the association of genetic variations of chondroitin sulfate proteoglycan 4 pseudogene 12（CSPG4P12）with the susceptibility to non-small cell lung cancer（NSCLC）and to reveal the role of CSPG4P12 in the development of NSCLC.Methods Chapter 1:This study was a group-designed case-control study,which included703 NSCLC patients and 703 normal controls.Genotyping of each single nucleotide polymorphism（SNP）in CSPG4P12 gene was performed using Taqman fluorescent probe assay.Unconditional logistic regression was used to calculate odd ratio（OR）and 95%confidence interval（CI）to evaluate the effect of each genetic variation on NSCLC susceptibility.SHEsis was used for haplotype analysis.The GTEx database was used to predict the association between SNP and expression of CSPG4P12.Chapter 2:Real-time quantitative PCR was used to detect the expression of CSPG4P12 in NSCLC cancer tissues and adjacent normal tissues.Cell proliferation ability was determined by CCK-8 assay and colony formation assay.Cell invasion,migration and adhesion ability were determined by transwell assay,cell scratch assay and cell-matrix adhesion assay.Fluorescence staining assay and transmission electron microscopic were used to detect cell apoptosis.Immunofluorescence and western blot were used to detect protein expression.Results Chapter 1:Individuals with CSPG4P12 rs8040855 GC genotype had a higher risk of NSCLC.Stratified analysis results showed that individuals carrying rs8040855 GC genotype had a lower risk of NSCLC among non-smokers（OR=0.36,95%CI=0.18～0.72,P<0.01）,females（OR=0.29,95%CI=0.12～0.72,P<0.01）and youngers（OR=0.43,95%CI=0.23～0.80,P<0.01）,but not among smokers（OR=0.63,95%CI=0.29～1.35,P=0.23）,males（OR=0.57,95%CI=0.31～1.06,P=0.08）and elders（OR=0.51,95%CI=0.22～1.19,P=0.12）.We also found that rs2880765 TT carriers had a lower risk of NSCLC.when compared with individuals carrying rs2880765 A allele,rs2880765 T allele carriers were more likely to suffer NSCLC among males（OR=1.42,95%CI=1.09～1.86,P=0.01）or heavy smokers（OR=1.81,95%CI=1.03～3.18,P=0.04）,but not among females（OR=0.93,95%CI=0.64～1.34,P=0.69）and light smokers（OR=1.21,95%CI=0.74～1.98,P=0.44）.The haplotype analysis showed that T_rs2880765C_rs8040855 contributed to higher genetic susceptibility to NSCLC and Ars2880765Grs8040855 to a lower genetic susceptibility,when Ars2880765Crs8040855was set as the reference.Results from GTEx presented that rs2880765 A>T variant was associated with the high expression of CSPG4P12,and rs8040855 C>G was associated with the low expression of CSPG4P12.Chapter 2:The expression level of pseudogene CSPG4P12 in NSCLC tissues was significantly lower than that in adjacent normal tissues（P<0.05）.CCK-8 assay and clone formation assay showed that CSPG4P12 inhibited the growth and proliferation of A549 cells（P<0.01）.Transwell assay,wound healing assay and cell-matrix adhesion assay showed that CSPG4P12 inhibited the migration,invasion and adhesion of A549 cells（P<0.01）.Fluorescence staining assay and transmission electron microscopic showed CSPG4P12 promoted cell apoptosis of A549 cells（P<0.01）.And the overexpression of CSPG4P12 resulted in mitochondrial cristae disappeared and vacuolated under transmission electron microscopic.Western blot and immunofluorescence showed that CSPG4P12 induced the expression of p53 and Bax proteins and inhibited Bcl2 protein（P<0.01）.Conclusions CSPG4P12 may affect the malignant phenotypes of NSCLC by p53/Bcl2/Bax mitochondrial apoptosis pathway and the genetic variants of CSPG4P12contributed to the susceptibility to NSCLC.Figure 10;Table 11;Reference 188...