To Investigate The Biological Role Of LCP2 In Lung Adenocarcinoma And Its Correlation With PD-L1 Expression

Objective: LCP2 has been identified to be ectopically expressed in a variety of tumor cells with prognostic value.However,the clinical significance and role of LCP2 in lung adenocarcinoma(LUAD)remains unclear.Studying the prognostic role of LCP2 in LUAD and exploring its biological function in lung adenocarcinoma cells provides the experimental basis for improving the survival rate of lung adenocarcinoma patients and screening immune-related prognostic markers.By examining the correlation between LCP2 and PD-L1 expression in LUAD and the role of LCP2 on PD-L1 expression in inflammatory pathways,the study provides a reference for the function of LCP2 in LUAD and the regulation mechanism of PD-L1.A potential research direction for optimizing the application of immune checkpoint antibody drugs in LUAD is provided.Methods: 1)The RNA-seq data of LCP2 gene in pan-carcinoma was used to analyze the expression of LCP2 m RNA in LUAD in different types of tumors by using the TIMER database,the expression of LCP2 m RNA in LUAD was analyzed by using the GEPIA database,and the protein expression level of LCP2 in LUAD and control tissues was evaluated by immunohistochemical staining data in human protein atlas.The pathological specimens of68 patients with lung adenocarcinoma were collected,and the expression of LCP2 was detected by IHC.According to the staining score,LPC2 was divided into low and high expression groups to explore the relationship between LCP2 expression difference and different clinical characteristics.Kaplan-Meier survival analysis was used to analyze the differences in overall survival(OS)between the two groups,and survival curves were drawn.RNA-seq data were extracted from the TCGA database for survival analysis and verification.The m RNA with positive correlation with LCP2 expression was screened out using the c Bio Protal database,and the genes with positive and negative correlation were analyzed by Linked Omics heat map in LUAD.The correlation between LCP2 and related immune checkpoint genes in LUAD was analyzed by the TIMER database.GO and KEGG analysis was used to explore the possible signaling pathway mediated by LCP2 coregulatory genes.The relative abundance of immune-infiltrating lymphocytes in lung adenocarcinoma samples was reconstructed using the deconvolution method.The expression differences and correlation between high and low LCP2 expression groups in various immune cell subtypes were evaluated.2)Cell lines with relatively high LCP2 expression were screened by Western-blot,lentivirus transfection and screening were used to construct LCP2-overexpressed H1299 stable cell line,The effects of LCP2 on the proliferation of LUAD cells were determined by CCK-8 assay and clonal formation assay.The effect of LCP2 on the invasion ability of lung adenocarcinoma cells was detected by Transwell invasion assay,the effect of LCP2 on the migration ability of lung adenocarcinoma cells was evaluated by wound healing assay,and the apoptosis protein was detected by flow cytometry to evaluate the apoptosis ability of LCP2 on lung adenocarcinoma cells.3)The expression relationship between LCP2 and PD-L1 in LUAD was analyzed based on TCGA database.The expression of LCP2 and PD-L1 was detected by IHC in 68 cases of lung adenocarcinoma.LCP2 overexpressed lung adenocarcinoma cell lines were constructed by lentivirus transfection experiment and transfection efficiency verification.q-PCR was used to detect the effect of LCP2 overexpression on PD-L1 m RNA in lung adenocarcinoma cell lines,and Western-blot was used to detect the effect of LCP2 overexpression on PD-L1 protein in lung adenocarcinoma cell lines.Flow cytometry was used to verify the induction effect of different concentrations of IFN-γ on the expression of PD-L1 on the surface of lung cancer cells and a co-culture system of human peripheral blood T lymphocytes and lung adenocarcinoma cells was established.Twelve cytokines including IL-6,IFN-γ,IFN-α,IL-2,IL-1β,TNF-α,IL-10,IL-5,IL-4,IL-17,IL-8 and IL-12P70 in cell supernatant were detected by flow fluorescence method.To observe the effects of LCP2 on IFN-γ and other cytokines released by T lymphocytes.Flow cytometry was used to detect the effect of LCP2 overexpressing lung adenocarcinoma cells co-culture with lymphocytes on the expression of PD-L1.Results: 1)Compared with normal tissues,LCP2 expression was up-regulated in 7 cancers and down-regulated in 5 cancers,respectively.The expression level of LCP2 m RNA in the 483 LUAD groups was significantly lower than that in the 347 normal controls,and the difference was statistically significant(P<0.05).Moreover,the protein expression level of LCP2 in LUAD was lower than that in the normal control group.The immunohistochemical method was used to detect LCP2 protein in 68 cases of lung adenocarcinoma.There were 22 high expression cases,accounting for 32.35%,and 46 cases of low expression,accounting for 67.65%.Expression of LCP2 in patients with lung adenocarcinoma was associated with clinical stage and lymph node metastasis(P<0.05).Kaplan-meier curve analysis showed that the OS of lung adenocarcinoma patients with high LCP2 expression was significantly higher than that of lung adenocarcinoma patients with low LCP2 expression,and the difference was statistically significant(P<0.05).Gene analysis showed that LCP2 was positively correlated with TCR signaling molecules,CD28-B7 signaling molecules,and multiple immune checkpoint molecules,including PD-L1 in patients with lung adenocarcinoma.GO analysis found that LCP2 may be associated with a large number of immune function enrichment,and KEGG analysis suggested that LCP2 was associated with CAMs Signaling,JAK/STAT Signaling,T Cell Receptor Signaling,and other inflammatory immune Signaling pathways.In lung adenocarcinoma,there were significant differences in the expression levels of various immune cell infiltrates and LCP2,and the expression of LCP2 was correlated with the abundance of T lymphocyte-dominated immune cell infiltrates.2)CCK-8 method showed that overexpression of LCP2 in H1299 cells significantly reduced cell growth rate compared with the control WT group and empty vector control GFP group(P<0.05).Clonogenesis experiments showed that LCP2 overexpressed in H1299 cells significantly reduced the number of clonogenesis compared with the WT group and empty vector control GFP group(P<0.05).Transwell invasion assay showed that compared with the control WT group and empty vector control GFP group,the number of cell lines with LCP2 overexpression was significantly reduced(P<0.05).Wound-healing experiments showed that the LCP2 overexpression group had a significantly lower cell convergence rate compared with the WT control group and empty vector control GFP group(P<0.05).Apoptosis protein detection showed that there was no significant difference in apoptosis rate among all groups.3)In lung adenocarcinoma,LCP2 ranked fourth among all molecules positively correlated with PD-L1 m RNA content(r=0.66,P < 0.05).Immunohistochemical results showed that LCP2 was positively correlated with PD-L1 protein level in lung adenocarcinoma patients(r=0.58,P<0.05).The m RNA and protein expression levels of PD-L1 in lung adenocarcinoma cells overexpressing LCP2 showed no statistical difference compared with WT and GFP groups.IFN-γ induced the expression of PD-L1 in lung adenocarcinoma cells,and the expression of PD-L1 increased with the increase of IFN-γ concentration.When lung adenocarcinoma cells were co-cultured with lymphocytes,IFN-γ,IL-2,IL-8,and TNF-α were increased after overexpression of LCP2 compared with the control group,and the expression rate of PD-L1 in lung adenocarcinoma cells was significantly increased(P<0.05).Conclusion: LCP2 is a potential prognostic marker of lung adenocarcinoma,which is involved in multiple immune signaling pathways and is associated with immune cell infiltration.Overexpression of LCP2 can inhibit the proliferation,invasion,and migration of lung adenocarcinoma cells.The expression level of LCP2 in lung adenocarcinoma is closely related to PD-L1,and overexpression of LCP2 can induce the expression of PD-L1 by promoting the release of cytokines such as IFN-γ by lymphocytes.