The Correlation Between CMTM6 And STUB1 In Regulating PD-L1 Expression In Gastric Cancer

Objective:To investigate the expression of programmed cell death 1 ligand 1(PD-L1),CKLF-like MARVEL Transmembrane Domain Containing 6(CMTM6)and STIP1homology and U-Box Containing Protein(STUB1)in gastric cancer(GC)tissues.(STUB1)in gastric cancer(GC)tissues,and analyze whether PD-L1,CMTM6 and STUB1 expression can be used as independent risk factors for GC prognosis,and verify The potential value of CMTM6 and STUB1 as tumor molecular markers in GC was verified.To elucidate the effects of CMTM6 and STUB1 on PD-L1 expression in GC cells and their potential mechanisms of action.Methods:(1)Differential expression of CMTM6,STUB1,PD-L1 and correlation with tumor-associated immune cells,MSI and TMB in GC patients were analyzed by R language and online database TIMER.(2)Clinicopathological data of 61 eligible GC patients were collected,and the expression of PD-L1,STUB1 and CMTM6 in GC tissues as well as paraneoplastic tissues was detected by immunohistochemical staining method(EnVision).Statistical analysis was performed to analyze the relationship between the expression of three proteins,PD-L1,STUB1 and CMTM6,and the clinicopathological characteristics of GC,as well as the correlation between the three proteins,and to analyze whether CMTM6,STUB1and PD-L1 were independent risk factors for GC prognosis.(3)Gastric normal cell line GES-1 and GC cell lines MKN-45 and SGC-7901 were cultured.RT-qPCR and WB method were used to detect the expression of CMTM6,STUB1 and PD-L1 in GC cell lines and gastric normal cell lines.GC cell lines SGC-7901 and MKN-45 were transfected with lentiviral CMTM6 silencing vector(sh-CMTM6 group),and SGC-7901 and MKN-45 cells transfected with negative control lentivirus were used as control group(NC group),and the expression of CMTM6 and PD-L1 mRNA and protein in control and experimental groups were detected by RT-qPCR and WB method,respectively.The STUB1 transient GC cell line(pcDNA-STUB1group)was constructed on CMTM6 lentivirus stable transfection strain with plasmid as vector and plasmid empty vector as control group(NC group),and STUB1,PD-L1 mRNA and protein expression in control and experimental groups were detected by RT-qPCR and WB method.The effects of CMTM6 and STUB1 on GC cell proliferation were investigated by in vitro CCK-8 assay.Results:(1)Bioinformatics analysis showed that the expression levels of CMTM6 and PD-L1mRNA in gastric cancer tissues were significantly higher than those in normal gastric tissues,while the expression levels of STUB1 mRNA in gastric cancer tissues were significantly lowe r than those in normal gastric tissues(P<0.05).There was a positive correlation between the mRNA expression of CMTM6 and PD-L1 in gastric cancer,and a negative correlation between the mRNA expression of STUB1 and PD-L1 in gastric cancer(P<0.05).TIMER database was used to analyze the correlation between the expressionof CMTM6,PD-L1,STUB1 and four k inds of infiltrating immune cells(B cells,macrophages,CD4~+T cells,CD8~+T cells)in gastric cancer.The results showed that the expression of CMTM6 was not correlated with B cells(r=-0.088,P<0.001),CD4~+T cells(r=0.106,P<0.001),CD8~+T cells(r=0.122,P<0.001)and macro phages(r=0.477,P<0.001)significant correlation.The expression of STUB1 was not correlat ed with B cells(r=-0.018,P<0.001),macrophages(r=0.014,P<0.001),CD4~+T cells(r=0.065,P<0.001),CD8~+T cells(r=0.119,P<0.001).The expression of PD-L1 was significantly correlate d with B cells(r=-0.287,P<0.001),CD8~+T cells(r=0.443,P<0.001),but not with CD4~+T cell s(r=0.004,P<0.001)and macrophages(r=0.076,P<0.001).Based on the analysis of the correl ation between CMTM6,STUB1,PD-L1 and MSI in GC,it is concluded that the expression of CMTM6 and PD-L1 in GC was related to the MSI(P<0.05),the expression of STUB1 w as not related to MSI(P>0.05).Bioinformatics showed that the expression of CMTM6 in G C was associated with both MSI and TMB(P<0.05).(2)Immunohistochemical staining showe d that the positive expression of PD-L1,CMTM6 and STUB1 in GC was 78.69%(48/61),77.05%(47/61)and 45.90%(28/61),respectively,and in normal gastric tissues the positive expr ession of PD-L1,CMTM6 and STUB1 was 32.79%(20/61),32.79%(20/61),and 77.05%(47/61).During the progression from normal gastric tissue to GC,the expression of PD-L1 and CMTM6 tended to increase,while the opposite was true for STUB1 protein(P<0.05).The exp ression of CMTM6 protein in GC tissue was significantly correlated with the patient’s age,de gree of infiltration,vascular invasion,and microsatellite status(P<0.05);The expression of ST UB1 protein in GC tissue was not correlated with any of the clinical features(P>0.05);PD-L1protein expression in GC tissues was significantly correlated with patients’age,tumor diamete r,degree of infiltration,lymph node metastasis,tumor-associated immune cells and microsatel lite status(P<0.05).Spearman correlation analysis of the expression of CMTM6,STUB1,and PD-L1 in GC tissues concluded that the expression of PD-L1 and CMTM6 were positively co rrelated(r=0.348,P=0.006);STUB1 was negatively correlated with the expression of PD-L1(r=-0.255,P=0.048).Kaplan-Meier method results showed that patients with high CMTM6 ex pression,high PD-L1expression,and low STUB1 expression in GC were associated with shor ter OS(P<0.05).(3)Cellular experiments showed that the expression of CMTM6,PD-L1 m R NA and protein in SGC-7901 and MKN-45 was significantly higher than that of GES-1(P<0.001);The expression of STUB1 mRNA and protein in SGC-7901 and MKN-45 was significan tly lower than that of GES-1(P<0.001).After knockdown of the target gene CMTM6,the expr ession of PD-L1 mRNA and protein in sh-CMTM6 group in both SGC-7901 and MKN-45 was significantly lower compared with NC group(P<0.001);After upregulation of STUB1,sh-CMTM6+pcDNA-STUB1 group PD-L1 in SGC-7901 and MKN-45 mRNA and protein ex pression were significantly reduced in sh-CMTM6 group compared to NC group(P<0.001).C CK-8 results showed that cell proliferation ability was significantly reduced in sh-CMTM6 gr oup compared to NC group(P<0.001);Cell proliferation ability was significantly reduced in s h-CMTM6+pcDNA-STUB1 group compared to sh-CMTM6 group(P<0.001).Conclusion:The combined detection of PD-L1,CMTM6,and STUB1 will provide a useful reference for the prognosis and immunotherapy of GC.