Study On The Mechanism Of PD-L1 Regulating The Proliferation And Metastasis Of Colorectal Carcinoma

Background: Colorectal carcinoma(CRC)is one of malignant tumor threatened human health.It is a common malignant tumor of digestive system and it is the third morbidity of malignant tumor in the western developed countries in the long run.In China,with the progress of society,the change of living environment and living habits,the incidence rate is increasing,and it has the characteristics of younger development.Currently the main treatments include surgical resection of the primary tumor,chemotherapy,combined with postoperative radiotherapy,immunotherapy in recent years.In spite of cetuximab as the representative molecular-targeted drugs in clinical application,the overall survival rate of patients with CRC has improved significantly,but the prognosis of some patients is still just passable,the transfer rate and the recurrence rate is still high.With the intensive study of the pathogenesis about CRC,there are a number of molecules have been shown to participate in the occurrence and development of CRC.Immune checkpoints including PD-L1,B7-H3,B7-H4,involved in tumor immune escape,are closely related with degree of malignancy and poor prognosis,can abnormal expression in a variety of human cancer cells and immune cells in tumor microenvironment.Among them,PD-L1/PD-1 pathway plays an important role in tumorigenesis and development.A number of studies have shown that PD-L1 has abnormal expression in many malignant tumor tissues,which is closely related to tumor immune tolerance,immune escape and prognosis.But there is a small amount of reports about the relationship between PD-L1 and biological behavior and prognosis of colorectal cancinoma existing consistent phenomenon.Some reported that PD-L1 expression has relationship with clinical pathological characteristics and prognosis in CRC.Some other reports showed that PD-L1 expression has no correlation with clinicopathologic factors and prognosis of patients,or the expression of PD-L1 had better prognosis.Therefore,in CRC,the relationship between PD-L1 and the biological behavior and its significance as a molecular marker to judge prognosis remains controversial.So it is necessary to further study on the expression of PD-L1 in CRC,and the biological effects of PD-L1 on proliferation,invasiveness and the transfer capability of CRC.The mutation of proto oncogene K-ras is closely related with the occurrence and development of CRC.K-ras gene mutation is an early event in carcinogenesis,it can directly activate PI3K/AKT/m TOR signaling pathway without rely on the upstream EGFR.PI3K/AKT/m TOR signaling pathway also play an important role in colorectal carcinogenesis.Some scholars have found that PI3K/AKT/m TOR signaling pathway involved in the regulation of PD-L1 mediated immune escape in pancreatic cancer.D ’Incecco A pointed out that the expression of PD-L1 has relationship with EGFR mutation in lung adenocarcinoma.But in CRC,the relationship between the expression of PD-L1 and K-ras gene mutation and its regulation mechanism is unclear.We conjectured that PD-L1 has the interaction between K-ras and PI3K/AKT/m TOR pathway.Therefore,further study on the expression of PD-L1 and K-ras gene mutation and the association with PI3K/AKT/m TOR pathway may aid the selection of immunotherapy drugs for CRC patients.To sum up,this study focused on the relationship between PD-L1 and its possible mechanism of interaction with K-ras gene mutation and PI3K/AKT/m TOR pathway in CRC.Objective: 1,To detect the expression of PD-L1 in CRC tissues,and analyse the relationship between the PD-L1 expression and clinicopathological characteristics of CRC.2,To analyse the effect of PD-L1 on the ability of colorectal cancer cell lines by inhibiting PD-L1 with sh RNA,and the killing effect on T cell.3,To understand the relationship between PD-L1 and K-ras gene mutation status and PI3K/AKT/m TOR signal pathway,to explore the impact of PD-L1 and related regulatory network in CRC.Methods:1,First of all,a total of 250 cancer tissues of CRC patients were collected from the First Affiliated Hospital of Nanchang University.All the patients were not received chemotherapy,radiotherapy,biological therapy,and had no history of other malignant tumor before surgery.Secondly,the expression of PD-L1 was detected by immunohistochemical method.Finally,the clinical and pathological data including patient age,gender,tumor size,gross type,location,degree of differentiation,histological type,lymph node metastasis,liver metastasis,lung metastasis,prognosis were collected.Appliing statistical methods to analyze the relationgship bwtween the expression of PD-L1 and its clinical significance.2,Through the construction of PD-L1 gene sh RNA lentiviral infection system,silencing PD-L1 gene expression in colorectal cancer cell lines,and then detect cell function through flow cytometry,crossed experiment,invasion experiment,CCK8 cell activity detection and tumor killing experiments,to verify the effect of low expression of PD-L1 on the proliferation,invasion and metastasis of colorectal cancer cells and the killing effect of T cells.3,Through the detection of K-ras gene mutation in colorectal cancer cells and tissues,understand the relationship between the expression of PD-L1 and K-ras gene mutation status.At the same time,using the RT-PCR and Western blot for the detection of PI3K/AKT/m TOR pathway molecules Akt,m TOR,S6K1 m RNA and protein expression levels in cells that had PD-L1 gene silencing,to understand the relationship between PD-L1 and K-ras gene mutation status and PI3K/AKT/m TOR signal pathway,to explore the impact of the expression of PD-L1 and related regulatory network in CRC.Results 1,The results of immunohistochemistry showed that the positive expression rate of PD-L1 was 44%.There has high expression status of PD-L1 in colorectal cancer tissues.The expression level of PD-L1 in cases with lymph node metastasis was significantly higher than that without lymph node metastasis(52.2% vs 39.2%),the difference was statistically significant(P<0.05).In cases with liver metastasis,the expression level of PD-L1 was significantly higher than that in patients without liver metastasis(55% vs 41.9%),the difference was statistically significant(P< 0.05).Follow-up data showed that 5 year survival rate of CRC was 60%(150/250),the average survival time was 55.4 months,median survival time was 48 months(range 1-84 months survival).5 years survival rate of PD-L1 positive group was 47.3%(52/110),the average survival time was 45.2 months,median survival time was 42 months.5 years survival rate of PD-L1 negative group was 70%(98/140),the average survival time was 57 months,the median survival time was 55 months.Kaplan-Meier curve and Log-rank test showed that the survival time of PD-L1 positive patients was significantly short than that of PD-L1 negative,statistically significant difference(P < 0.05).Cox proportional hazard model showed that,PD-L1 expression is related to survival.In addition,univariate analysis also found that there has relationship between the degree of tumor differentiation,lymph node metastasis and survival.Multivariate survival analysis showed that PD-L1 expression is related to survival.In addition,multivariate analysis also showed that lymph node metastasis is correlated with prognosis.2,Compared with empty lentivirus group and control group,cell apoptosis significantly increased after PD-L1 gene silencing,and the percentage of cells in G1 phase increased significantly.Transwell invasion assay and crossed experiment showed that the low expression of PD-L1 can inhibit the invasion and migration.The results of CCK8 showed that the low expression of PD-L1 can inhibit cell proliferation ability.3,Among the 250 cases,89 cases were mutant type of K-ras gene,the mutation rate was 35.6%,the major mutation sites were located in codon 12.In cases with lymph node metastasis,liver metastasis and lung metastasis,the mutation rate of K-ras gene was significantly higher than that without lymph node metastasis,without liver metastasis or without lung metastasis,the difference was statistically significant(P<0.05).The positive rate of PD-L1 in patients with K-ras gene mutation was significantly higher than that in wild-type,the difference was statistically significant(P<0.05).There had Akt,m TOR,S6K1 m RNA and protein expression in cells with K-ras wild type and mutated type.The expression of Akt,m TOR,S6K1 m RNA and protein are higher in K-ras mutated type cells than that in wild type.After silencing of PD-L1,The expression of Akt、m TOR、S6K1 m RNA and protein were decreased,but compared to wild type,the decreasing expression are more obvious.in mutated type.Conclusion: 1,PD-L1 had high expression in CRC.PD-L1 was significantly correlated with lymph node metastasis,liver metastasis.The patients with high expression of PD-L1 had worse prognosis.PD-L1 can be used as an independent indicator of judging prognosis of CRC.2,Low expression of PD-L1 can effectively inhibit the malignant biological behavior of colorectal cancer cells,and can inhibit the proliferation,invasion and migration ability of colorectal cancer cells in vitro,and promote apoptosis of cancer cells.3,The K-ras mutation rate was 35.6%,the major mutation site was located in codon 12.CRC patients with K-ras mutations were more likely to develop lymph node metastasis,liver metastasis,and lung metastasis.4,The expression of PD-L1 was closely related to the mutation of K-ras gene,and the status of K-ras gene may affect the expression of PD-L1.5,In CRC,PD-L1 affect the activation status of PI3K/AKT/m TOR pathway to some extent,suggesting that PD-L1 may be involved in the regulation of PI3K/ AKT/m TOR signaling pathway and was related to the mutation of K-ras gene.