The Role Of PTEN In Immune Escape Of Mouse Melanoma And Its Molecular Mechanism

Objective:Malignant Melanoma?MM?,a highly malignant tumor that frequently occurs in the skin,is also commonly found in oral mucosa in East Asia,particularly China.MM has two major characteristics:high metastasis rate and poor prognosis.Metastasis in its early stage can be transmitted through blood and lymphatic metastasis,whose incidence has been sharply increasing recently.With ongoing rapid progression in Tumor Molecular Immunology,scientists gradually recognize that the occurrence and development of melanoma are closely related to the immune function of patients.For melanoma patients,complex and heterogeneous tumor micro-environment establishes inside the body,resulting in rapid reproduction of the tumor cells.Investigating the immune escape mechanism of melanoma could therefore provide valuable implications for clinical immunotherapy,prognosis judgement,and curative effect evaluation of patients.Phosphatase and Tension Homolog Deleted on Chromosome 10?PTEN?is the first tumor suppressor gene ever found to have bispecific phosphatase activity.PTEN-encoded protein is a lipid phosphatase activity protein that participates in regulating many crucial signaling pathways in cells through the process of phosphatase,for instance PI3K/AKT signaling pathway,and inhibites the growth,invasion,and metastasis of tumors through a variety of ways,promoting the apoptosis of tumors.In this study,the PTEN gene of mouse melanoma cell line B16F10 was knocked out by the CRISPR/Cas9?Clustered Regulatory Interspace D Short Palindromic Repeats CRISPR-Associated Proteins 9?system.To observe and compare the tumor size of the experimental and controlled groups,?1?a mouse melanoma model with PTEN gene knockout and?2?a mouse melanoma model without PTEN gene knockout were initiated,respectively,along with the relative number of Cytotoxic T Lymphocyte?CTL?and Natural Killer cell?NK?in the spleen of the two groups.The research primarily aims at exploring the specific pathway of immune escape of PTEN-inactivated melanoma and revealing the role of PTEN-inactivated melanoma in immune escape.Methods:1 Experimental materials1.1 Mouse melanoma high metastatic cells?B16F10?:purchased in Zhongqiao Xinzhou Company.1.2 C57BL\6 mice:from the Animal Center of Hebei Medical University.?Certificate no.11400700331927?2 Experimental methods2.1 The high-metastasis cell lines of mouse melanoma with PTEN gene knockout were obtained by CRISPR/Cas9 system.Immunohistochemistry?IHC?and Western Blot?WB?were used to detect the expression of PTEN protein and identify the highly metastatic melanoma cell lines of mice knocked out of PTEN gene.2.2 Flow CytoMetry?FCM?was used to detect the relative number of CTL cells and NK cells in spleen cells of mice in(PTEN^+/+-B16F10)group,(PTEN^-/--B16F10)group and PBS group.Results:1.DNA sequencing results of(PTEN^-/--B16F10)cells showed that double knockout was due to the unmatched sequence of gRNA binding sites,single point deletion and single peak map.Immunohistochemical and Western Blot results showed that PTEN protein was not expressed in(PTEN^-/--B16F10)cells.2.Western Blot results showed that PTEN protein was not expressed in tumor tissues of mice in(PTEN^-/--B16F10)group.3.The tumor volume of mice in(PTEN^-/--B16F10)group and(PTEN^+/+-B16F10)group was measured.The results showed that(PTEN^-/--B16F10)group was larger than(PTEN^+/+-B16F10)group on the 7th,12th,17th and 22nd day after inoculation of tumor cells.There were significant differences in each time period.?P<0.05?.4.Flow CytoMetry?FCM?assay showed that the number of NK cells in spleen of mice in(PTEN^-/--B16F10)group,(PTEN^+/+-B16F10)group and PBS group showed that on the 7th,12th,17th and 22nd day after inoculation of tumor cells,there was no significant difference in each time period.?P>0.05?.5.Flow CytoMetry?FCM?assay showed that the number of CTL cells in spleen of mice in(PTEN^-/--B16F10)group,(PTEN^+/+-B16F10)group and PBS group showed that on the 7th day and on the 12 th day of tumor cell inoculation,the(PTEN^-/--B16F10)group was lower than(PTEN^+/+-B16F10)group,but there was no significant difference?P>0.05?and on the 17th day and 22nd day of tumor cell inoculation(PTEN^-/--B16F10)group was lower than(PTEN^+/+-B16F10)group,there was significant difference.?P<0.05?.Conclusion:1.The tumor-bearing model of melanoma in mice with PTEN gene deletion was successfully constructed.2.In melanoma of mice with PTEN gene deletion,the growth rate of tumors was significantly accelerated.3.In mouse melanoma,while the deletion of PTEN gene resulted in the decrease of CTL cells in spleen,the number of NK cells remained unchanged.4.In mouse melanoma,the deletion of PTEN gene resulted in the low immunity of mice,the mechanism of which was due to the decrease in the number of CTL cells.