A Study Of The Role And Molecular Mechanism Of Long Non-coding RNA HITT In Regulation Of Anti-tumor Immunity

Programmed Death Ligand 1(PD-L1)is an important immune checkpoint molecule with widely attention.PD-L1 is abnormally high expressed in various tumors and PD-L1 binds to PD-1 on the surface of activated T cells,transmits inhibitory signals to T cells,promoting immune evasion of tumor cells.Blocking PD-1 and PD-L1 signals is the most representative new type of immune checkpoint blocked(ICB)treatment method.This method has been approved for clinical treatment of melanoma,non-small cell lung cancer,Hodge’s lymphoma,head and neck cancer and other tumors,and has achieved significant clinical therapeutic effects.However,clinical research found that only 10-30 % of tumor patients benefited from this therapy,and the expression level of PD-L1 in the tumor microenvironment was related to the reactivity of PD-L1 antibody treatment.Therefore,fully revealing the expression regulation mechanism of PD-L1 not only helps to predict the efficacy of ICB,but also provides clues for the establishment of new ICB treatment methods.Studies have found that abnormal expression of long non-coding RNA(lncRNA)is related to tumors.Recent evidence shows that lncRNA is widely involved in the interaction between tumor cells and the immune microenvironment.Therefore,lncRNA may have important targeting or therapeutic value for improving the effect of tumor immunotherapy.Our laboratory has identified a new lncRNA gene named HITT(HIF-1 α Inhibitor at translation level),through analysis gene expression profiles in various tumor tissues,it is found that HITT is significantly downregulated in various tumor tissues.Therefore,HITT may be related to the occurrence and development of tumors.Based on this,this article deeply explores the anti-tumor function of HITT,and finds that the growth of breast cancer in mice is significantly inhibited after the 4T1 tumor cells HITT overexpression.After receiving anti-CD8 antibody treatment,the tumor growth significantly increases in mice.At the same time,it is found that this treatment significantly inhibits the HITT mediated anti-tumor effect,indicating that the inhibition of tumor cell growth by HITT mainly relies on CD8+T cells.In addition,the co-culture experiment between tumor cells and T cells finds that overexpression of HITT in tumor cells can promote CD8+T cell activity in the co-culture system and increase CD8+T killing effect on tumor cells.In order to further explore how HITT participates in the regulation of anti-tumor T cell immunity,proteomics data after HITT knockout are obtained and analyzed.The results show that PD-L1 is significantly increased after HITT is down regulated,suggesting that PD-L1 may be a potential regulatory target of HITT in promoting anti-tumor T cell immunity.Indeed,after adding anti-PD-1 antibodies to the co-culture system between tumor cells and T cells,or knocking down the expression of PD-L1 in tumor cells,CD8+T cell killing activity against tumor cells mediated by HITT is disappeared.Overexpression of PD-L1 can reverse the increased CD8+T cell activity mediated by HITT overexpression.The above results indicate that HITT promotes antitumor T cell immunity by regulating PD-L1.On this basis,this article further explores the molecular mechanism of HITT inhibitory PD-L1 expression.The results indicate that HITT does not affect the m RNA and protein stability of PD-L1.However,HITT inhibits PD-L1 translation in a PD-L15,UTR dependent manner.Through GO database screening and subsequent experimental verification,it is found that HITT collaborates with RGS2 to regulate PD-L1 translation.Further molecular mechanism studies find that HITT can form binary ternary complexes with RGS2,PD-L1-5,UTR.HITT binds to PD-L1-5,UTR 97-105 nt regions through its1134-1142 nt region;HITT binds to the C(80-212 aa)terminal F194,Q196,and D197 key sites of RGS2 through its 1080-1130 nt interval;The 28-36 nt interval of PD-L1-5,UTR binds to the C(80-212 aa)terminal K175,R176,and S179 key sites of RGS2.The construction of multiple mutants has demonstrated that the formation of this ternary complex is crucial for HITT and RGS2 mediated PD-L1 inhibitory effects.The study finds that overexpression mutant HITT,which lost its regulatory function on PD-L1 in breast cancer tumor cells,has a significantly lower ability to inhibit tumor growth than full-length HITT.The above results indicate that HITT inhibits the growth of breast cancer through PD-L1.Subsequently,the potential therapeutic effect of HITT functional fragments is studied.It is found that intratumor injection of HITT functional fragments packaged with lentivirus can significantly inhibit tumor growth and promote the therapeutic effect of anti-PD-1 antibody.In addition,this article further explores the regulation of HITT on tumor immunotherapy sensitivity and the impact of mice prognosis and survival.The results show that overexpression of HITT in breast cancer tumor cells can significantly increase the activation of tumor infiltrating CD8+T cells,prolong the life cycle of mice,improve the survival rate of breast cancer mice.Thus,HITT promotes the therapeutic effect of anti-PD-1 antibody on breast cancer in mice.In conclusion,this article reveals that HITT can inhibit tumor growth by inhibiting the translation of PD-L1 and promoting the activity of anti-tumor T cell immunity,thus providing new ideas and clues for clinical treatment of anti-PD-1/PD-L1 antibodies.