The Role Of LncRNA-ANRIL In Regulating Proliferation And Apoptosis Of Adult T-cell Leukemia Cells And Its Molecular Mechanism

Adult T-cell leukemia is a kind of malignant lymphoid tumors caused by Human T-cell leukemia virus type 1.The molecular mechanism about its pathogenesis is not clearly known yet.Study of the mechanism of ATL,especially the mechanism of malignant proliferation of leukemia cells,has become the hot topic of this field.More and more studies have shown that a new class of tumor regulatory factor long noncoding RNA plays an important role in the process of tumor growth,proliferation and apoptosis.Long noncoding RNA is RNA that could not be translated to protein,and its transcript length is longer than 200 nucleotides.ANRIL is a kind of long noncoding RNA,and has been widely reported that it play critical role in liver cancer,gastric cancer,esophageal cancer and others.However,its expression and function in ATL are not clear,which needs further investigation.In order to verify whether ANRIL plays an important role in ATL,we investigated in three levels:cell,molecule and in vivo.The main contents of this study are shown as follows:Part ?:ANRIL is overexpressed in ATL cellsFirstly,the level of ANRIL mRNA was detected by RT-PCR in 7 kinds of ATL cell lines MT-1,MT-2,MT-4,C8166,ATL-2,ATL-T,TL-Oml and a HTLV-1 negative cell Jurkat.The results showed that the ANRIL gene was highly expressed in ATL.We further confirmed the high expression of ANRIL in ATL cells using qRT-PCR.Part ?:ANRIL induces proliferation of ATL cellsIn order to study the effect of ANRIL on the malignant proliferation of ATL and its molecular mechanism,our research used lentivirus mediated gene silencing technique to knock down the expression of ANRIL.Firstly,we constructed the lentiviral vector of shANRIL,and using HEK-293FT cells to package the control lentivirus and the recombinant lentivirus,then infected ATL-T and ED cells respectively.Positive cells were screened by puromyein.After confirming ANRIL silence by qRT-PCR,we use MTT and clone formation assay to study cell proliferation.The result shown that knock down of ANRIL inhibit the proliferation of control group ATL-T and ED cells.Moreover,the ANRIL knocking-down cells was apoptosis.In addition,we also detected the cell cycle,and the results showed that ED cells were arrested in G1 phase after ANRIL silencing.Part ?:The mechanism of ANRIL silencing inhibits tumor cell growth and promotes cell apoptosisIn order to explore the molecular mechanism of ATL eell apoptosis after ANRIL silencing,we used western blot to analysis the expression of Caspase proteins.After silencing the expression of ANRIL,our research found the cleavage of Caspase-3,-7,-9 and PARP in ATL cells.The expression of apoptosis-associated proteins AIF were increased.The proliferation related genes such as E2F1,C-Myc was reduced,KLF2 was up-regulated.The NF-?B pathway in ATL cells is continuously activated.To investigate whether ANRIL is involved in the regulation of NF-?B signaling pathway,the effect of ANRIL on NF-?B signaling pathway was detected.The results showed that ANRIL alone did not promote the activation of NF-?B signaling pathway,but ANRIL could significantly activate the Tax-mediated NF-?B signaling pathway.RelA(p65)is a key component of the classical NF-?B signaling pathway.Our research farther studied whether ANRIL could also promote the p65-induced classical NF-?B signaling pathway activation.As showed in the results,ANRIL and EZH2 could enhance the activation of NF-?B signaling pathway by p65 respectively.Compared with other groups,the cells expressed ANRIL,EZH2 and p65 showed more significant activation of the NF-?B signaling pathway.It indicates that ANRIL,EZH2 and Tax(or p65)synergistically activated the NF-?B signaling pathway.These results suggest that ANRIL silencing can inhibit the growth of tumor cells and promote cell apoptosis.These effects are associated with the expression of apoptosis related proteins and regulation of NF-?B signaling pathway.Part ?:Mouse tumorigenesis experimentIn order to further confirm the role of ANRIL in vivo,our study used ANRIL silenced ED cells shANRIL#1 ED and control group ED cells subcutaneous injected into SCID mouse.Three weeks later,the mouse were killed.The results showed the tumor size and weight are smaller in shANRIL#I ED group than the control group,indicating that the tumor formation ability was significantly reduced after ANRIL silencing.The expression of E2F1 KLF2 and C-Myc were down-regulated in ANRIL knock-out mice.In conclusion,our studies confirmed that ANRIL was highly expressed in ATL,and demonstrated ANRIL promotes the proliferation of ATL cells,and inhibit cell apoptosis.In vivo experiments further demonstrated that ANRIL can promote the proliferation of ATL cells.This study provides a basis for the mechanism of adult T cell leukemia,providing a new target for the treatment of ATL,and providing a new idea and strategy for the clinical treatment of adult T-cell leukemia.