Application of long-chain non-coding RNA LAMP5-AS1 to MLL-R leukemia
A RNALAMP5-AS1, long-chain non-coding technology, applied in the field of biomedicine, can solve the problems that the degradation of lncRNA and MLL fusion protein has not been reported, and achieve the effect of prolonging the life cycle
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Embodiment 1
[0038] Example 1 LAMP5-AS1 expression analysis and evaluation of its clinical value
[0039] The present invention discovers an lncRNA LAMP5-AS1 that is specifically highly expressed in MLL-R leukemia and lowly expressed in normal physiological conditions and other types of leukemia. In order to further confirm the expression specificity of LAMP5-AS1 in MLL-R leukemia, we re-collected a batch of bone marrow samples from the First Affiliated Hospital of Sun Yat-sen University for detection and analysis, including MLL wild-type (MLL-wt) leukemia samples163 cases, and 53 cases of MLL-R leukemia samples. All sample collection was approved by the Ethics Committee of Sun Yat-Sen University and informed consent was obtained from the patients. LAMP5-AS1 was specifically detected by extracting RNA and using qRT-PCR technology. The primer sequences of the qRT-PCR used are as follows:
[0040] Forward primer sequence: 5'-CACTGAACGGATCTCAAACC-3' (SEQ ID NO: 2);
[0041] Reverse primer...
Embodiment 2
[0043] Example 2 Functional identification of LAMP5-AS1 in MLL-R leukemia
[0044] In order to solve the core problem of LAMP5-AS1 participating in the regulation of MLL-R leukemia, this example intends to further study the function of LAMP5-AS1 on MLL-R leukemia. MLL-R leukemia cell lines, THP1 (MLL-AF9) and MV4-11 (MLL-AF4) were selected as the research objects. Through siRNA interference technology, two different siRNA sequences were designed for LAMP5-AS1.
[0045] Forward sequence of siRNA-1: 5'-CUGACAAAGUGCCGUCCAA dTdT-3' (SEQ ID NO: 4);
[0046] Reverse sequence of siRNA-1: 5'-UUGGACGGCACUUUGUCAG TdTd-3' (SEQ ID NO: 5);
[0047] Forward sequence of siRNA-2: 5'-GAGGCAAGACGAAGAAAGU dTdT-3' (SEQ ID NO: 6);
[0048] Reverse sequence of siRNA-2: 5'-ACUUUCUUCGUCUUGCCUC TdTd-3' (SEQ ID NO: 7).
[0049] In the above two MLL-R leukemia cell lines, LAMP5-AS1 was knocked out respectively, and the cell proliferation was detected by CCK-8 assay. Experimental results such as f...
Embodiment 3
[0059] Example 3 LAMP5-AS1 targets MLL fusion protein
[0060]Whether at the cellular level or in vitro mouse experiments, LAMP5-AS1 significantly affected the function of MLL fusion gene leukemia cells. So, can this lncRNA directly regulate the expression of MLL fusion protein and thus participate in this type of disease? To investigate this question, the expression levels of MLL fusion proteins were examined by inhibiting LAMP5-AS1 by RNA interference in THP1 and MV4-11 cells. The experimental results found that knocking down LAMP5-AS1 in the MLL cell line, the protein levels of MLL-AF9 and MLL-AF4 were significantly reduced, but the mRNA of the MLL fusion protein had no significant effect ( Figure 4 A and B), suggesting that LAMP5-AS1 probably plays a role by mediating the stability or degradation of MLL fusion protein. On the other hand, in the tumor samples of NOD-SCID mice subcutaneously inoculated with shRNA Molm13, we also found that knocking down LAMP5-AS1 signific...
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