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. 2024 Jan 15;16(1):342-355.
doi: 10.62347/TXKA6586. eCollection 2024.

LncRNA AL645608.3 mediates malignant progression of acute myeloid leukemia

Jin-Hua Yan  1 , Kai-Qiong Liao  2 , Ling Yao  3 , Jian-Lan Chen  2 , Li-Fang Xiong  2 , Xu-Zhang Tao  4
Affiliations

LncRNA AL645608.3 mediates malignant progression of acute myeloid leukemia

Jin-Hua Yan et al. Am J Transl Res. .

Abstract

Objective: To investigate the role of lncRNA AL645608.3 in the malignant progression of acute myeloid leukemia (AML) cells and explore relevant molecular mechanisms.

Methods: The expression level of AL645608.3 was measured in AML cell lines (THP-1, HL-60, KG-1, and AML-193) via real-time quantitative polymerase chain reaction (RT-qPCR). Small hairpin RNA (shRNA) and open reading frame of AL645608.3 were cloned into lentiviral vectors and were infected into THP-1 and AML-193 cells. The expression of casitas B-lineage lymphoma (CBL), interferon regulatory factor 6 (IRF6), and interferon beta 1 (IFNB1) was detected through RT-qPCR, and western blot. Co-immunoprecipitation (Co-IP) on IRF6 was conducted. Matrix metalloprotease-9 (MMP-9) activity was evaluated via gelatin zymography assay.

Results: LncRNA AL645608.3 was expressed in the four AML cell lines (THP-1, HL-60, KG-1, and AML-193). Silencing AL645608.3 mitigated the expression of IRF6 and IFNB1 but elevated the expression of CBL in THP-1 cells. Oppositely, AL645608.3 overexpression up-regulated the expression of IRF6 and IFNB1 but decreased the expression of CBL in AML-193 cells. Co-IP results proved that AL645608.3 could directly mediate IRF6 activity in THP-1 and AML-193 cells. MMP-9 activity was decreased by AL645608.3 knockdown and was improved by AL645608.3 overexpression in AML-193 cells.

Conclusion: AL645608.3 is expressed in different AML cell lines, and mediates the expression of CBL, IRF6, IFNB1, and MMP-9. These findings might deepen our comprehension of the molecular mechanisms underlying AML.

Keywords: AL645608.3; CBL; IFNB1; IRF6; MMP-9; acute myeloid leukemia; lncRNA.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
The expression of lncRNA AL645608.3 in various AML cells. A. CCK-8 for detecting the cell viability of THP-1, HL-60, KG-1, and AML-193 cell lines. B. RT-qPCR for measuring the expression of AL645608.3 in the above cell lines. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.
Figure 2
Figure 2
Construction of lentiviral vectors for silencing AL645608.3 in THP-1 cells. A. Design of shRNAs of AL645608.3. B, C. Cellular morphology and fluorescence intensity of THP-1 cells under light microscope or immunofluorescence microscope. Magnification, 200×. D. RT-qPCR for evaluation of AL645608.3 expression in THP-1 cells with infection of pLVX-shRNA2 lentiviral vectors. ns, P>0.05; ***P<0.001; ****P<0.0001.
Figure 3
Figure 3
Construction of lentiviral vectors for overexpressing AL645608.3 in AML-193 cells. A. Design of the open reading frame of AL645608.3. B, C. Cellular morphology and fluorescence intensity of AML-193 cells under light microscope or immunofluorescence microscope. Magnification, 200×. D. RT-qPCR for evaluation of AL645608.3 expression in AML-193 cells with infection of pLVX-IRES-ZcGreen1 lentiviral vectors. ns, P>0.05; ****P<0.0001.
Figure 4
Figure 4
Expression of IRF6 and CBL in diverse AML cells. A. RT-qPCR for detection of the mRNA level of IRF6 in THP-1, HL-60, KG-1, and AML-193 cell lines. B. RT-qPCR for measuring the mRNA level of CBL in the above AML cells. C-E. Western blot of the protein levels of IRF6 and CBL in the above AML cells. ns, P>0.05; **P<0.01; ***P<0.001; ****P<0.0001.
Figure 5
Figure 5
Effects of AL645608.3 on the mRNA expression of IRF6, CBL and IFNB1 in AML cells. A-C. The mRNA expression of IRF6, CBL and IFNB1 in THP-1 cells infected with sh-AL645608.3 lentiviruses. D-F. The mRNA expression of IRF6, CBL and IFNB1 in AML-193 cells infected with AL645608.3 overexpression lentiviruses. ns, P>0.05; ****P<0.0001.
Figure 6
Figure 6
Effects of AL645608.3 on the protein expression of IRF6, CBL and IFNB1 in AML cells. A. Representative western blot images for IRF6, CBL and IFNB1 in THP-1 cells with sh-AL645608.3 lentiviruses infection and in AML-193 cells with AL645608.3 overexpression lentiviruses infection. B-D. Western blot for the detection of protein levels of IRF6, CBL and IFNB1 in THP-1 cells infected with sh-AL645608.3 lentiviruses. E-G. Western blot for the detection of protein levels of IRF6, CBL and IFNB1 in AML-193 cells infected with AL645608.3 overexpression lentiviruses. ns, P>0.05; **P<0.01; ***P<0.001; ****P<0.0001.
Figure 7
Figure 7
AL645608.3 directly enhanced IRF6 expression in AML cells. A. Co-IP for IRF6 in THP-1 cells with infection of sh-AL645608.3 lentiviruses as well as AML-193 cells with infection of AL645608.3 overexpression lentiviruses. B-E. Quantification of IRF6 and ubiquitin expression in above THP-1 and AML-193 cells. ns, P>0.05; **P<0.01; ***P<0.001; ****P<0.0001.
Figure 8
Figure 8
MMP-9 was activated in AML cells with AL645608.3 overexpression. A, B. Gelatin zymography assay for MMP-9 in THP-1, HL-60, KG-1, and AML-193 cell lines. C-E. Gelatin zymography assay for MMP-9 in THP-1 cells with infection of sh-AL645608.3 lentiviruses and AML-193 cells with infection of AL645608.3 overexpression lentiviruses. ns, P>0.05; ****P<0.0001.
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