Long Noncoding RNA (lncRNA) Small Nucleolar RNA Host Gene 5 (SNHG5) Regulates Proliferation, Differentiation, and Apoptosis of K562 Cells in Chronic Myeliod Leukemia

Abstract

BACKGROUND Human chronic myelogenous leukemia (CML) is a hematopoietic stem cell disorder with high malignant and invasive activity. lncRNA SNHG5 has been reported to be upregulated in CML. However, whether it affects the proliferation, differentiation, and apoptosis in CML cells is still unknown. This study investigated the role of SNHG5 in CML and revealed the potential mechanism. MATERIAL AND METHODS K562 cells were transfected with shRNA, and the expression level of SNHG5 was assessd by quantitative RT-PCR. The proliferation ability was determined by CCK-8 assay. Western blot analysis was performed to detect protein expressions related to cell proliferation, differentiation, and apoptosis. Cell apoptosis rate was analyzed by flow cytometry. The DNA methylation level was determined by methylation-specific PCR (MSP). RESULTS Our results show that inhibition of SNHG5 induced by RNA interference significantly inhibits K562 cells proliferation and induces cell differentiation with the increased expression of CD42b, CD11b, CD14, GATA-1, and ß-globin. Flow cytometry analysis indicated that inhibition of SNHG5 significantly induced cell apoptosis with decreased expression of Bcl-2 and increased expression of Bax and cleaved capase-3. Additionally, Western blot and MSP analyses confirmed that inhibition of SNHG5 increased the expression of DR4 gene through suppressing its methylation. CONCLUSIONS Inhibition of SNHG5 suppressed K562 cell proliferation through inducing the differentiation and apoptosis by inhibiting methylation of DR4. Therefore, downregulated SNHG5 could play a key role in CML progression, and might provide a new strategy for the treatment of CML.

Figure 1

K562 cells were transfected with SNHG5-shRNA. The interference efficiencies in K562 cells were detected by RT-PCR. Data are expressed as the mean± SD of at least 3 experiments. * p<0.05, **p<0.01 vs. control.

Figure 2

Inhibition of SNHG5 significantly suppressed K562 cell growth. Cell proliferation ability of K562 cells was analyzed by CCK-8 assay at 24 h, 48 h, and 72 h (A). Western blot analysis was performed to examine the protein expression of cyclin E1 (B). Western blot analysis was performed to examine the protein expression cyclin-dependent kinase (CDK2) and p27 (C). Data are expressed as the mean ±SD of at least 3 experiments. * p<0.05, ** p<0.01, *** p<0.001 vs. control.

Figure 3

Inhibition of SNHG5 induced the differentiation of K562 cells. Western blot analysis was performed to detect the protein expression of CD42b, CD11b, CD14 (A), GATA-1, and β-globin (B). Data are expressed as the mean ±SD of at least 3 experiments. ** p<0.01, *** p<0.001 vs. control.

Figure 4

Inhibition of SNHG5 induced cell apoptosis as shown by flow cytometry analysis (A) and a quantification of cell apoptosis proportion was generated (B). Data are expressed as the mean ±SD of at least 3 experiments. *** p<0.001 vs. control.

Figure 5

Inhibition of SNHG5 significantly upregulated the expression of Bax and cleaved caspase-3, but downregulated the expression of Bcl-2 compared to the control in K562 cells. Data are expressed as the mean ±SD of at least 3 experiments. ** p<0.01, *** p<0.001 vs. control.

Figure 6

Inhibition of SNHG5 changed the expression and methylation of DR4. The protein expression of DR4 was determined by Western blot (A) and the mRNA level of DR4 gene was analyzed by qRT-PCR (B). The methylation of DR4 CpG sites was analyzed by methylation-specific PCR (MSP) analysis in K562 cells (C). Data are expressed as the mean ±SD of at least 3 experiments. *** p<0.001 vs. control.