KIAA0101 knockdown inhibits cell proliferation and induces cell cycle arrest and cell apoptosis in chronic lymphocytic leukemia cells

Abstract

Background: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with intense cytogenetic aberrations. Importantly, our recent report indicated that thyroid hormone receptor interactor 13 (TRIP13) is a potential new therapeutic target in CLL. In this study, we predicted 20 TRIP13-related genes and found that KIAA0101 is a novel gene that regulates cell proliferation and the cell cycle of CLL cells.

Methods: CD19⁺ B cells were isolated from the peripheral blood of 26 CLL patients and 6 healthy donors through magnetic cell sorting. Cell proliferation was assessed by the CCK-8 assay. The mRNA and protein levels of genes were examined through RT-qPCR and western blot assays, respectively. Cell cycle and cell apoptosis were measured through Annexin V-based flow cytometry and the caspase 3/7 activity assay. Potential targets of KIAA0101 were identified through microarray analysis. 20 TRIP13 related genes was predicted by Ingenuity Pathway Analysis (IPA). KIAA0101-regulated functions and molecular pathways were predicted through IPA.

Results: KIAA0101 knockdown had the strongest inhibitory effect on CLL cell proliferation among the 20 TRIP13-related genes. KIAA0101 was highly expressed in CD19⁺ B cells of CLL patients. KIAA0101 knockdown induced cell cycle arrest and cell apoptosis, and inhibited FOXO1, MYD88, and TLR4 expression in CLL cells.

Conclusions: Taken together, we demonstrated that KIAA0101 plays a critical role in cell proliferation and the cell cycle of human CLL cells. KIAA0101 knockdown induced cell apoptosis, and reduced FOXO1, MYD88, and TLR4 expression, and may therefore be used as a therapeutic target of CLL.

Keywords: Chronic lymphocytic leukemia (CLL); KIAA0101; apoptosis; cycle; proliferation.

Figure 1

Effects of knockdown of 20 TRIP13-related genes on Granta-519 cell proliferation. (A) The mRNA abundances of 20 TRIP13-related genes predicted by the IPA database in Granta-519 cells were examined through RT-qPCR. The expression levels of all genes were normalized to SAPCD2. (B,C,D,E,F) Granta-519 cells were infected with lentiviruses targeting the above-mentioned 20 genes or control lentiviruses (shctrl). The above-mentioned 20 genes were divided into 5 groups (i.e., chromosome maintenance (B), DNA repair (C), cell division (D), macrophage polarization (E), and others (F) according to their molecular functions. Cell proliferative ability was assessed through the CCK-8 assay at the indicated time points after infection. ^nsP>0.05, *P<0.05, **P<0.01, ***P<0.001. CLL, chronic lymphocytic leukemia; BTK, Bruton’s tyrosine kinase; TRIP13, thyroid hormone receptor interactor 13; IPA, Ingenuity Pathway Analysis; RT-qPCR, real-time quantitative PCR; MACS, magnetic cell sorting; NC, negative control; CCK-8, Cell Counting Kit-8; aRNA, amplified RNA; MYD88, myeloid differentiation factor 88; TLR, toll-like receptor; FOXO, Forkhead box O.

Figure 2

KIAA0101 expression was markedly up-regulated in CD19⁺ B cells isolated from the peripheral blood of CLL patients. The mRNA expression level of KIAA0101 was examined through RT-qPCR in CD19+ B cells isolated from the peripheral blood of 6 healthy people and 26 CLL patients. *P<0.05. CLL, chronic lymphocytic leukemia; RT-qPCR, real-time quantitative PCR.

Figure 3

Potential functions and molecular mechanism investigations of KIAA0101. (A) Heatmap of differentially expressed genes (|fold change| >1.5, P<0.05) in Granta-519 cells infected with shKIAA0101 lentiviruses versus cells infected with shctrl lentiviruses (green: down-regulated; red: up-regulated). shKIAA0101 and shctrl groups contained 3 replicates. Genes and samples were displayed in rows and columns, respectively. (B) The top 15 disease and molecular function terms significantly enriched by differentially expressed genes. (C) The top 20 molecular functional pathways significantly enriched by differentially expressed genes.

Figure 4

Knockdown of KIAA0101 inhibited cell proliferation and induced cell cycle arrest in Granta-519 and JVM-2 cells. (A,B,C,D,E,F) Granta-519 and JVM-2 cells were infected with shctrl or shKIAA0101 lentiviruses. (A,B) On day 3 after infection, mRNA and protein levels of KIAA0101 were measured through RT-qPCR and western blot assays, respectively. (C,D) At the indicated time points (1, 2, 3, 4, or 5 days) after infection, cell proliferative activity was detected by the CCK-8 assay. (E,F) On day 3 after infection, cell distribution patterns in different phases of the cell cycle were measured by flow cytometry after PI staining. *P<0.05, **P<0.01, ***P<0.001. RT-qPCR, real-time quantitative PCR; PI, propidium iodide.

Figure 5

KIAA0101 knockdown induced cell apoptosis in Granta-519 and JVM-2 cells. (A,B,C,D,E,F) Granta-519 and JVM-2 cells were infected with shctrl or shKIAA0101 lentiviruses. (A,C) On day 3 post lentivirus infection, relative caspase 3/7 activity was determined by ELISA. (B,D) On day 3 post lentivirus infection, Bcl-2 protein level was detected by western blot assay. (E,F) On day 3 post lentivirus infection, cell apoptotic rate was assessed by flow cytometry. ***P<0.001.

Figure 6

KIAA0101 knockdown triggered the notable down-regulation of FOXO1, MYD88, and TLR4 protein levels in Granta-519 cells. (A) The interaction networks of KIAA0101 (PCLAF) and some KIAA0101 downstream genes of interest (BAX, FOXO1, CCND1, BRCA1, CDKN1A, CAST, CDK6, MYD88, TCF4, TLR4, and P53) were established using the Ingenuity Pathway Analysis (IPA) database. (B) The effects of KIAA0101 knockdown on the expression of FOXO1, MYD88, TCF4, and TLR4 were examined through western blot assays on day 3 after lentivirus infection in Granta-519 cells infected with shctrl or shKIAA0101 lentiviruses.