CD81 inhibits the proliferation of acute megakaryoblastic leukemia cells

Cell culture

Human AMKL cell line UT-7 was purchased from Fuheng Cell Center, Shanghai, China. All cells were maintained in Roswell Park Memorial Institute 1,640’s medium (RPMI 1640, Fuheng Corporate, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, Inc., Logan, UT, USA) and 1% penicillin/streptomycin (Sangon Biotech Co., Ltd, Shanghai, China). Cells were incubated in a thermostatic incubator at 37 °C with 5% CO₂.

Screening of the best target sequence

The siRNA targeting CD81 was synthesized by Gencefe Corporate, Jiangsu, China. The siRNA-1 (CD81-human-498), siRNA-2 (CD81-human-168), and siRNA-3 (CD81-human-636) that target distinct regions of the CD81 mRNA, which helps rule out off-target effects and sequences, are shown in Table S1. UT-7 cells were transfected with siRNAs using Lipo8000 (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Real-time polymerase chain reaction (RT-PCR) analysis was conducted to evaluate the interfering efficiency. The sense sequence of the CD81 primer was 5′-TGACCCGCAGACCACCAAC-3′. The siRNA-2 sequence was chosen for further research.

Total RNA isolation and quantitative real-time polymerase chain reaction analysis

Total RNA from the AMKL cell line UT-7 was isolated using Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. A Smartspec Plus (Bio-Rad, Hercules, CA, USA) was used to investigate the quality and concentration of RNA, and 5 µL total RNA (concentration: 25%) was used to synthesize cDNA using a 1st strand cDNA synthesis kit (Aidlab Biotechnologies Co., Ltd., Beijing, China). SYBR green reagent (Aidlab, China) was used to perform qPCR analysis on an ABI7500. Relative mRNA expression level was quantified using the 2^−ΔΔCt method.

Construction of stable CD81 knockdown cell line

For stable knockdown of the CD81 gene, LV-shCD81 (a lentiviral vector encoding a short hairpin RNA (shRNA) specifically designed to knock down CD81 expression) lentiviral particles were purchased from Shanghai GenePharma, Shanghai, China. AMKL cells were infected with the indicated lentivirus. Stable cell lines were selected using puromycin (0.4 µg/mL, 72 h).

Cell proliferation assay

A Cell Counting Kit-8 (CCK8, CTCC, Jiangsu, China) was used to examine the proliferation ability of AMKL cells. Cells were seeded into a 96-well plate at a density of 5 × 10³ cells per well and cultured for 48 h. We then added 10 µL CCK8 reagents into each well and incubated them for 2 h at 37 °C. The Smartspec Plus (Bio-Rad, Hercules, CA, USA) was used to measure the absorbance at the wavelength of 450 nm.

Detection of cell cycle

When the cells were cultured to the logarithmic stage, the cell concentration was adjusted to 2×10⁵ cells/mL. Cells were seeded into a six-well plate at a density of one mL per well and cultured for 48 h. The cells were washed with phosphate-buffered saline (PBS) and 70% ethanol was added slowly to pre-washed cells while vortexing and fixed overnight at 4 °C. The cells were then washed with PBS again, RNaseA (100 µg/mL) was added, and they were incubated for 30 min at 37 °C. Propidium iodide (PI, 50 µg/mL) was added into the cells and incubated at 37 °C for 30 min without light. The cells were washed with PBS and the cell cycle was detected using a flow cytometer.

Detection of apoptosis

The cells were incubated for 48 h at 37 °C/5% CO₂. The AMKL cells were then washed with PBS. The cells were suspended in 1 ×binding buffer, stained with Annexin V-FITC and PI, and apoptosis was detected using a flow cytometer (the flow cytometry raw data were deposited in FlowRepository at FR-FCM-Z9ZQ.

Statistical analysis

Data were analyzed using GraphPad Prism. A paired t-test was performed to compare two samples. P-values <0.05 were considered statistically significant.

Construction of stable CD81 knockdown cell line

To screen the best target sequence, we transfected UT-7 cell line with three CD81 siRNA (siCD81). We compared the interfering efficiency of the control group (CK), siNC (a non-targeting siRNA used as a negative control), siRNA-1, siRNA-2, and siRNA-3. The expression of CD81 in UT-7 cells was detected by RT-PCR. As shown in Fig. 1, siRNA-2 demonstrated the best knockdown efficiency and its sequence was therefore selected for further research.

After 72 h of viral infection, the infection efficiency was greater than 80% when the multiplicity of infection (MOI) was 100 (Fig. 2). In this condition, cells with shCD81 significantly downregulated CD81 expression when compared with UT-7 cells (Fig. 3).

CD81 knockdown inhibits proliferation, inducts apoptosis, and decreases S phase cells of AMKL cell line UT-7

We investigated whether CD81 knockdown would inhibit the proliferation of AMKL cells. The AMKL cell line UT-7, including the CK, shRNA negative control (NC), and CD81-shRNA knockdown groups, was cultured and its proliferation capacity was assessed.

To further confirm the effects of CD81 expression on leukemic cell proliferation, UT-7 cells were transfected with shRNAs against CD81 and the knockdown efficiencies were validated by RT-PCR. As shown in Fig. 4, cells with reduced CD81 expression exhibited lower proliferation rates in CCK-8 assays.

Figure 5 shows that the knockdown of CD81 in UT-7 was associated with an induction of apoptosis detected by increased Annexin V. The P value was less than 0.0001 when comparing the shCD81 and NC groups.

Figure 6 shows that CD81 knockdown inhibited cell proliferation with decreased S phase cells. Inversely, the proportions of G1 and G2 phase cells were upregulated in shCD81 AMKL cells.

Collectively, knockdown of CD81 significantly suppressed proliferation rate of AMKL cells.