Lenti-shRNA-mediated FAM54A knockdown suppresses the proliferation and induces apoptosis of human Burkitt lymphoma cells CURRENT POSTED

Background Burkitt lymphoma is a kind of non-Hodgkin B-cell-derived malignancy, derived from germinal center B cells. FAM54A has been proved to be involved in various physiological and pathological processes of cancers, but the biological function of FAM54A in Burkitt lymphoma remains unclear. Thus, the aim of our research was to elucidate the roles of FAM54A in proliferation, apoptosis and cell cycle of Burkitt lymphoma. Methods Burkitt lymphoma cell line (Namalwa) was chosen to perform the following experiments. FAM54A-shRNA and negative control-shRNA lentivirus that were synthesized, respectively by Qiagen were used to transfect targeted cells in order to knockdown FAM54A or as negative control. Then, cell proliferation, cell cycle and cell apoptosis were detected by using MTS assay, propidium iodide staining and Annexin V-APC staining, respectively. Results Our results showed that high expression of FAM54A protein was found in Namalwa cell line. Furthermore, MTS analysis exhibited that knockdown of FAM54A obviously inhibited cell proliferation in Namalwa cells. What’s more, cell cycle analysis showed that knockdown of FAM54A induced Namalwa cell apoptosis and arrested cell cycle in G2/M phase. Conclusions These findings suggest that FAM54A is essential for Namalwa cell proliferation and may be a potential therapeutic target for the treatment of Burkitt lymphoma.

were designed based on FAM54A sequence (Accession no. NM_001099286). After evaluation and determination by a design software, the target sequence (5'-AATTGTGGAAATGCAGGAA-3') from the full-length sequence of FAM54A was selected as the most suitable interference target by Genechem company (Shanghai, China). After detecting of knockdown efficiency, the stem-loop oligonucleotides were synthesized and inserted into lentivirus-based GV115-GFP vectors with ageI/EcoRI sites (Genechem, shanghai, China). Lentivirus particles were prepared as a previous report [11].

Cells transfection
For lentivirus transfection, the target cells were cultured in a 6-well microplate, and then FAM54A-shRNA lentivirus and negative control (NC)-shRNA lentivirus was added according to a multiplicity of infection (MOI). After 72 hours of transfection, the cells were observed by a fluorescence microscope (NIB900, Olympus, Japan). After 120 hours of transfection, the cells were collected to detect the knockdown efficiency by WB.

Cell cycle analysis
Cell cycle distribution was analyzed using propidium iodide (PI) following the manufacturer's instructions as previously described [13]. Briefly, Namalwa cells that were transfected with FAM54A-shRNA or NC-shRNA lentivirus were collected, washed twice with ice-cold phosphate-buffered saline (PBS), fixed with ice-cold 70% ethanol at 4˚C for 1 h, and stained with PI (Genechem, Shanghai, China). Finally, cell cycle analysis was performed by flow cytometry. Each experiment was performed in triplicates.

Cell apoptosis detection
The apoptosis rate of cells was determined by Annexin V-APC stainning according to the kit protocols (Santacruz, USA). The Namalwa cells (6×10 5 cells/well) were plated into 6-well plate after infection with FAM54A-shRNA or NC-shRNA lentivirus. After 5 days of cultivation. They were washed, collected, and resuspended in PBS buffer and the cell concentration was adjusted to 1×10 6 /ml. Then, the 5 μl Annexin V (0.1 μg/μl) was added into 100 μl cell suspension and incubated at room temperature in dark for 30 min on ice. The cells given different treatment were subjected to Fluorescent-Activated Cell Sorting (FACS) analysis. All experiments were performed in triplicates.

Statistical analysis
The statistical analysis was performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). Student's t-test was used for raw data analysis. For all statistical tests, p-values less than or equal to 0.05 were considered to be statistical significance.

FAM54A protein was highly expressed in human BL cell line
The expression level of FAM54A protein in human BL cell line (Namalwa) and human renal epithelial cells (293T) were examined by WB. As depicted in Fig. 1, the expression of FAM54A at the protein level was significantly high in the Namalwa cells.

Determination of knockdown efficiency by WB
Knockdown efficiency was determined by detecting FAM54A protein expression level in infected 293T cells with FAM54A-shRNA or NC-shRNA lentivirus by WB. Compared to NC-shRNA lentivirus, the expression level of the protein in 293T cells transfected with FAM54A-shRNA lentivirus was greatly decreased, which indicated a efficient knockdown of the target gene (Fig. 2). GAPDH was used as a loading control.

Lentivirus-mediated knockdown of FAM54A in the human BL cell line Namalwa
To explore the function of FAM54A, we knocked down FAM54A in namalwa cell line. After 3 days of transfection, fluorescence imaging technology was applied to reflect the transfection results of cells.
Results showed that the percentage of infected cells was >50% for both the FAM54A-shRNA or NC-shRNA lentivirus (Fig. 3).

Knockdown of FAM54A inhibits human BL cell proliferation in vitro
In order to study the effect of FAM54A on cell growth, FAM54A-shRNA lentivirus was transfected into Namalwa cells to silence the gene. Then, cells expressing FAM54A-shRNA or NC-shRNA lentivirus were implanted into 96-well plates and analyzed by MTS for 5 days. The effect of FAM54A downregulation on the proliferation of Namalwa cells was detected by MTS assay. Compared to NC, the absorbance of FAM54A-shRNA-transfected Namalwa cells at 490 nm was significantly lower (Fig. 5), which meant that the speed of proliferation of FAM54A-shRNA lentivirus-transfected cells was slower than that of NC (p<0.05). Thus, the experimental data showed that the downregulation of FAM54A noticeably suppressed cell proliferation.

Knockdown of FAM54A in Namalwa cells increases cell apoptosis
In order to detect whether the expression of FAM54A protein affects the apoptosis of BL cells, we knock out FAM54A in Namalwa cells. Annexin V staining and flow cytometry were applied to check apoptosis rate (Fig. 6A). As shown in Figure 6B, detailed data showed that apoptosis rate of FAM54A-shRNA lentivirus-transfected cells was significantly higher than that of NC (NC 2.97±0.02% vs. FAM54A-shRNA 4.93 ±0.07%, p < 0.001). These results implied that the lack of the FAM54A protein expression is a critical factor of cell apoptosis in Namalwa cells.

Knockdown of FAM54A renders cell cycle arrest in human BL cell in vitro
In order to determine the detailed mechanism that FAM54A knocdown led to cell proliferation inhibition, cell cycle distribution of FAM54A-shRNA or NC-shRNA lentivirus-transfected cells was analyzed by flow cytometry (Fig. 7A)

Discussion
In the world, BL is one of the most common causes of cancer-related death, because it is difficult to be perceived in early stage and the disease has developed to late stage when it is diagnosed accurately, so the best treatment time may not be utilized [14]. Therefore, early diagnosis and targeted therapy are key to better prognosis of patients with BL. Additionally, FAM54A, aslo named as Mitochondrial fission regulator 2 (MTFR2) or DUF729 domain-containing protein 1 (DUFD1) plays an important role in regulating the structure and function of mitochondria. FAM54A protein is also related to membrane-enriched subcellular fractions, including mitochondria. It has been proven that inhibition of Dufd1 expression in testicular germ cell lines can seriously impair oxygen consumption, suggesting that the gene is necessary for mitochondrial respiration [15]. Therefore, the downregulation of First of all, we measured the expression level of FAM54A protein in Human BL cell line (Namalwa) and the results exhibited that expression level of FAM54A protein was very high in cancer cell line. So, we speculated that it probably exerted a role to promote cell growth in BL and acted as an oncogene.
Next, we transfected the Namalwa cells using FAM54A-shRNA lentivirus, which significantly downregulated the endogenous expression level of FAM54A protein. Then, the cell proliferation viability was detected by MTS assay. Our results showed that knockdown of FAM54A obviously leaded to a inhibition in cell proliferation in BL. Furthermore, results of cell cycle and apoptosis analysis disclosed that downregulation of FAM54A negatively regulated BL progression by inducing apoptosis and blocking cell cycle in Namalwa cells.

Conclusions
Although, detailed reference literatures on FAM54A are very limited because few studies are carried out to explore it carefully, our present study states that FAM54A protein is expressed highly in human BL cell line. Knockdown of FAM54A suppresses cell proliferation by increasing cell cycle arrest and inducing cell apoptosis. These findings not only elucidate the effects of FAM54A on BL, but also can provide a novel strategy and idea used for improvement of prognosis of BL and promote the development of early diagnosis and targeted therapy of human BL. However, the detailed molecular mechanism involved in effects of FAM54A on BL still needs further to be further investigated.

List Of Abbreviations
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request

Competing interests
The authors declare that they have no competing interests  The expression level of FAM54A protein in 293T cells and Namalwa cells. WB was used to analyze FAM54A protein expression level in both cell lines. GAPDH was used as a loading control.