Inhibition of BPHL inhibits proliferation in lung carcinoma cell lines

Abstract

Background: Lung cancer is one of the most common human malignant tumors and the leading cause of cancer death worldwide. Biphenyl hydrolase-like (BPHL) is a gene encoding the human BPHL enzyme, a serine hydrolase that catalyzes the hydrolytic activation of amino acid ester prodrugs of nucleoside analogs such as valacyclovir and valganciclovir. However, the role of BPHL in lung cancer is still unknown.

Methods: In this study, we assessed the effect BPHL knockdown on the proliferation, apoptosis, colony formation, metastasis, and cell cycle of cancer cells. BPHL knockdown NCI-H1299 and A549 cells demonstrated decreased proliferation, as measured by Celigo cell counting. The MTT assay results were consistent with Celigo cell counting. Caspase 3/7 activity increased significantly in the NCI-H1299 and A549 cells after shBPHL knockdown. Decreased colony formation in the NCI-H1299 and A54 cells after shBPHL knockdown, as measured by crystal violet staining. Transmigration assay using a Transwell demonstrated that there were significantly fewer migrating cells in the lower chamber in the BPHL knockdown NCI-H1299 and A549 cells. Cell cycle analysis by Propidium Iodide (PI) staining and fluorescence activated cell sorter (FACS). We also explored the effect of BPHL knockdown on tumor growth in a mouse model of tumor implantation in nude mice.

Results: We found that the knockdown of BPHL gene expression by short hairpin RNA (shRNA) leads to a decrease in proliferation, colony formation, and metastasis and an increase in apoptosis in two lung adenocarcinoma (LUAD) cell lines in vitro. BPHL knockdown induces decreased tumor growth, colony formation, and metastasis; increased apoptosis; and altered cell cycle destruction. BPHL knockdown results in decreased tumor growth in vivo. Moreover, BPHL knockdown A549 cells demonstrated slower growth compared to control cells upon implantation in nude mice, confirming the in vitro findings.

Conclusions: In this study, the data indicate that BPHL potentially promotes proliferation, inhibits apoptosis, and increases colony formation and metastasis in lung cancer. Overall, our study suggests that BPHL may be a gene that promotes tumor growth in lung cancer.

Keywords: Biphenyl hydrolase-like (BPHL); inhibits proliferation; lung carcinoma cell lines.

Figure 1

Bioinformatics analysis of TCGA database to identify BPHL as a disease-associated gene. (A) The TMM was used for data standardization, and statistical analysis of the paired samples was conducted. The BCV was observed for quality control, and then normal samples (tissues adjacent to cancer) and cancer samples could be clearly separated at Dim1. (B) Log2 was used (Cancer/Normal) for statistical analysis of the multiple paired samples, and the filtering criteria was set as ≥1 or ≤−1 to estimate the dispersion. Genes with a statistical test P value less than 0.05 are considered as differentially expressed genes that meet the null hypothesis (marked in red in the figure); differentially expressed genes that do not meet the null hypothesis are marked as black dots. (C) The differential expression of the original BPHL gene data in each TCGA RNA-seq sample was expressed by a line chart. The vertical axis is the original expression data of each sample, and the horizontal axis is the cancer and adjacent tissues. Each line indicates the data of one sample, and the trend of the line shows the gene changes in all samples. FC, fold change; CPM, counts per million; BPHL, biphenyl hydrolase-like; TCGA, The Cancer Genome Atlas; TMM, Trimmed Mean of M-values; BCV, biological coefficient of variation.

Figure 2

Screening human lung carcinoma cell lines expressing BPHL. (A) Three common human lung carcinoma cell lines, A549, NCI-H1975, and NCI-H1299, were chosen to assess their BPHL gene expression using qPCR. GAPDH was used as reference gene. (B) NCI-H1299 and A549 cells were transduced with GV115-RNAi-BPHL lentivirus (shBPHL) and the control lentivirus (shCtrl). At 72 hours post-transduction, the cells displayed good viability. The transduction efficiency had reached 80%, as measured by the percentage of fluorescence-positive cells. (C) mRNA levels were measured by qPCR to confirm the efficiency of the knockdown. (D) Western blot demonstrated that there was a significant decrease in BPHL at the protein level compared to the controls. **, P<0.01 compared with shCtrl and shBPHL lentivirus treatment group. BPHL, biphenyl hydrolase-like; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; STDEV, standard deviation; qPCR, quantitative polymerase chain reaction.

Figure 3

BPHL knockdown leads to decreased tumor growth, colony formation, and metastasis, increased apoptosis, and altered cell cycle distribution. (A) BPHL knockdown NCI-H1299 cells demonstrated decreased proliferation, as measured by Celigo cell counting. (B) A549 of BPHL knockdown demonstrated decreased proliferation, as measured by Celigo cell counting. (C) The MTT assay results were consistent with Celigo cell counting. (D) Caspase 3/7 activity increased significantly in the NCI-H1299 and A549 cells after shBPHL knockdown. (E) Decreased colony formation in the NCI-H1299 and A549 cells after shBPHL knockdown, as measured by crystal violet staining. (F) Transmigration assay using a Transwell demonstrated that there were significantly fewer migrating cells in the lower chamber in the BPHL knockdown NCI-H1299 and A549 cells (Giemsa stain, ×200). (G) Cell cycle analysis by PI staining and FACS. **, P<0.01 compared with shCtrl and shBPHL lentivirus treatment group. BPHL, biphenyl hydrolase-like; PI, propidium iodide; FACS, fluorescence activated cell sorter; OD, optical density.

Figure 4

The results of tumor growth in vivo. (A) Gross appearance of mice that received inoculation of BPHL knockdown cells and control cells. (B) Gross appearance of tumors from BPHL knockdown cells and control cells. (C) Sizes of tumors from BPHL knockdown cells and control cells. (D) Weights of tumors from BPHL knockdown cells and control cells. NC, mice that received inoculation of BPHL control cells; KD, mice that received inoculation of BPHL knockdown cells. BPHL, biphenyl hydrolase-like.