Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR‐424‐3p and targeting PTEN/PI3K/Akt pathway

Abstract Lung cancer is the leading cause of death in individuals with malignant disease. Non‐small‐cell lung cancer (NSCLC) is the most common type of lung cancer, and chemotherapy drugs such as cisplatin are the most widely used treatment for this disease. Baicalein is a purified flavonoid compound that has been reported to inhibit cancer cell growth and metastasis and increase sensitization to chemotherapeutic drugs via different pathways. Therefore, we assessed the effects of baicalein on the proliferation, apoptosis and cisplatin sensitivity in the NSCLC A549 and H460 cell lines and determined the pathways through which baicalein exerts its effects. Baicalein was slightly toxic to normal human bronchial NHBE cells but inhibited growth, induced apoptosis and increased cisplatin sensitivity in A549 and H460 cells. Baicalein down‐regulated miR‐424‐3p, up‐regulated PTEN expression and down‐regulated expression of PI3K and p‐Akt in A549 and H460 cells. Dual‐luciferase reporter assay demonstrated that PTEN is a target gene of miR‐424‐3p, and overexpression of miR‐424‐3p or silencing of PTEN partially attenuated the effects of baicalein on A549 and H460 cells. Taken together, we concluded that baicalein inhibits cell growth and increases cisplatin sensitivity to A549 and H460 cells via down‐regulation of miR‐424‐3p and targeting the PTEN/PI3K/Akt pathway.

studies. 8,9 In addition to lung cancer, baicalein also inhibits the growth and metastasis of prostate cancer cells, 10 the invasion of gastric cancer cells, 11 the migration, adhesion and invasion of breast cancer cells, 12 and induces apoptosis and autophagy in hepatocellular carcinoma cells. 13,14 In addition, some studies have demonstrated the effects of baicalein on cisplatin sensitivity via different pathways in various cancer cells. [15][16][17] Baicalein has also exhibited a wide range of anti-inflammatory effects associated with airway injury, liver injury and rheumatoid arthritis. [18][19][20] In summary, baicalein has the potential to become an ideal adjuvant therapy in the treatment of cancer.
Previous studies of baicalein have identified several pathways, such as the ROS/AMPK pathway in lung cancer, 21 the caveolin-1/Akt/mTOR pathway in prostate cancer, 10 the p38 signalling pathway, 11 the PTEN/ Akt/HIF-1a signalling pathway 22 in gastric cancer, the NF-jB signalling pathway in ovarian cancer, 23 the Wnt/b-catenin pathway in breast cancer, 24 the PI3K/Akt pathway in oesophageal squamous cell carcinoma 25 and the ERK pathway in hepatocellular carcinoma. 26 Phosphatase and tensin homolog (PTEN) is known as a tumour suppressor gene that has been reported to affect cancer cell behaviour in various cancers. [27][28][29][30] PTEN may inhibit cellular proliferation, growth and survival through the PI3K/Akt/mTOR pathway. 31,32 There is also evidence that baicalein may exert its effects via the PTEN/Akt pathway. 22,33 MicroRNAs (miRNAs) are small non-coding RNAs with lengths of [19][20][21][22][23][24][25] nucleotides that regulate the translation or degradation of target mRNA in the human body. 34 MiRNAs play an important role in the proliferation, invasion and apoptosis of malignant tumour cells and may affect resistance to cisplatin. [35][36][37] In addition, pathways that link PTEN and miRNAs exist in cancers. [38][39][40][41] In this study, we assessed the effects of baicalein on proliferation, apoptosis and cisplatin sensitivity in NSCLC A549 and H460 cell lines. We also determined changes in miRNA expression caused by baicalein treatment using the miRNA microarray and validated the hypothesis that baicalein affects the miRNA-PTEN/PI3K/Akt pathway. Our study indicated that baicalein may inhibit cell growth and increase cisplatin sensitivity to A549 and H460 cells by downregulating miR-424-3p and targeting the PTEN/PI3K/Akt pathway.

| Cells and reagents
Non-small-cell lung cancer A549 and H460 cell lines and the normal human bronchial epithelial (NHBE) cell line were purchased from the

| Clone formation assay
Soft agar clone formation assay was used to measure the colonyforming ability of cells. Following high-pressure steam sterilization, 1.2% and 0.7% agarose gels (Low melting gel; Solarbio, Beijing, China) were put into a water bath (55°C) for preparation. Agarose (1.2%) plus an equal volume of 29 DMEM (20% FBS + 2% penicillin-streptomycin) was added to the lower layer of a 6-cm dish and allowed to solidify at room temperature. Following transfection, cells were dissociated and suspended in 37°C DMEM plus 20% FBS at a density of 5 9 10 4 /mL. Next, 100 lL of cell suspension (5000 cells) was suspended into a 1:1 solution of 0.7% agarose and 2 9 DMEM (3 mL total) in the upper layer of growth agar. Different concentrations of baicalein (0, 40 lmol/L) were added into the upper layer of growth agar, and cells were incubated for 14 days. After incubation, cells were fixed with a combination of 10% methanol and 10% acetic acid, and then stained with 1% crystal violet (Solarbio, Beijing, China). The number of colonies containing more than 50 cells was determined using an optical microscope (Olympus, Tokyo, Japan).

| Cell apoptosis assays
The Annexin V-FITC/PI Apoptosis Detection Kit (Solarbio, Beijing, China) was used to detect apoptotic cells. Cells were collected 24 hours after treatment, washed with PBS (Solarbio, Beijing, China) and resuspended in binding buffer (500 lL) in an Eppendorf (EP) tube. Annexin V-FITC and PI (5 lL) were added into binding buffer.
After mixing, the EP tube was kept away from light for 5-15 minutes at room temperature. Flow cytometry (BD, San Diego, CA, USA) was used to identify cells of normal status, early apoptosis, late apoptosis and death; FITC was detected using channel FL1, and PI was detected using channel FL3.

| Statistical analysis
Each experiment was repeated three times. Statistical analysis was performed with SPSS 21 software, and data were expressed as mean AE standard deviation (S.D.). One-way ANOVA was carried out to compare three or more groups; Student's t test was used to compare two independent groups. The IC50 of cisplatin was calculated using the normal probability conversion method and probit regression analysis. A P-value of <.05 was considered statistically significant.

F I G U R E 3 Baicalein down-regulates miR-424-3p expression in A549 and H460 cells. (A) A549 cells treated with DMSO or 40 lmol/L
baicalein for 24 h were collected for RNA extraction and miRNAs microarray analysis. Hierarchical clustering was performed to display the differentially expressed miRNAs with a fold change of ≥2.0 and a P-value of ≤.05. (B) qRT-PCR was performed to validate the differential expression of miR-424-3p in NSCLC A549 and H460 cells. (C) qRT-PCR was performed to validate the differential expression of miR-377-3p in NSCLC A549 and H460 cells. (D) qRT-PCR was performed to validate the differential expression of miR-1224-5p in NSCLC A549 and H460 cells. *P < .05 3 | RESULTS

| Baicalein inhibits cell proliferation, promotes apoptosis and increases cisplatin sensitivity in A549 and H460 cells via up-regulation of PTEN and suppression of the PI3K/Akt pathway
To evaluate the antiproliferative effects of baicalein, A549 and H460 cells were treated with 0 or 40 lmol/L baicalein for up to 72 hours.

| Baicalein down-regulates miR-424-3p in A549 and H460 cells
To determine the molecular mechanisms behind the effects of baicalein in NSCLC cells, we performed miRNA microarray analysis in A549 cells treated with different concentrations of baicalein (0, and 40 lmol/L) for 24 hours. As shown in Figure 3A and Table 1, 33 miRNAs were differentially expressed, with a greater than twofold change in cells treated with 40 lmol/L baicalein as compared to DMSO controls (P < .05). Of the 33 differentially expressed miRNAs, 24 were down-regulated and 9 were up-regulated. Interestingly, the microarray results indicated that miR-424-3p was significantly downregulated, with a greater than fivefold change.
To validate the data obtained from the miRNA microarray, we examined three of the differentially expressed miRNAs in NSCLC  Figure 3B, C), and miR-1224-5p was up-regulated ( Figure 3D) in A549 and H460 cells with baicalein treatment at different concentrations, which is consistent with the results from the miRNA microarrays. Thus, we confirmed that the microarray data were reliable and that miR-424-3p was down-regulated by baicalein treatment.

424-3p
We suggested that miR-424-3p may mediate the effects of baicalein on the PTEN pathway according to the above results. Thus, we pre-

| DISCUSSION
Baicalein is one of major flavonoids found in Scutellaria baicalensis.
Previous studies have indicated that baicalein inhibits cell growth   Baicalein has potential as an adjuvant therapy in NSCLC.

This work was supported by a grant named School-Hospital Joint
Fostering Fund of Zhengzhou University (23230016) from the First Affiliated Hospital of Zhengzhou University.

CONFLI CT OF INTEREST
The authors confirm that there is no conflict of interest.

AUTHORS' CONTRIBUTI ONS
GJZ and CYL designed the study; CYL, HQW, SSC and HL carried out part of experiments; and CYL wrote the manuscript and performed the statistical analysis. All authors have approved the final manuscript.