Piperlongumine inhibits the growth of non-small cell lung cancer cells via the miR-34b-3p/TGFBR1 pathway

Background Non-small cell lung cancer is a common type of lung cancer. Piperlongumine (PL), which is extracted from the roots of piperaceae plant, long pepper, and peppercorn, is an alkaloid amide that inhibits tumor growth and metastasis. However, whether it affects lung cancer cells remains unclear. Methods We assessed the effects of PL on the proliferation and apoptosis of A549 and H1299 NSCLC cell lines. Results PL was mildly toxic to normal human bronchial epithelial cells and significantly suppressed growth and facilitated apoptosis of A549 and H1299 cells. It also upregulated microRNA (miR)-34b-3p and downregulated the transforming growth factor beta type I receptor (TGFBR1). The dual-luciferase reporter assay showed that TGFBR1 is a target gene of miR-34b-3p. Silencing of miR-34b-3p or overexpression of TGFBR1 partially attenuated the effects of PL on A549 and H1299 cells. Conclusions PL inhibits proliferation and induces apoptosis of A549 and H1299 cells by upregulating miR-34b-3p and modulating TGFBR1 signaling pathway. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-020-03123-y.


Background
Lung cancer (LC) is a major contributing factor to the cancer-related death worldwide and has a high incidence and mortality rate [1]. The two main types of LC are small cell lung cancer and non-small cell lung cancer (NSCLC). Despite continuous improvements of medical treatment and technology, the vast majority of NSCLC patients are diagnosed with late-stage disease and the 5year survival rate is low [2]. At present, LC treatment has rapidly developed, but there are many side effects and the prognosis still remains poor [3]. Therefore, there is an urgent need to identify new targets and methods for early diagnosis in order to improve the 5-year survival rate of patients.
MicroRNAs (miRNAs) are non-coding RNAs that regulate the expression of several genes [4,5] through complementary binding to the 3′ untranslated region (UTR) of target gene mRNAs [6,7]. It is reported that miRNAs participate in cancer progression via regulation of cell cycle, cell proliferation, and cell invasion etc. [8,9]. For example, miR-221 and miR-222 inhibits the growth of LC [10,11], and miR-493 promotes the sensitivity of cancer cells to cisplatin [12]. The abnormal expression of miRNAs in LC (high or low) may lead to tumor heterogeneity and tumorinitiating cell behaviors [13].
Piperlongumine (PL) exists naturally in nature and plays an anticancer role in a variety of tumors [14]. It has been shown to inhibit cell growth and induce apoptosis of cancer cells, such as breast cancer [15], human melanoma [16], head and neck cancer [17], human prostate cancer [18], pancreatic cancer [19], gastric cancer [20], and NSCLC [21]. Furthermore, Lad [22,23] and Mizuno et al [24] found that PL is involved in the occurrence and development of multiple cancers through regulating different signaling pathways. Mizuno et al [24] reported that miR-34b-3p can inhibit the occurrence and development of LC.
In this study, we performed a miRNA microarray assay in NSCLC cells and found the differential expression of miR-34b-3p after PL treatment. In addition, we also assessed the effects of PL on the proliferation and apoptosis of A549 and H1299 NSCLC cell lines.

Cells and reagents
The NSCLC cell lines A549, H1299, H520, and SPC-A-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). A549 and H1299 cells were transfected with lentivirus containing miR-34b-3p or NC fragment to obtain stable expression of miR-34b-3p/NC (Hanbio, Shanghai, China). The PL was purchased from Sigma (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO) (Sigma) and stored at 4°C for subsequent experiments.

Cell transfection
Transient transfection was conducted using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's instructions. Cells were collected for subsequent experiments at different time points after transfection.

CCK-8 assay
A549 and H1299 cells were seeded into 96-well plates. After treatment for 0, 24, 48, 72 h, 10 μL CCK-8 reagent (Dojindo, Kumamoto, Japan) was added to each well and incubated for another 1-2 h followed by measuring the optical density values to assess cell growth.

Cell apoptosis assay
Apoptotic cell numbers were detected by Annexin V-FITC/PI Apoptosis Detection Kit (Solarbio, Beijing, China) in accordance with the kit instructions. Annexin V-FITC and PI were added to the binding buffer and mixed well followed by being added into cells. After 30 min incubation at room temperature under dark, a flow cytometer (BD Biosciences, San Diego, CA, USA) was used to measure cell apoptosis.

Transwell assay
A549 and H1299 cells were cultured in 6-well plates. After treatment with PL or transfection for 24 h, 5 × 10 4 cells were seeded in the top compartment of 8 μm pores transwell culture inserts (Corning Life Sciences, Corning, NY, USA) in the presence of serum-free medium, and the experiment was performed following the instructions. Three independent experiments were performed.

Xenograft mouse model
Female BALB/c nude mice of 4-6 weeks old (n = 12) were purchased from the Beijing Vital River Laboratory Animal Technology Center (Beijing, China). Three groups of A549 cells (miR-34b-3p, NC, and noninfected) were subcutaneously injected into the dorsal scapular region of the mice at a density of 2 × 10 6 . After tumors were visible (about 1 week), the tumor-bearing nude mice were intraperitoneally injected with PL at a dose of 5 mg/kg (dissolved in 0.01% DMSO) twice per week for 3 weeks in the PL group with non-infected A549 cells, and the other two groups were injected with the same amount of 0.9% saline. The tumor volumes were monitored every week after tumor cell injection. After 4 weeks, the nude mice were sacrificed by Automated CO2 Delivery System. In brief, After the animals were put into the Automated CO 2 Delivery System, the CO 2 was infused into the box at the rate of 10-30% of the volume of the box per minute. When the animals were determined to be immobile, not breathing and pupil dilation (about 5 min), the CO 2 was turned off and observed for 2 min to determine the death of the animal. The tumors were stripped and weighed, and immunohistochemistry (ki-67) and the TUNEL assay were performed. The study protocols were approved by the Animal Experimental Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

Immunohistochemistry
The processed paraffin sections were incubated with primary antibody against Ki-67 (1:50 dilution, AF1738; Beyotime, Beijing, China) followed by incubation with secondary antibodies according to the instructions.

TUNEL assay
The TUNEL Assay Kit (C1088; Beyotime) was purchased, the experiment was conducted according to the manufacturer's instructions, and the TUNEL staining was observed under a fluorescence microscope.

Luciferase reporter assay
The wild-type (Wt) TGFBR1 3′-UTR and mutant type (Mt) TGFBR1 3′-UTR fragments were amplified by overlapping PCR. Then the two fragments were inserted into the pmirGLO promoter vector (Promega) to generate recombinant Wt TGFBR1 and Mt TGFBR1 plasmids, respectively. 293 T cells were seeded in 24-well plates Statistical analysis SPSS 21.0 software was used for processing data. Each experiment was repeated three times. Data with normal distribution were presented as mean ± standard deviation. One-way analysis of variance was used to compare the difference among three or more groups; Student's ttest was used to compare difference between two independent groups. P < 0.05 was considered to be statistically significant.

PL upregulates miR-34b-3p expression in LC cell lines
To study the anti-tumor mechanism of PL, miRNA microarray analysis was performed in A549 cells after treatment with PL. We found that there were 74 differentially expressed miRNAs with more than 3-fold changes, of which 27 were upregulated and 47 were downregulated. In addition, miR-34b-3p showed a 14fold change increase in the 10 μM PL group compared with the 0 μM PL group (Table 1, Fig. 1a, b). To further confirm these results, miR-34b-3p expression was detected by quantitative PCR (qPCR) after total RNA extraction from PL-treated A549, H1299, H520, and SPC-A-1 NSCLC cells. Compared with cells in the 0 μM PL group, miR-34b-3p expression was consistently  Fig. 1c). These results showed that PL treatment increased the expression of miR-34b-3p.

PL treatment and upregulation of miR-34b-3p inhibits proliferation and invasion of A549 and H1299 LC cell lines
To evaluate the tumor-suppressing effects of miR-34b-3p and PL on LC, we constructed a lentiviral expression vector expressing miR-34b-3p or miR-negative control (NC), and then transfected separately into A549 and H1299 cells. Increased expression of miR-34b-3p after infection of the miR-34b-3p expressing lentivirus was found as demonstrated by qPCR, which indicated successful transfection (Fig. 2a). The CCK-8 assay showed that both upregulation of miR-34b-3p and PL inhibited cell proliferation (P < 0.05; Fig. 2b, c). In addition, a significant apoptosis-promoting effect was found after 10 μM PL treatment (P < 0.05); however, upregulation of miR-34b-3p had little effect on cell apoptosis (P > 0.05; Fig. 2d and e). In the transwell experiments, PL treatment and upregulation of miR-34b-3p effectively decreased the invasion ability of LC cells (P < 0.05; Fig. 2f). Overall, these data indicated that upregulating miR-34b-3p and PL treatment inhibited the proliferation and invasion of A549 and H1299 cells in vitro.

miR-34b-3p overexpression and PL treatment inhibits the growth of A549 xenograft tumors in vivo
To compare the effects of upregulated miR-34b-3p and PL on the proliferation of tumors in vivo, we performed a nude mouse xenograft experiment. The tumor volume was significantly reduced in the inoculated nude mice of miR-34b-3p group and PL group compared with mice of the miR-NC group after 2, 3, and 4 weeks (P < 0.05; Fig. 3a, b). In addition, the tumor weight in the miR- 34b-3p and PL groups showed a significant decrease (P < 0.05; Fig. 3c). Immunohistochemical staining of Ki-67 in the xenograft tumor tissue showed that the positive staining of Ki-67 in the tumor cells was significantly lower in the miR-34b-3p and PL groups than that in the miR-NC group (P < 0.05; Fig. 3d). We also investigated the effect of miR-34b-3p and PL on tumor apoptosis using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. As shown in Fig. 3e, the number of apoptotic cells in the PL group was significantly higher than that in the miR-NC and miR-34b-3p group. Taken together, the in vivo results were consistent with in vitro assays indicating that both PL and miR-34b-3p inhibits the proliferation of NSCLC cells and xenograft tumor growth, but only PL promotes tumor cell apoptosis.

miR-34b-3p targets TGFBR1
The result of bioinformatics analysis through TargetScan indicated that the TGFBR1 mRNA shared a complementary region with miR-34b-3p, and thus may be a potential target of miR-34b-3p (Fig. 4a). We verified this through dual-luciferase reporter assays and found that in the miR-34b-3p mimics and wild-type pmirGLO co-transfection group, the fluorescence activity was significantly reduced compared to the other three groups (P < 0.05; Fig. 4b), suggesting that miR-34b-3p bound to the TGFBR1 mRNA 3'UTR and decreased firefly luciferase activity. Moreover, Western blotting showed an apparent decrease in TGFBR1 protein expression after miR-34b-3p mimics transfection (P < 0.05; Fig. 4c). Taken together, these data showed that TGFBR1 is a target gene of miR-34b-3p.
Overexpression of TGFBR1 attenuates the inhibitory effects of PL on the proliferation and migration of A549 and H1299 cells To further demonstrate the role of PL/miR-34b-3p/ TGFBR1 axis in LC cells, we constructed an expression vector of TGFBR1 lacking the 3 'UTR (named pcDNA3.1-TGFBR1) which was transfected into PL-treated A549 and H1299 cells (combination group). The CCK-8 assay showed that compared to cells with PL treatment alone, the combination group showed few inhibitory effects on both A549 and H1299 cells (Fig. 5a, b), indicating that overexpression of TGFBR1 partially restored the inhibitory effects on PL-induced cell proliferation. In addition, the migration effects of PL on tumor cells were attenuated after TGFBR1 overexpression (Fig. 5c). Western blot analysis showed that PL treatment alone significantly decreased TGFBR1 protein expression which was rarely affected in the combination group (Fig. 5d, e).

Discussion
There is evidence that PL has anti-tumor effects in the progression of LC through various pathways, including the Akt [25] and nuclear factor-kappa B (NF-κB) signaling pathways [26]. PL suppresses NF-κB signaling pathway by directly binding to the DNA binding site of NF-κB p50 subunit and decreasing nuclear translocation of NF-κB p65 subunit, and inhibits Akt signaling pathway by suppressing phosphorylation of Akt in lung tumor cells. Moreover, PL can regulate expressions of c-Mycregulated miRNAs (miR-27a, miR-20a, and miR-17) by decreasing c-Myc via an epigenetic pathway [14]. Here, we also supposed that PL may upregulate miR-34b-3p by some transcription factors induced transcriptional regulation. However, the related mechanisms are quite complicated and involve several pathways, thus needing further exploration. If the anti-tumor effects of PL are confirmed in the clinic, it will certainly be beneficial to patients. In this study, we showed that PL inhibited the proliferation of NSCLC cell lines (A549 and H1299), upregulated the expression of miR-34b-3p, and reduced the expression of TGFBR1. It has been reported lower miR-34b-3p level in cancers, such as LC, pancreatic cancer, and prostate cancer [14,27,28]. miR-34b-3p plays a role in tumor inhibition, such as pancreatic cancer [29], oral cancer [30], bladder cancer [31], cholangiocarcinoma [32], breast cancer [22], cervical cancer [33], ovarian cancer [23], and colorectal cancer [34]. In LC studies, the abnormal expression of miR-34b-3p in H1299 and A549 cells was shown to represses cell proliferation, cell cycle progression, and promote cell apoptosis by targeting cyclin-dependent kinase 4 [35]. Consistently, our study found that PL/miR-34b-3p/ TGFBR1 axis influenced cell growth, invasion, and apoptosis of A549 and H1299 cells. TGFBR1 is a component of the transforming growth factor beta (TGF-β)/SMAD signaling pathway, which can regulate cell growth, differentiation, and migration [36]. TGF-β (tumor promoter or inhibitor) plays a key role in tumorigenesis [37]. TGFBR1 participates in cellular processes, differentiation, and apoptosis [38,39]. The activation or overexpression of TGFBR1 is found in various types of cancer. For example, it can enhance the migration and invasion of breast cancer cells and promote the invasion and metastasis of colorectal cancer [39][40][41][42]. Furthermore, TGFBR1 affects tumor invasion and metastasis through the epithelial-mesenchymal pathway [43]. Together, these results indicate that TGFBR1 inhibition may be a novel therapeutic target for tumor treatment, consistent with previous results showing that downregulation of TGFBR1 suppresses cell proliferation, migration, and invasion in NSCLC [44]. In this study, the expression of TGFBR1 was affected after PL treatment and TGFBR1 knockdown inhibited cell proliferation and promoted cell apoptosis, consistent with PL-induced upregulation of miR-34b-3p expression. The overexpression of TGFBR1 repressed the proliferation and invasion of A549 and H1299 cells, suggesting that PL affected growth and apoptosis of NSCLC cells, and upregulated miR-34b-3p by targeting TGFBR1.

Conclusions
PL upregulates the expression of miR-34b-3p in NSCLC cell lines and inhibit cell proliferation and migration through TGFBR1 signaling pathway.