MiR‐21 improves invasion and migration of drug‐resistant lung adenocarcinoma cancer cell and transformation of EMT through targeting HBP1

Abstract This study was aimed at the investigation of the effects of miR‐21 on drug resistance, invasion, migration, and epithelial–mesenchymal transition (EMT) of lung adenocarcinoma cells and the related molecular mechanisms. Cell viability of A549 cell line was measured by MTT assay. Wound healing assay and transwell assay were, respectively, employed to examine cell migration and invasion abilities. The cells were transfected with miR‐21 mimic or inhibitor using Lipofectamine 3000. The target relationship between miR‐21 and HBP1 was confirmed by luciferase reporter gene assay. Western blot and qRT‐PCR were used to examine the expression of HBP1 and EMT‐related molecules. Compared with A549 cells, drug resistance of A549/PTX cells and A549/DDP cells were obviously stronger. A549/PTX cells and A549/DDP cells had stronger ability of migration and invasion compared with parental A549 cells. Meanwhile, EMT of A549/PTX and A549/DDP was significantly higher than that of A549 cells. MiR‐21 promoted migration, invasion, and EMT of human lung adenocarcinoma cancer cells. Our experiment also verified the target relationship between miR‐21 and HBP1. MiR‐21 may affect migration and invasion ability of drug‐resistant lung adenocarcinoma cells by targeting HBP1, therefore modulating EMT.


Introduction
Lung cancer is the leading cause of cancer-related mortality worldwide. The number of deaths from lung cancer in middle urbanized areas of China in 2013 was 36,200 with mortality of 47.79/100,000 [1]. The proportion of lung adenocarcinoma, a major subtype of non-small-cell lung cancer (NSCLC) [2], has raised about 40% among lung cancer since 2013 [3]. Great progress has been made in early diagnosis, surgical techniques, and targeted therapy, whereas the prognosis for patients with NSCLC remains poor with 15% of 5-year survival rate [4]. The classification of lung adenocarcinoma has been revised to incorporate important new elements by the World Health Organization (WHO), which has prognostic significance and might help predict therapeutic approaches [5]. The poor outcome is mainly accounted for by chemoresistance, an inevitable occurrence in lung adenocarcinoma [6]. Li et al. reported that cisplatin (DDP) or paclitaxel (PTX) drug exposure gave rise to lung adenocarcinoma cells with aggressiveness and metastatic potential [7]. Thus, it is of the utmost importance for deeper understanding of novel molecular mechanisms to overcome drug resistance in lung adenocarcinoma cells.

ORIGINAL RESEARCH
MiR-21improvesinvasionandmigrationofdrug-resistant lungadenocarcinomacancercellandtransformationofEMT throughtargetingHBP1    MiRNAs play an important part in various biological processes such as cell proliferation, migration, differentiation, and apoptosis [8,9]. Beyond the involvement in physiological processes, accumulating evidence strongly suggests that the dysregulation of miRNAs may bring about the initiation and progression of cancer, which makes them valuable cancer biomarkers [10]. In vivo and in vitro studies revealed that miR-21 could serve as a diagnostic or prognostic marker for human malignancies such as tongue squamous cell carcinoma (SCC), NSCLC, chronic lymphocytic leukemia, and pancreatic cancer [11][12][13][14][15]. Besides, miR-21 overexpression is associated with EGFR-TKI-resistant human lung adenocarcinoma cell line [16]. However, the role of miR-21 and how it works in lung adenocarcinoma remain unclear.
HMG box transcription factor 1 (HBP1) is a member of the high-mobility group protein family, which is a transcriptional repressor and contains 513 amino acid residues of polypeptide [17,18]. By regulating transcription of various genes, HBP1 has been found to participate in multiple cell progressions including cell cycle inhibition, terminal differentiation, senescence induction, and tumor suppression in a variety of tissues and cell types [19][20][21]. HBP1 pathway could also be modulated by other factors, therefore influencing cancer development. For instance, HBP1-p53-Srebp1c pathway could be regulated by miR-21 to affect hepatocellular carcinoma progression [22]. Nevertheless, no study has thoroughly elaborated the role of HBP1 during lung adenocarcinoma development.  Epithelial-mesenchymal transition (EMT) is the conversion of epithelial cells to mesenchymal cells, in which cells undergo physiological or pathological changes including the loss of cell polarity and cell-cell adhesion as well as the acquisition of migratory and invasive properties [23]. Thus, EMT has recently been recognized to be highly responsible for carcinoma progression in several types of cancer, including non-small-cell lung cancer (NSCLC) [24]. On the other hand, the EMT-induced stemness endows cancer cells with the ability to overexpress chemoresistancerelated genes, leading to multiple drug resistance in cancer treatment. Both the development of drug resistance and the occurrence of EMT are negative effects induced by chemotherapy. It has been reported that EMT is associated with reduction of drug sensitivity and acquisition of resistance in lung adenocarcinoma [25]. Taken together, several studies have demonstrated that EMT not only enhances the metastatic potentials of cancer, but also participates in the development of chemoresistance [26].
Our study herein was conducted to evaluate the biological roles of miR-21/HBP1 in lung adenocarcinoma growth, migration, and invasion, and to identify HBP1 as a target of miR-21. We also tried to find the association of EMT and the above two factors miR-21/HBP1. The regulatory mechanism between HBPI and miR-21 found in our study might be a therapeutic target for patients with NSCLC, which would further control the recurrent and improve the prognosis of lung adenocarcinoma.

MaterialsandMethods
Celllinesandcellculture Human lung cancer cell line A549 was obtained from BeNa Culture Collection (BNCC; Beijing, China). The cell line was confirmed by short tandem repeat profiling and tested for mycoplasma contamination. Paclitaxel (PTX) was purchased from Beijing Pharmaceutical (Beijing, China) and cisplatin (DDP) was procured from Qilu Pharmaceutical (Jinan, Shandong, China). Cells were cultured in RPMI 1640 (Gibco, Gaithersburg, MD) containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin, subcultured every 3-4 days, and incubated at 37°C in a humidified environment. A549 cells were continuously cultured with gradient concentration of PTX and DDP for more than 12 months until the cells showed the drug resistance against 200 μg/mL of PTX or 1000 μg/mL of DDP. Thereafter, PTX-resistant A549 (A549/PTX) cell line and DDP-resistant A549 (A549/DDP) cell line were screened out and prepared for subsequent experiments.

Cellproliferationassay
After transfected with miR-21 mimic or miR-21 inhibitor, A549 cells were first harvested, seeded into 96-well plates (7 × 10 3 cells/ well), and incubated overnight. Next, the cells were treated with different concentrations of PTX or DDP and then 20 μL of MTT (Sigma-Aldrich, St. Louis, MO) was added into each well and incubated for 4 h. Afterward, the supernatant was discarded and 150 μL of dimethyl sulfoxide (DMSO) was added into each well. After the crystals were dissolved, the cells were observed at 24 h, 48 h, and 72 h under a microscope, and half maximal inhibitory concentration (IC 50 ) and resistance index (RI) were calculated. The optical density (OD) was measured at 490 nm.
Woundhealingassay A549 cell, A549/PTX cells, and A549/DDP cells were put on the six-well plate 24 h post-transfection and then cultured for 24 h until 90% confluence. A scratch was created using a sterile The migration distance of A549 cells in miR-21 mimic group was more than that in mimic NC group (×100). (B) The number of migrated and invaded A549 cells in miR-21 mimic group was more than that in mimic NC group (×200). *P < 0.05, compared with mimic NC group.
MiR-21/EMT/HBP1 Promotes Lung Adenocarcinogenesis C. Su et al. 200 mL micropipette tip. The cells were subsequently rinsed twice with serum-free medium to remove cell debris. Afterward, the cells continued to be incubated in an incubator at room temperature. The scratch healing areas of cells were observed among different transfection groups and during different time points. Finally, three randomly selected fields were photographed by an inverted microscope (BD Biosciences, San Jose, CA).

Transwellassay
A 24-pore transwell chamber (Corning Inc., NY, USA) with polycarbonate membrane filter covered with the gelatin package was used to measure the migration ability of A549 cells, A549/PTX cells, and A549/DDP cells. After diluted with 100 μL serum-free medium, Matrigel (BD Biosciences) was used to cover the bottom membranes of transwell chamber. After that, 2.5 × 10 4 cells were inoculated onto the upper chamber. The serum and growth factors such as chemokines were added in the lower chamber and incubated overnight. The noninvading cells on the transwell chamber were wiped off with cotton swab after incubation, while the migratory or invasive cells were fixed with 4% paraformaldehyde and then stained with Giemsa stain (Keygentec, Jiangsu, China). The migratory or invasive cells were counted through a microscope (×400).

RNAextractionandreversetranscription quantitativepolymerasechainreaction
For the reverse transcription quantitative polymerase chain reaction (qRT-PCR) analysis of miRNAs, total RNA including miRNA from the tissues and cultured cells was extracted through mirVana ™ miRNA isolation kit (AM1560; Ambion, Austin, TX) following the producer's guideline. After being reverse transcribed by miScript Reverse Transcription II Kit (Qiagen), specialized qRT-PCR kits (Qiagen) for miRNA were utilized to measure expression levels of miR-21 mRNA. U6 small nuclear RNA was used as an internal control. For the qRT-PCR analysis of mRNAs, total RNA was isolated from harvested cells using Trizol (Invitrogen) followed by further purification with RNeasy mini kit and RNase-free DNase Set (Qiagen, Shanghai, China) in accordance with the instructions. To determine the mRNA levels in A549, A549/PTX, and A549/DDP cells, total RNAs were treated with DNase I (AM1906; Ambion) to eliminate genomic DNA contamination, and then reversely transcribed using Advantage RT-4PCR kit   Table 1. Results were calculated using 2 −ΔΔCt method.

Differentialexpressionanalysis
Total RNA for each of the three kinds of cultured cells (A549, A549/PTX, and A549/DDP cells) was divided into two parts, one for miRNA sequencing and the other for mRNA sequencing using the Illumina HiSeq2000 sequencing system at the BGI in Shenzhen, China. The Illumina HiSeq2000 system was a high-throughput sequencing technology based on massive parallel sequencing of millions of fragments using proprietary reversible terminatorbased sequencing chemistry, the same sequencing principle used in the Illumina Genome Analyzer II system. The RNA sequencing process was performed according to the manufacturer's protocols as well as standardized protocols developed by the core laboratory at the BGI. Differentially expressed mRNAs and miRNAs were screened out from the expression data of the above three kinds of cell lines, with each kind including five groups of cells. Fold-change (FC) value >2 and P < 0.05 were the screening conditions to identify the genes and miRNAs with differential expression, using the R project for statistical computing. On the other hand, data for prognostic and survival analysis of patients were obtained from The Cancer Genome Atlas (TCGA) database (https://tcga-data.nci.nih.gov/tcga/).
Statisticalanalysis SPSS version 20.0 statistical software (SPSS, Chicago, IL) was applied for statistical analyses. Experimental data were calculated as mean ± SD. Statistical differences between groups were compared by one-way ANOVA and statistical differences among groups were compared by Student's t-test. P < 0.05 denoted a statistical significance.

EstablishmentofA549drug-resistantcell lines
To measure the drug resistance of A549 cell lines, we cultured cells with PTX or DDP in different concentrations (0.0, 0.2, 0.4, 0.8, 1.6, and 3.2 mmol/L) for 24 h, 48 h, and 72 h. The result of MTT assay showed that the drug resistance of A549/PTX cells and A549/DDP cells were obviously stronger than that of A549 cells (Fig. 1), which indicated that the drug-resistant cell   (Tables 2 and  3). PTX or DDP drug-resistant cell line established successfully was isolated for the preparation for follow-up experiments.

Migration,invasion,andEMTofA549/PTX andA549/DDPcelllines
Compared with parental A549 cells, stronger ability of migration and invasion was observed in A549/PTX and A549/DDP cells by the detection of wound healing assay and transwell assay (all P < 0.05, Fig. 2A and B). The expression of EMT markers was further measured by the method of western blot and RT-PCR. The results showed that compared with A549 cell lines, the expression of epithelial adhesion molecules E-cadherin and βcatenin in A549/PTX and A549/DDP cells was dramatically downregulated, while the expression of mesenchymal markers except for Twist (Vimentin, MMP-2, MMP-9, Snail, Slug, and ZEB1) was significantly upregulated (all P < 0.05, Fig. 3A and B).

MiR-21promotedmigrationandinvasionof lungadenocarcinomacancercells
In five cases of A549, A549/PTX, and A549/DDP cells, expression of 515 miRNAs was upregulated, while expression of 361 miRNAs was downregulated (Fig. S1A). The expression of miR-21 in drug-resistant A549/PTX and A549/DDP cells was significantly increased by 1.997 times (P = 3.04E-18). The heat map showed the miRNAs with the most significant differential expression (Fig. S1B). TCGA analysis displayed that patients with higher level of miR-21 had poorer prognosis and shorter survival time (Fig. S1C). qRT-PCR showed that the expression of miR-21 in A549/PTX cells and A549/DDP cells was obviously upregulated compared with A549 cells (both P < 0.05, Fig. 4A). Our experiment results showed that miR-21 mimic dramatically enhanced the expression of miR-21 in A549 cells (P < 0.05, Fig. 4B), and miR-21 inhibitor effectively suppressed the expression of miR-21 in A549/PTX cells and A549/DDP cells (both Ps < 0.05, Fig. 4C and D). MiR-21 mimic improved the ability of invasion and migration of A549 cells (all Ps < 0.05, Fig. 5A and B), and miR-21 inhibitor weakened the ability of invasion and migration of A549/PTX cells (all Ps < 0.05, Fig. 6A and B) and A549/DDP cells (all Ps < 0.05, Fig. 7A and B). These results showed that miR-21 was the upstream regulation factor of invasion and migration of lung adenocarcinoma cancer cells, drug sensitive or drug resistant.

MiR-21reducedtheexpressionlevelofHBP1 bytargetingHBP13′UTRdirectly
In five cases of A549, A549/PTX, and A549/DDP cells, expression of 8378 mRNAs was upregulated, while expression of 10,952 mRNAs was downregulated (Fig. S1D). The expression of HBP1 in drug-resistant A549/PTX and A549/DDP cells was significantly decreased by 3.45 times (P = 1.33E-10). The heat map showed the mRNAs with the most significant differential expression (Fig. S1E). TCGA analysis displayed that patients with higher level of HBP1 had better prognosis and longer survival time (Fig. S1F). The fact that HBP1 was the potential target gene of miR-21 had been authenticated through miRNA online prediction database (miRNA.org and TargetScan) (Fig. 12A). The result of western blot experiment showed that compared with parental A549 cells, the protein level of HBP1 in A549/PTX cells and A549/DDP cells was much lower (Fig. 12B). Luciferase assay showed that after the cotransfection of miR-21 mimic and PGL3-HBP1-wt, the luciferase intensity in cells decreased (P < 0.05), while the cotransfection of miR-21 mimic and PGL3-HBP1-mut did not show any significant difference from the blank group (P > 0.05, Fig. 12C). According to the detection of western blot, miR-21 mimic contributed to the reduction of HBP1 protein level in A549 cells, and miR-21 inhibitor upregulated HBP1 protein level in A549/PTX and A549/DDP cells (Fig. 12D). We have figured out how miR-21/HBP1/EMT pathway influenced drug-resistant lung adenocarcinoma cells, which is illustrated in Figure 12E that miR-21 affected migration and invasion ability of drug-resistant lung adenocarcinoma cells by targeting HBP1, therefore modulating transformation of EMT.

Discussion
In this study, we have revealed that miR-21 played an important part in the invasion and migration ability of drug-sensitive or drug-resistant lung adenocarcinoma cancer cells through targeting HBP1. The IC 50 of A549/PTX cells and A549/DDP cells were obviously higher than that of A549 parental cells. Meanwhile, we also found that A549/PTX and A549/DDP cells had stronger ability of migration and invasion compared with parental A549 cells. Enforced overexpression of miR-21 promoted the formation of EMT phenotype in parental A549 cells, whereas the knockdown of miR-21 reversed the EMT phenotype in A549/PTX cells or A549/DDP cells. HBP1 was an inhibitory target gene of miR-21 and their expression levels were negatively related.
MiR-21 level was significantly higher in nine gastric cancer cell lines than in normal gastric mucosal epithelial cell line GES-1 [27]. Meanwhile, miR-21 expression in clear cell renal cell carcinoma (ccRCC) cells was significantly higher compared with that in the HK-2 cell line (an immortalized proximal tubule epithelial cell line from normal adult human kidney) [28]. Besides, a recent study has specifically identified dysregulated miRNAs in NSCLC using next-generation sequencing (NGS) technology, among which miR-21 or miR-188 overexpression correlates with a negative prognosis [29]. The present study results demonstrated that miR-21 level in PTX-and DDP-resistant A549 cells was significantly higher than in A549 cells. Additionally, miR-21 could promote the propagation and metastasis of gastric cancer cell line SGC-7901 in vitro [27,30]. Similarly, miR-21 upregulation increased the viability and facilitated the metastasis of HepG2 cells (hepatic cancer cell line) [31]. Besides, miR-21 promotes migration and invasion of glioma cells lines U87, A172, T98, and U343 [32]. In the current study, miR-21 overexpression promoted A549 cells metastasis and miR-21 underexpression led to the repression of cell migration and invasion.
On the other hand, miRNAs are emerging as critical core regulators of drug resistance, acting by modulating EMT and cancer-related immune responses [33]. MiR-21 overexpression facilitates ccRCC sphere formation, in which the elevated levels of stem cell-related transcription factors and EMT markers have been seen, and therefore promotes ccRCC tumorigenesis [28]. In addition, miR-21 inhibited TGF-β1induced EMT in gastric cancer, leading to the progression of gastric cancer [34]. The knockdown of miR-21 repressed TGF-β1induced fibrogenic EMT in hepatic fibrosis cell line QSG-7701 [35]. It is also notable that upregulated miR-21 by nicotine intake promoted EMT in esophageal cancer cell line EC9706, therefore inducing the aggressiveness of esophageal cancer [36]. We found Figure 11. The effect of miR-21 inhibitor on EMT of A549/DDP cells. (A) MTT assay indicated that the cell viability of A549/DDP cells transfected with miR-21 inhibitor was decreased. (B) According to the results of western blot, A549/DDP cells transfected with miR-21 inhibitor increased the expression level of E-cadherin and βcatenin and decreased the expression level of EMT-related markers except Twist. (C) qRT-PCR verified that A549/DDP cells transfected with miR-21 inhibitor increased the expression level of E-cadherin and βcatenin and decreased the expression level of EMT-related markers except Twist. *P < 0.05, compared with inhibitor NC group.
MiR-21/EMT/HBP1 Promotes Lung Adenocarcinogenesis C. Su et al. that the overexpression of miR-21 promoted the formation of EMT phenotype in parental A549 cells, whereas the underexpression of miR-21 reversed the EMT phenotype in PTX-or DDP-resistant A549 cells. EMT has been recognized to be involved in acquiring drug resistance and cell aggressiveness in cancer. The knockdown of Linc-ROR led to the decrease of EMT of docetaxel-resistant lung adenocarcinoma cells to sensitize drug-resistant cells to chemotherapy [37]. The acquisition of pemetrexed resistance enhances EMT in vivo and in vitro [38]. DDPresistant nasopharyngeal carcinoma cells acquired EMT characteristics, which could be responsible for the DDP resistance of nasopharyngeal carcinoma cells [39]. In addition, chemoresistant NSCLC cells displayed higher mobility and more invasive capabilities as well as enhancement of EMT [40]. Based on previous discoveries and our results, we thus speculated that miR-21 might contribute to the EMT within lung adenocarcinoma cells and induce drug resistance. This has been investigated in other studies. For example, miR-21 sustained EMT, promoted the tumor immune microenvironment formation, and conferred resistance to neoadjuvant trastuzumab and chemotherapy of HER2-positive breast cancer cells [33]. Similarly, Zhen et al. found that miR-21 induced EMT and gemcitabine resistance in breast cancer cells [41].
HBP1, a tumor suppressor, has been shown to contribute to cell senescence and block tumorigenesis in several cancers [42], including lung adenocarcinoma (H1299 [p53−/−], A549), hepatocellular carcinoma (HepG2), and osteogenic sarcoma (U2OS). In addition, a previous study of Chan et al. investigated the influence of HBP1 on invasive oral cancer [43]. Suppression of HBP1 could also promote human breast cancer cell migration and invasion [44]. Taken together, alterations of the HBP1 transcriptional repressor were associated with the invasion and migration ability of cancer cells [45,46], which was consistent with our experiments result. The activity of HBP1 is modulated through various mechanisms such as the activation of Wnt/β-catenin signaling [47], the targeting of PI3K/FOXO pathway [48], and the regulation of abnormal gene promoter methylation [49]. Furthermore, a previous study revealed that HBP1 enhanced radiosensitivity and overcame acquired radioresistance after radiotherapy [50]. In the study herein, we investigated and verified the targeted relationship between HBP1 and miR-21-3p via dual luciferase report system. Based on the above studies, we may safely pose a hypothesis that miR-21/HBP1 axis played a significant role in invasion, migration, and transformation of EMT of drug-resistant lung adenocarcinoma cancer cells.

Conclusion
In conclusion, the present study demonstrated that miR-21 was upregulated in lung adenocarcinoma cell lines with or without drug resistance. Moreover, miR-21 promoted cell propagation, migration, invasion, and EMT transformation of A549 cells. Finally, we verified that miR-21 could directly bind to the 3′ UTR of HBP1, which was found to be an important functional mediator of miR-21 in A549 cells. Overall, our findings indicate that miR-21 functions as a tumor facilitator in lung adenocarcinoma through targeting HBP1 and may be a promising candidate for miR-based therapy against lung adenocarcinoma. Western blot assay showed that the protein expression level of HBP1 in A549/PTX cells and A549/DDP cells was lower than that in parental A549 cells. (C) The relative luciferase activity in HBP1 3′ UTR-wt and miR-21 mimic cotransfection group was significantly lower than that in mimic NC group, whereas there was no significant difference in the relative luciferase activity between HBP1 3′ UTR-mut and miR-21 mimic cotransfection group and mimic NC group. *P < 0.05, compared with mimic NC group. (D) The result of western blot showed that miR-21 mimic reduced the protein level of HBP1 in A549 cells, while miR-21 inhibitor raised the protein expression of HBP1 in A549/PTX cells and A549/DDP cells. (E) The mechanism scheme showing miR-21/HBP1/EMT signaling pathways in A549 cells was illustrated.