ORIGINAL RESEARCH ARTICLE
The suppressive effects of miR-16-5p on lung cancer by targeting Fermitin family member 2AbstractLung cancer is one of the most severe causes contributing to tumor-related mortality. MicroRNAs (miRNAs), an evolutionarily conserved class of small noncoding RNA, can manipulate the expressions of endogenous cancer-related genes and are implicated in the progression of wide types of tumors. Although it has been reported that miRNA-16-5p (miR-16-5p) can suppress cell growth in many cancers, the biological mechanisms of miR-16-5p in lung adenocarcinoma are not to be fully elucidated. In the present study, we first detected the level of miR-16-5p and Fermitin family member 2 (FERMT2) in clinical samples and cultured cell lines. A negative correlation between miR-16-5p and FERMT2 was observed, implying there may be a potential link about their regulation. The hypothesis was further tested and confirmed by bioinformatics analysis and dual luciferase reporter assay. In addition, we also found that the transfections of miR-16-5p mimics or inhibitors can alter the biological characteristics of lung cancer cells remarkably accomplished by the expression variance of FERMT2 in vitro and in vivo. Taken together, this study demonstrated that miR-16-5p plays as a tumor suppressor in lung adenocarcinoma by targeting FERMT2. The present study provided a novel insight into the critical roles of miRNAs in the development of lung cancer which has potential applications for clinical treatment of lung cancer.KEYWORDS lung cancer, miR-16-5p, FERMT2, apoptosis, cell cycle1  INTRODUCTIONLung cancer is one of the most severely malignant tumors and the leading cause of cancer-associated mortality worldwide. It has been estimated that non-small cell lung cancer (NSCLC) accounts for ~80% of all clinical lung cancer cases. To date, surgery is the most effective treatment for NSCLC. However, most patients are diagnosed at the later or metastatic stages and lost the chance for operation. The 5-year survival rate after diagnosis is only maintained at 16.6%(Lewis et al., 2018). Because of the difficulties in early diagnosis of the disease, NSCLC is still a significant challenge for public health.  miRNAs, a class of small noncoding RNAs that approximately 17 to 25 nucleotides in length, can promote the degradation of mRNAs or inhibit their translation by binding to the 3’-untranslated regions (3’-UTRs) of mRNAs, contributing to mRNA degradation (Bentwich et al., 2005). To date, about 2500 human miRNAs have been identified (Kozomara&Griffiths-Jones, 2011). It has been well-known that quite a few miRNAs can serve as practical and safe markers for cancer detection (Li et al., 2018; Yan et al., 2017). Similarly, some miRNA signatures have successfully been applied in lung cancer screening or diagnosis (Croce, 2009). As a tumor suppressive factor, miRNA-16-5p (miR-16-5p) has been identified to be deregulated in multiple types of malignant cells, such as breast cancer (Li et al., 2018; Qu et al., 2017), retinal leukostasis (Ye et al., 2016), chordoma (Zhang et al., 2018), pituitary adenoma (Renjie&Haiqian, 2015), gastric carcinoma (Wang et al., 2017b), prostate cancer (Hao et al., 2016), ovarian cancer (Pero-Gascon et al., 2018; Yan et al., 2017), colon cancer (Shi et al., 2014) and chronic lymphocytic leukemia (Pekarsky&Croce, 2015). However, the role of miRNA-16-5p in lung cancer has not yet been fully elucidated.Cell adhesion to the cellular matrix is crucial for cells immobilization, especially in solid tissues. At the beginning of the tumor, cells begin to aggregate and adhere to the substratum. With the development of the tumor, cells invade into the cellular matrix, thus allowing cancer to acquire metastasis characteristics (Kloeker et al., 2004). FERMT2, also named as Kindlin2, is a focal adhesion protein involved in tumor development and progression through the interact with integrins, especially integrin beta1 (Rognoni et al., 2016). Although there are many studies reported that FERMT2 prompted epithelial-mesenchymal transition (EMT) which closely related to tumor migration and invasion (Yang M et al., 2014), the mechanisms of its regulation need to be further investigated.In the current study, the hypothesis was tested that the changes of FERMT2 expression can be regulated in the miRNA level in lung adenocarcinoma. Bioinformatics analysis was first performed to reveal FERMT2 as a potential target of miR-16-5p. The level of miR-16-5p and FERMT2 was detected in clinical samples and cultured cell lines. A negative correlation between miR-16-5p and FERMT2 was observed. The luciferase reporter assays further confirmed that FERMT2 is a direct target of miR-16-5p. Also, our results showed that the overexpression and knockdown of miR-16-5p could influence the biological characteristics of lung cancer cells remarkably accomplished by the expression changes of FERMT2. Furthermore, the transfection of FERMT2 without 3’ UTR reversed the effects of miR-16-5p on lung cancer cells. Our study provided evidence that miR-16-5p exhibited an anti-cancer effect by targeting FERMT2.2  MATERIALS AND METHODS2.1  Ethics Statement The Institutional Review Board for Human Research at Binzhou Medical University approved access to patient samples and an anonymous analysis of data. Written informed consent was obtained from all participants, and all procedures were approved by The Ethics Committee of Binzhou Medical University. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Binzhou Medical University. Animal care protocols conducted were in accordance with the IACUC guidelines to minimize pain and discomfort to animals.2.2  Clinical samplesAll samples were obtained from Yantai Shan Hospital, Binzhou Medical University, Shandong Province, China. The informed consents were obtained from the patients enrolled in this research. The lung cancer tissues and matched normal tissues were derived from adult patients (6 males and 6 females) who had pathologically diagnosed as lung adenocarcinoma and no any treatments prior to surgery, including radiotherapy, chemotherapy and immune therapy. All the tissues were frozen and stored at -80?C for further analyses.2.3  Cell cultureThree human lung cell lines, including human bronchial epithelial (HBE), A549 and H1299) were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai Institute of Biochemistry and Cell Biology) and were cultured in RPMI-1640 medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, USA) incubator with a 5% CO2 at 37 ?C. 293T cells were purchased from ATCC (Chinese Academy of Sciences, Shanghai) cultured with DMEM medium containing10% fetal bovine serum at 37 ?C under 5% CO2.2.4  Transfection Cells (2×105) were seeded into 6-well plates and cultured 12-15h until reaching about 70?80% confluence. The scram miR-16-5p mimics and miR-16-5p inhibitors were synthesized by Shanghai GenePharma Co., Ltd (Shanghai, China). Transfections were performed using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) After incubation for 6-8 h, the medium was replaced with RPMI-1640 containing 10% FBS.2.5  Cell proliferation assays For colony formation assays, 1.5×103 cells with or without treatment were seeded into each 10-cm plate. The plates were fixed with methanol and stained with crystal violet about two weeks later. Then, the number and size of colonies was analyzed. For MTT assays, the detection was performed according to the instrument of the manufacturer (Beyotime Biotechnology, cat. no. C0009). Briefly, 6×103 cells with or without treatment were seeded into 96-well plates. After cultured for 48 h, 10 µl reagent was added and incubated for 4 h. After 150 µl detergent reagent was added into wells, the absorbance was detected in the microplate reader at 570 nm wavelength.2.6  Apoptosis and cell cycle analysis by flow cytometry For cell apoptosis analysis, cells with different treatments were harvested at 48 h after the oligonucleotides were transfected into lung cancer cells, and washed with ice-cold PBS, suspended the cells with buffer then double stained with Annexin V-FITC (5 ?g/ml) and PI (5 ?g/ml). The assay was performed using Annexin V-FITC/PI (BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA). For cell cycle analysis, cells with different treatments were washed with ice-cold PBS twice and then fixed with 70% ethanol (v/v) overnight at -20?C. Fixed cells were resuspended in PBS containing RNase A (50 ?g/ml) for 30 min,   then added PI (50 ?g/ml). Finally, both the apoptosis and cell cycle were analyzed using a flow cytometer (FACS FC500; Beckman Coulter, Brea, CA, USA).2.7  Cell migration and invasion assaysThe cell migration and invasion were tested by transwell chamber assay respectively. Briefly, cells (5×105) in 100 µl serum-free medium were added into the upper chamber of 24-well plates directly. Serum-containing media (20% FBS, 600 µl) acted as chemo-attractants in the lower chambers. After incubation for 48 h, cells in the upper chamber were carefully removed using a cotton plug. The cells at the bottom of the membrane were fixed with methanol, stained with crystal violet, and then imaged with a microscope (Olympus, Tokyo, Japan). Ten randomly selected fields were examined and the average numbers of invaded or migrated cells were calculated.2.8  Quantitative-polymerase chain reaction (q-PCR)For miR-16-5p expression analysis, small RNAs were isolated from cells from different groups using RNAiso for small RNA reagent (Takara Biotechnology Co. Ltd. Dalian, China). Human 5S rRNA served as the positive control. For the expression analyses of related genes, total RNA was isolated using TRIzol reagent. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. Real-time quantitative PCR was carried out using SYBR-Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an RG3000 System (Qiagen GmbH, Hilden, German). The reaction protocol was as follows: 95?C for 10 min, followed by 40 cycles of 95?C for 10 sec and annealing 60?C for 20 sec, and then extension at 72?C for 30 sec. The primers used in this study are listed in Table 1. All experiments were performed in triplicate.2.9  Western blot analysis Total proteins were extracted lung carcinoma cells by RIPA (Solarbio, Beijing, China). Subsequently, SDS-PAGE was performed followed by transferring to PVDF membrane (Sigma-Aldrich, USA). After blocking with skimmed milk, primary antibodies against FERMT2 (Abcam, cat. no. ab127745) were incubated with PVDF membrane overnight at 4 ?C. The Rabbit antibodies (LI-COR, C60405-05, USA) was added to PVDF membrane after primary incubation. Finally, the signals of protein expression were detected using Tanon 5200. The antibodies used were as follows: rabbit antibodies against GAPDH (Bioworld Technology, cat. no. AP0063), Rb (Bioworld Technology, cat. no. BS1311), p-Rb (Bioworld Technology, cat. no. BS4164), Bcl-2 (Bioworld Technology, cat. no. BS1511), Bax (Bioworld Technology, cat. no. BS2538), caspase-3 (Cell Signaling, cat. no. 9662), caspase-8 (Cell Signaling, cat. no. 9748) and caspase-9 (Cell Signaling, cat. no. 9502). GAPDH was used as an internal control.2.10  Plasmid construction and luciferase reporter assayPrediction of miRNA target genes was performed using the TargetScan 5.2 (http://www.targetscan.org/) databases. The sequence of FERMT2 (NM_006832.3) was obtained from GenBank. The primers to amplify the 3’ UTR of FERMT2 (wild type or mutant) were commercially synthesized (Shanghai GenePharma Co., Ltd.). The DNA from 293T cells served as the amplification template. Then, either wild-type FERMT2 or its mutant fragment were inserted in the vectors of pMIR-REPORT miRNA Expression Reporter Vector System (RN: R10032.4 Guangzhou RibBio Co., LTD) and were termed FERMT2-3’-UTR-WT and FERMT2-3’-UTR-Mut, respectively. The 293T cells were plated in 96-well plates at a density of 7×103 cells in 100 ?l medium per well in triplicate. Then, the cells were transfected with miR-16-5p mimics/scram control (50 nM, Suzhou GenePharma), miR-16-5p inhibitor/scram control (100 nM, Suzhou GenePharma) together with wild-type/mutated luciferase reporter vector using Lipofectamine 2000. According to the protocol of the manufacturer, cells were harvested after 48 h, and then the luciferase activities were detected by Luci-Pair Duo-Luciferase Assay kit 2.0 (GeneCopoeia, Iin, Rockville , USA).2.11  Tumor growth in nude miceThe A549 cells (1×106) with different treatment were resuspended in 100 ?l of sterile PBS and then subcutaneously injected into the back of nude mice (the left for the scram group and the right for the mimics group, respectively). The tumor growth curve was made to determine the effects of miR-16-5p on cancer formation. Tumor volumes were measured every three days. The formula V (mm3) = (L×W2)/2 was used to calculate the volume, where L and W indicate the long and short axes of the tumor, respectively. At the end of the study (day 30), mice were sacrificed by cervical dislocation and tumor masses were removed and weighed.2.12  Statistical analysis All experiments were performed at least in triplicate, and all data are expressed as the mean ± standard error (SE). The differences in the mean values of the variables were compared using an unpaired, two-sided, Student’s t-test. One-way analysis of variance (ANOVA) was used to analyze the differences among multiple groups. Correlations were calculated with Spearman’s rank correlation coefficient. All statistical analyses were performed using SPSS 22.0 software package (IBM Corp., Armonk, NY, USA). For all tests, p values less than 0.05 were considered statistically significant.3  RESULTS3.1  The decreased expressions of miR-16-5p in lung adenocarcinomaTo determine whether there are differences in the levels of miRNA expression between cancer and normal tissues, the small RNAs from 12 lung adenocarcinoma samples and their normal counterparts were isolated. The results of quantitative real-time PCR indicated that the expressions of miR-16-5p are significantly decreased in lung adenocarcinoma compared with those in normal tissues (Figure 1a). Subsequently, we further detected the expression levels of miR-16-5p in three cell lines (HBE, A549 and H1299) respectively. Similarly, the expressions of miR-16-5p were reduced obviously in A549 and H1299 cells compared with those in HBE cells (Figure 1b). In contrast, the results of quantitative real-time PCR and Western blot showed the expressions of FERMT2 in mRNA and protein levels were much higher in cancer tissues than those in the normal tissues (Figure 1c and 1d). Statistical analysis about the results of Western blot showed significant differences between the two groups (Figure 1e). These data implied that there may have an inverse correlation between miR-16-5p and FERMT2 expressions during the tumorigenesis of lung adenocarcinoma, which has been verified by the correlation analysis (Figure 1f).3.2  The suppressiveeffect of miR-16-5p onFERMT2 expression To determine the potential link between the expression levels of miR-16-5p and FERMT2 in lung cancer, bioinformatics analysis was performed to search for potential targets of miR-16-5p with TargetScan. The predict results showed that there were partial complementary sequences between miR-16-5p and the 3’-UTR of FERMT2, indicating that FERMT2 might be a potential target of miR-16-5p (Figure 2a).Subsequently, the mimics or inhibitors of miR-16-5p were designed and transfected into A549 and H1299 cells for further investigation. The quantitative real-time PCR indicated they can influence the expression of miR-16-5p significantly in both cells (Figure 2b and 2c). It has also been investigated that whether the transfections of miR-16-5p mimics or inhibitors could alter the expression of FERMT2. The results demonstrated that FERMT2 can be significantly downregulated by miR-16-5p overexpression at protein levels. Conversely, the inhibition of miR-16-5p could increase the FERMT2 expression (Figure 2d). These data further suggested that FERMT2 is a potential target of miR-16-5p.To verify whether FERMT2 was a direct target of miR-16-5p, the dual luciferase reporter vectors named as FERMT2-3’-UTR-Wt and FERMT2-3’-UTR-Mut were constructed and co-transfected into 293T cells along with the mimics or inhibitors of miR-16-5p for 48 h. The results of dual luciferase reporter assays revealed that miR-16-5p mimic significantly suppressed the luciferase activity of FERMT2-3’-UTR-Wt. In contrast, the miR-16-5p inhibitor increase the luciferase activity of FERMT2-3’-UTR-Wt (Figure 2e). However, there was no significant difference in FERMT2-3’-UTR-Mut transfected cells (Figure 2f), implying miR-16-5p can regulate the FERMT2 expression through binding to its 3’-UTR.3.3  miR-16-5p acts as an inhibitor on cell proliferation and colony formationAs the stimulative effects of FERMT2 on cancer progression and the inverse correlation between miR-16-5p and FERMT2, we next investigate the impacts of miR-16-5p on cell proliferation and colony formation. The results of colony formation assay demonstrated that the upregulation of miR-16-5p significantly inhibited and the capacity of colony formation in miR-16-5p-mimic-treated A549 cells compared with the scram cells (Figure 3a). In contrast, the capcity of colony formation miR-16-5p-inhibitor-treated A549 cells enhanced compared with the scram group (Figure 3b). Consistently, similar results were observed in H1299 cells (Figure 3c and 3d). For MTT assay, we observed obvious decrease of cell viability in both A549 and H1299 cells treated with miR-16-5p-mimic. Oppositely, the cell viability increased significantly in these cells treated with miR-16-5p inhibitors (Figure 3e and 3f). Overall, these data indicated that miR-16-5p might act as a suppressive regulator for the proliferation of A549 and H1299 cells.3.4  The influences of mir-16-5p on the cell apoptosis and cell cycle distributionTo evaluate the influences of miR-16-5p on the status of apoptosis and cell cycle distribution, flow cytometry was performed. As shown in Figure 4a and 4c, significant increases of apoptosis were observed in A549 and H1299 cells treated with miR-16-5p mimics. However, the apoptotic rates decreased obviously after the treatment of miR-16-5p inhibitors in both A549 and H1299 cells (Figure 4b and 4d). To further delineate the mechanism by which miR-16-5p-regulated apoptosis in these cells, the expressions of some apoptosis-related genes were assessed by Western blot. The results showed that, in both cells, the cleaved-caspase3, 8, 9 and Bax increased obviously after the transfection with miR-16-5p mimics at 48 h, while Bcl-2 was significantly downregulated (Figure 4e and 4f). In contrast, the opposite trends were observed in both cells treated with the miR-16-5p inhibitors (Figure 4e and 4f). These findings demonstrated that the miR-16-5p acts as a facilitating factor for the apoptosis of lung adenocarcinoma cells.For cell cycle analyses, the  results showed  that, after the treatment of miR-16-5p mimic, the proportions of cell population in G0/G1 increased significantly in both A549 and H1299 cells compared with those in the scram group (72.13% VS 65.20% in A549 cells and 66.41% VS 57.58% in H1299 cells, respectively) (Figure5a and 5c).   Conversely, in the cells transfected with miR-16-5p inhibitors, no cell arrest at G0/G1 phase was observed (67.34% VS 78.48% in A549 cells and 69.90% VS 79.03% in H1299 cells, respectively) (Figure 5b and 5d). Moreover, we found the significant decrease of p-Rb in both A549 and H1299 cells after the treatment of miR-16-5p mimics, despite of the increased Rb expression (Figure 5e). Similarly, the opposite results were observed in the inhibitor groups (Figure 5f). These findings suggested that miR-16-5p exhibits an inhibitory effect on cell cycle and the status of Rb may involved in at least in part.3.5 miR-16-5pinhibits cell migration and invasion via FERMT2To detect the effects of miR-16-5p on the migration and invasion of A549 and H1299 cells, transwell assay and rescue assay were performed. In the migration assays, Figure 6a and 6b showed that the number of migrated cells decreased significantly in the miR-16-5p-mimics transfected groups compared with the scram group, while the number of the migrated cells in the co-transfected group of miR-16-5p mimics and FERMT2-without-3’UTR has no significant difference compared with the scram group. Similar results were observed in the invasion assays (Figure 6c and 6d).3.6  miR-16-5p inhibits lung cancer growth in vivo To further explore the anti-tumor effects of miR-16-5p in vivo, tumor growth after the subcutaneous inoculation of A549 cells (1×106) with different treatments was evaluated in nude mice. The tumor volumes of different groups were measured on day 1, 6, 12, 18, 24, and 30, respectively. As shown in Figure 7a and 7b, the tumor volumes from miR-16-5p-mimics-treated mice were significantly reduced compared with those from the scram group. At the endpoint (day 30), mice were sacrificed and tumors were removed and weighed. The data showed that the tumor weights in the miR-16-5p-mimics-treated group were markedly decreased than those in the scram group (1.59±0.28g VS 0.67±0.38g) (Figure 7c and 7d).4  DISCUSSIONLung cancer is the most leading cause of cancer-related mortality, accounting for ~1.2 million deaths every year (Crino et al., 2010). Despite of huge advances in lung cancer biology and some improvements in treatment strategies, it is still the fact that the therapeutic efficacy of lung cancer remains dismal. Nowadays, with the more recent research, numerous evidence has indicated miRNA changes are closely related to cancer biology, including cells growth, differentiation, proliferation and metabolism (Cai et al., 2018; Cui et al., 2018; Ha, 2011; Paladini et al., 2016).miR-16 was first recovered in chronic myelogenous leukemia, and defined as a kind of oncogene genes (Cimmino et al., 2005). In contrast, it is interesting that miR-16-5p, as a subgroup of miR-16, is generally thought to be a critical tumor-suppressive miRNA. Many studies reported that miR-16-5p could modulate the cell cycle, inhibit cell proliferation and invasion, as well as promote cell apoptosis (Hanniford et al., 2015; Rinnerthaler et al., 2016; Zhang et al., 2018). Also, a majority of literature demonstrated that miR-16-5p can suppress cell growth in multiple cancer types, including chronic lymphocytic leukemia (Hanniford et al., 2015), prostate cancer (Bonci et al., 2008), hepatocellular carcinoma (Guo et al., 2009), breast cancer (Xu et al., 2010), ovarian cancer (Bhattacharya et al., 2009), gastric cancer (Xia et al., 2008), and multiple myeloma (Corthals et al., 2010). In this study, we first tested miR-16-5p expression in lung cancer samples and found that it was significantly down-regulated in lung cancer samples compared with healthy tissues. The same results were observed in cultured cells, such as normal bronchial epithelial cells (HBE cells) and lung cancer cells (A549 and H1299 cells). These data suggested that miR-16-5p may act as a tumor suppressor in lung cancer. To elucidate the potential functions of miR-16-5p in tumorogenesis and development of lung cancer, a series of evaluations about gain of function and loss of function were performed to observe the effects of miR-16-5p on the biological characteristics of A549 and H1299 cells. Our data demonstrated that miR-16-5p could inhibit the proliferation, invasion, migration and induce apoptosis in lung adenocarcinoma cells. Furthermore, in the xenograft model of nude mice, we found that the overexpression of miR-16-5p can also suppress tumor growth in vivo.It is well established that miRNA can regulate gene expression by recognizing the 3’-UTR of their target genes leading to mRNA decay or translation repression. Undoubtedly, it is the expression changes of target genes of miRNA further trigger the downstream signal cascades related to a broad range of biological processes. Therefore, the identification of the target genes of miRNAs is pivotal to elucidate the underlying mechanisms. To date, there are several studies about the important role of miR-16-5p in cancer development. For example, it has been reported that miR-16-5p inhibits chordoma cell proliferation, invasion and metastasis by targeting Smad3 (Zhang et al., 2018).  Similarly, in breast carcinoma, Qu and his colleagues found the suppressive roles of miR-16-5p in the proliferation and invasion by targeting VEGFA (Qu et al., 2017). In contrast, the molecular mechanisms of miR-16-5p in the suppression of the growth of lung adenocarcinoma cells are not elucidated.In this study, the bioinformatic analysis indicated that FERMT2 might be a possible target of miR-16-5p. This hypothesis was further validated by the luciferase reporter assay and related transfection tests. FERMTs are evolutionarily conserved focal adhesion proteins that interact with integrin protein family, especially integrin beta1 (Kloeker et al., 2004; Rognoni et al., 2016). In the FERMT family, FERMT2 was the first member to be discovered. Numerous studies showed that FERMT2 is involved in tumor development and progression, such as breast cancer (Sossey-Alaoui et al., 2018; Yu et al., 2013), pancreatic adenocarcinomas (Yoshida et al., 2017), colorectal cancer cells (Lin et al., 2017; Ma et al., 2013; Ren et al., 2015), prostate cancer (Sossey-Alaoui&Plow, 2016),  hepatocellular carcinoma (Fan et al., 2015; Lin et al., 2017) and esophageal cancer (Wang et al., 2017a; Zhang et al., 2015). Furthermore, a series of studies demonstrated that some miRNAs could participate in the regulation of FERMT2 expression. For instance, a previous report showed that miR-200b resulted in the inhibition of EMT and metastasis through targeting FERMT2 (Sossey-Alaoui et al., 2018; Zhang et al., 2014). Similarly, it has been reported that miR-138 inhibited the metastasis of prostate cancer via the specific suppression of FERMT2 (Selth et al., 2012; Sossey-Alaoui&Plow, 2016).Some limitations in the present study should be mentioned for future investigations. First, the exact mechanisms and signaling pathways involved in the anticancer actions of miR-16-5p have not been fully elucidated. The more detailed understanding about the influences of cellular signal pathway induced by miR-16-5p will be beneficial to its better applications in lung cancer therapy. Secondly, in this study, we only performed the assay of tumor formation in animal models. Considering the high metastasis potential of lung cancer,  more proper methods, such as the tail vein injection, could be performed in further studies to explore whether miR?16-5p could influence lung cancer metastasis in vivo.  Thirdly, the dynamic changes of the miR-16-5p level were only analyzed between normal and lung cancer tissues. 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