#These authors contributed equally to this work.
Increasing evidence indicates that aberrant expressions of some microRNAs are associated with cancer progression. However, the roles and biological mechanisms of miRNA-16-5p in human non-small cell lung cancer (NSCLC) are not to be well studied. Here, we validated that the expression of miR-16-5p was decreased significantly in NSCLC samples and cell lines. The correlation between the clinicopathological features of NSCLC and the miR-16-5p expression showed that the expression of miR-16-5p in non-small cell lung cancer was linked with the advanced TNM stage, positive lymph node metastasis, with short overall survival (OS). Also, a negative correlation between miR-16-5p and Fermitin family member 2 (FERMT2) was observed, implying there may be a potential link about their regulation. The hypothesis was further confirmed by in-silico analysis and dual-luciferase reporter assay. Moreover, we demonstrated that the transfections of miR-16-5p mimics could alter some biological characteristics of NSCLC cells remarkably accomplished by the expression variance of FERMT2
Lung cancer is one of the most severe malignant tumors and one of the leading causes of cancer-related deaths in the world. Non–small-cell lung cancer (NSCLC) accounts for about 80% of clinical cases of lung cancer. 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% (
MiRNA is a small non-coding RNA molecule with a length of about 17–25 nucleotides to boost the degeneration of mRNAs or suppress the mRNA translation by binding to the 3’-untranslated regions (3’-UTRs) of mRNAs, thus promoting the degradation of mRNA (
Cell adhesion to the cellular matrix is crucial for cell 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 (
In this study, we identified the expression of miR-16-5p and FERMT2 in clinical samples and cultured cell lines and then performed the correlation between the miR-16-5p and some clinicopathological parameters in NSCLC patients. As a negative correlation between miR-16-5p and FERMT2 was established, we further tested the hypothesis that FERMT2 could be guided by miR-16-5p in NSCLC cells. Bioinformatics analysis was further performed to verify FERMT2 as one probable target of miR-16-5p. The luciferase reporter assays further corroborated that FERMT2 is a direct target of miR-16-5p. Also, our results exhibit that the overexpression of miR-16-5p could influence some biological characteristics of NSCLC cell lines. Furthermore, the cotransfection of FERMT2 without 3’-UTR rescued the effects of miR-16-5p expression in NSCLC cells. The present study provided evidence that miR-16-5p exhibited an anti-cancer effect by targeting FERMT2 in NSCLC.
All experiments and assays for human samples and cell tissue were performed at Binzhou Medical University. Access to the relevant de-identified patient tissues and analysis of patient data was approved by the Institutional Review Board for Human Research at Binzhou Medical University. Written informed consent was obtained from all individual participants and the project was approved by the Ethics Board of Binzhou Medical University (approval number: 2019-23); Animal procedures are carried out in strict accordance with guidelines and procedures of the Institutional Animal Care and Use Committee (IACUC) of Binzhou Medical University (No. 2019-23).
In total, fifty-nine eligible NSCLC patients (33 males and 26 females) were enrolled at Yantai Shan Hospital, Binzhou Medical University, from June 2011 to July 2012. The lung cancer tissues and matched healthy tissues were derived from adult patients who had pathologically diagnosed as NSCLC and no adjuvant therapies before surgery, including chemotherapy, immune therapy, and radiotherapy. All patients were followed up after the operation, and their clinicopathological features were summarized and analyzed in
For
During transfections, both A549/H1299 cells were treated with miR-16-5p scram (Scram group, 50 nM), miR-16-5p mimics (Mimics group, 50 nM), and miR-16-5p mimics + FERMT2-without-3’-UTR (Rescue group, 50 nM), respectively. Briefly, cells were seeded into 6-well plates at a density of 200,000 cells/well and allowed to attach overnight until reaching about 70–80% confluence. The scram and mimics of miR-16-5p were synthesized by Shanghai GenePharmaCo., Ltd. (Shanghai, China). Cell transfections were performed with Lipofectamine™ 2000 (Life Technologies; Thermo Fisher Scientific, Inc.) using the manufacturer’s protocol. After incubation for 8 h, the medium was replaced with the standard culture medium containing 10% FBS. After 48 h, cells with different treatments were harvested for further analyses.
For colony formation assays, 1.5 × 103 cells with different treatments were seeded into each 10-cm plate. The plates were fixed with 100% methanol and stained in 20% ethanol containing 0.2% crystal violet dye two weeks later. Then, the numbers of colonies were analyzed. For MTT assays, the detection was performed according to the instrument of the manufacturer (Beyotime Biotechnology, cat. no. C0009). Briefly, 6 × 103 cells were seeded into 96-well plates in triplicate and treated with 10 μL reagents for further incubation 4 h. After 150 μL detergent reagents were added into wells, the absorbance was detected in the microplate reader at 570 nm wavelength.
For apoptosis analysis, cells with different treatments were harvested after 48 h, and the oligonucleotides were transfected into NSCLC cells, then washed twice with cold PBS, suspended the cells with the buffer then double-stained with Annexin V-FITC (5 μg/mL) and PI (5 μg/mL) (BD Pharmingen; NJ, USA). For cell cycle analysis, cells with different treatments were washed with cold PBS and then fixed in cold 75% ethanol (v/v) followed by 1h incubation on ice and 24 h incubation at −20°C. Fixed cells were resuspended in PBS containing RNase A (50 μg/mL) for 30 min, then added PI (50 μg/mL). Finally, both the apoptosis and cell cycle were analyzed by FACS (Beckman Coulter Flow Cytometer, CA, USA).
Transwell assay was used to detect the migration and invasion abilities of A549/H1299. Briefly, 1 × 106 cells in 100 µL serum-free medium were added into the upper chamber of 24-well plates directly. Serum-containing media (20% FBS, 600 µL) served as a chemical attractant in the lower chambers. The upper chamber was taken after 48 h, and non-migrated cells were swapped by a cotton plug. Cells at the bottom of the membrane were then fixed with methanol stained with crystal violet and observed the sample under a microscope (Olympus, Tokyo, Japan). The number of invasive or migrated cells was discerned by calculating ten randomly selected fields of stained cells with ImageJ software.
For miR-16-5p expression analysis, small RNAs were isolated from cells from different groups with RNAiso Plus (Takara Biotechnology. Dalian, China) in which human 5S rRNA acted as the positive endogenous references. For the expression analyses of related genes, total RNA was isolated using TRIzol reagent. qPCR was performed with SYBR-Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an RG3000 System (Qiagen GmbH, Hilden, German), GAPDH was used as an endogenous control. The relative gene expression levels were calculated as relative quantification (2−ΔΔCt) method. All experiments were performed in triplicate. The primers used in this study are listed below:
miR-16-5p: F5’-ACACTCCAGCTGGGTAGCAGCACG TAAATA-3’
R5’-AACATGTACAGTCCATGGATG-3’
5S rRNA: F5’-GTTGTCTCCTGCGACTTCA-3’
R5’-GGTGGTCCAGGGTTTCTTA-3’
FERMT2: F5’-TATCATTTTATCATGAAAACTTCTT-3’
R5’-GACTTTATCACCCGTTTA-3’
GAPDH: F5’- GTTGTCTCCTGCGACTTCA-3’
R5’- GGTGGTCCAGGGTTTCTTA-3’
Total protein was extracted from the lung carcinoma cells by RIPA lysis buffer (Solarbio, Beijing, China) and then quantified. Subsequently, the protein was separated by 10% SDS-PAGE, followed by transferring to the PVDF membrane (Sigma-Aldrich, USA) and then blocked in 5% non-fat skimmed milk at room temperature for 1 h. The PVDF membrane was incubated with the primary antibodies (1:1000) overnight at 4°C. The rabbit antibodies (LI-COR, C60405-05, USA) were added to the PVDF membrane after primary incubation. Finally, Tanon 5200 was used to detect the protein expression signal. The primary antibodies for detection were as follows: rabbit antibodies against FERMT2 (Abcam, cat. no.ab127745), GAPDH (Bioworld Technology, cat. no. AP0063), cleaved caspase-3 (Cell Signaling, cat. no. 9661), cleaved caspase-8 (Cell Signaling, cat. no. 9748) and cleaved caspase-9 (Cell Signaling, cat. no. 9505), Bcl-2 (Bioworld Technology, cat. no. BS1511), Bax (Bioworld Technology, cat. no. BS2538), p-Rb (Bioworld Technology, cat. no. BS4164), MMP-2 (Thermo Scientific, cat. no. MA5-14186), MMP-9 (Thermo Scientific, cat. no. MA5-13595), GAPDH as a loading control for protein normalization. The intensity of each protein in Western blot was normalized to internal control of GAPDH with ImageJ software and expressed as a ratio of the densitometric value.
Prediction 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 was 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 triplicate in 100 μL/well, then transfected with miR-16-5p scram/mimics (50 nM, Suzhou GenePharma) together with wild-type/mutated luciferase reporter vector by Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific. CA, USA). Cells were harvested after 48 h, and the luciferase activities were detected by Luci-Pair Duo-Luciferase Assay kit 2.0 (GeneCopoeia, Rockville, USA).
The A549 cells (1 × 106) with different treatments were resuspended in 150 μL PBS and then injected subcutaneously 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 observe the effects of miR-16-5p on tumor formation and growth
All detections were performed at least in triplicate, and all data are represented as the mean ± standard error (SE). The statistical comparisons were analyzed using Student’s
Firstly, the small RNAs from NSCLC samples and their healthy counterparts were isolated to determine the differences in miRNA expression between cancer and normal healthy tissues. The results of qPCR indicated the expressions of miR-16-5p is decreased in NSCLC tissues compared with that in healthy counterparts (
(A) A comparison of the relative expression level of miR-16-5p in normal and tumor tissues determined by qRT-PCR. (B) Correlation of miR-16-5p expression with overall survival (OS) of NSCLC patients. Among 59 patients with NSCLC, the overall survival was plotted to compare OS between patients with low miR-16-5p and those with high miR-16-5p (
To evaluate whether miR-16-5p can be used as an index of prognosis in patients with NSCLC, the association of miR-16-5p expression and the critical clinicopathological indexes of patients was discussed. All 59 patients were classified based on the median expression level of miR-16-5p into the elevated group (N = 23) and low group (N = 36). The Chi-squared analysis showed that some of the clinicopathological parameters were not correlated with miR-16-5p expression in NSCLC patients, such as age, gender, smoking history, tumor size, and cancer histology (
Clinicopathological features | Patients (n = 59) | miR-16-5p in NSCLC patients | ||
---|---|---|---|---|
Low (n = 36) n (%) | High (n = 23) n (%) | |||
Age (years) |
22 | 13 (36.1) | 9 (39.1) | 0.815 |
>60 | 37 | 23 (63.9) | 14 (60.9) | |
Gender | 0.642 | |||
Male | 33 | 21 (58.3) | 12 (52.2) | |
Female | 26 | 15 (41.7) | 11 (47.8) | |
Smoker | 0.909 | |||
Non | 20 | 12 (33.3) | 8 (34.8) | |
Previous or current | 39 | 24 (66.7) | 15 (65.2) | |
Tumor size | 0.964 | |||
≦3 cm | 31 | 19 (52.8) | 12 (52.2) | |
>3 cm | 28 | 17 (47.2) | 11 (47.8) | |
TNM stage | 0.003 | |||
Early (I/II) | 20 | 7 (19.4) | 13 (56.5) | |
Advanced (III/IV) | 39 | 29 (80.6) | 10 (43.5) | |
Pathology | 0.498 | |||
Adenocarcinoma | 25 | 14 (38.9) | 11 (47.8) | |
Squamous cell carcinoma | 34 | 22 (61.1) | 12 (52.2) | |
Node metastasis | <0.001 | |||
Negative | 26 | 9 (25.0) | 17 (73.9) | |
Positive | 33 | 27 (75.0) | 6 (26.1) |
Note: Abbreviations: NSCLC, non-small cell lung cancer; TNM, tumor (T), the extent of spread to the lymph nodes (N), and the presence of metastasis (M).
Bioinformatics analysis was first carried out by TargetScan to find out the potential target of miR-16-5p. The prediction results show that there are some complementary sequences between 3’-UTR of miR-16-5p and FERMT2, indicating that FERMT2 may be a prospective target of miR-16-5p (
To elucidate the possible roles of miR-16-5p in NSCLC, the potential mechanistic link between miR-16-5p and FERMT2 was further determined. To this end, we additionally detected the miR-16-5p expression in three cell lines (HBE, A549, and H1299), respectively. The results showed that miR-16-5p was obviously reduced in A549/H1299 cells compared with that in HBE cells (
(A) The binding sequences of miR-16-5p and FERMT2 were predicted with TagetScan 5.2. (B) Luciferase reporter assay was performed in 293T cells co-transfected with miR-16-5p mimics and FERMT2-3’-UTR-WT/FERMT2-3’-UTR-MUT. (C) The levels of miR-16-5p expression in HBE, A549 and H1299 cells, tested by qRT-PCR analysis. (D) The expressions of miR-16-5p in A549 and H1299 cells transfected with miR-16-5p mimics. (E) The mRNA levels of FERMT2 expression in A549 and H1299 cells transfected with miR-16-5p mimics. (F) The protein levels of FERMT2 expression in A549 and H1299 cells transfected with miR-16-5p mimics. (G) The quantified analyses of the FERMT2 protein level in A549 and H1299 cells transfected with miR-16-5p mimics detected by Western blot. *
We next investigate the role of miR-16-5p in cell proliferation and colony formation. The results from colony formation assay uncover that the upregulation of miR-16-5p notably inhibited the capacity of colony formation in miR-16-5p-mimic-treated A549/H1299 cells compared with the scram cells. Furthermore, the cotransfection of FERMT2 without 3’-UTR can significantly rescue the suppressive competence of miR-16-5p on colony formation of both cells (
(A) Colony formation assays in A549 and H1299 cells transfected with miR-16-5p scram, mimics, and rescue (mimics + FERMT2-without-3’-UTR), respectively. (B) Cell colonies were counted and quantified by the histogram. (C) The MTT assays were performed to assess the viability of A549 and H1299 cells transfected with miR-16-5p scram, mimics, and rescue (mimics + FERMT2-without-3’-UTR), respectively. *
The effects of miR-16-5p on cell apoptosis and cell cycle distribution were observed by flow cytometry. As shown in
The data of cell cycle analysis showed that the cells proportion of A549 and H1299 cells in G0/G1 was higher significantly than that from the scram group under miR-16-5p simulation. Conversely, in both cells cotransfected with FERMT2-without-3’-UTR, cells at the G0/G1 phase decreased substantially in both A549 and H1299 cells compared with those in the scram group (
To study the effects of miR-16-5p on the invasion of A549/H1299 cells, transwell assays were carried out.
(A) The percentages of apoptotic cells were detected in A549 and H1299 cells transfected with miR-16-5p scram, mimics and rescue (mimics + FERMT2-without-3’-UTR), respectively. (B) The percentages of apoptotic cells were quantified by the histogram. (C) The distributions of the cell cycle were detected in A549 and H1299 cells transfected with miR-16-5p scram, mimics and rescue (mimics + FERMT2-without-3’-UTR), respectively. (D) The distributions of the cell cycle were quantified by the histogram. (E) The transwell assays were performed to assess invasion capacity in A549 and H1299 cells transfected with miR-16-5p scram, mimics and rescue (mimics + FERMT2-without-3’-UTR), respectively. (F) The invaded cells were counted and quantified by the histogram. *
To further delineate the mechanisms underlying the miR-16-5p-regulated apoptosis, cell cycle, and invasion in these cells, the expressions of critical genes were assessed by Western blot. As shown in
(A) The apoptosis-, cell cycle- and invasion-related proteins were detected in A549 cells transfected with miR-16-5p scram, mimics and rescue (mimics + FERMT2-without-3’-UTR), respectively. (B) The quantified analyses of related proteins, detected by Western blot in A549 cells and indicated by the histogram. (C) The apoptosis-, cell cycle- and invasion-related proteins were detected in H1299 cells transfected with miR-16-5p scram, mimics and rescue (mimics + FERMT2-without-3’-UTR), respectively. (D) The quantified analyses of related proteins, detected by Western blot in H1299 cells and indicated by the histogram. C: cleaved, p-Rb: phosphate Rb, MMP: matrix metalloprotein. *
As the results from
(A) Comparison of tumor volume in tumor-bearing mice between the miR-16-5p mimics group and the scram group. The tumor volumes in different groups were measured on days 1, 6, 12, 18, 24, and 30, respectively. (B) The representative photos of tumor-bearing mice on day 30. The black arrows indicated the growing tumor after the inoculation on day 30. (C) The removed tumors on day 30 from mice with different treatments were shown. (D) The histograms represent the quantitative data of tumor weights between groups. (E) The miR-16-5p expression levels in each removed tumors. (F) The levels of FERMT2 protein in each removed tumor detected by Western blot. (G) The analysis of miR-16-5p expression between scram and mimics group. (H) The analysis of FERMT2 protein levels between scram and mimics group.*
Lung cancer has been the leading cause of cancer-related mortality, in which NSCLC accounts for 80–85% of cases (
In the present study, we set out to detect miR-16-5p expression in NSCLC samples and noticed that it was significantly down-regulated in the tissue of NSCLC compared with healthy counterparts. Also, a significant inverse correlation between miR-16-5p and FERMT2 expressions was revealed in NSCLC samples. The same results were observed in cultured lung cancer cells, such as A549 and H1299 cells. Moreover, miR-16-5p expression in NSCLC patients was associated with some clinicopathological features, such as advanced TNM stages, positive lymph node metastasis, as well as short overall survival. These data demonstrated that miR-16-5p presented the anti-cancer effect of miR-16-5p in NSCLC. To further elucidate whether the miR-16-5p potential functions in NSCLC tumorigenesis and development via FERMT2, a series of assays were carried out to observe the functional changes of miR-16-5p on the biological characteristics with or without of FERMT2-without-3’-UTR cotransfection in A549/H1299 cells. Moreover, in the xenograft model of nude mice, it was found that the induced expression of miR-16-5p can also inhibit tumor proliferation
MiRNA can regulate the target gene expression by recognizing the 3’-UTR, leading to mRNA of target gene 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 specific miRNA target genes is pivotal to elucidate the underlying mechanisms. To date, there have been several studies published recently about the predominant role of miR-16-5p in cancer deterioration. For example, miR-16-5p is reported to suppress the string tumor cells proliferation, invasion, and metastasis by targeting Smad3 (
FERMTs are evolutionary conserved, focal adhesion proteins that interact with the integrin family, in particular, the integrin beta1 (
Some limitations in the present study should be mentioned for future investigations. First, the number of NSCLC patients enrolled in this study is limited. Thus, more extensive clinical trials should be warranted to make a more robust and convincing conclusion about the relation between miR-16-5p and NSCLC development and prognosis. 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 summary, our study revealed that miR-16-5p showed noticeable anti-cancer effects for NSCLC in cell lines, animal models, and available clinical samples, which may involve in the abilities to the apoptosis induction, the proliferation suppression, and the inhibition of invasion. Moreover, definite evidence about the regulatory role of miR-16-5p targeting FERMT2 was also demonstrated. Our results provide further insight and new candidate targets for the promising therapeutic application in the treatment of NSCLC.
non–small-cell lung cancer
microRNAs
overall survival
miRNA-16-5p
Fermitin family member 2
glyceraldehyde 3-phosphate dehydrogenase
3’-untranslated region
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
epithelial-mesenchymal transition
quantitative-polymerase chain reaction
fluorescence activated cell sorting.