The present study was conducted between August 1, 2014 and July 31, 2015 at the Inpatient Department of Medical Oncology, Yantai Shan Hospital, The Teaching Hospital of Binzhou Medical University (Yantai, China). The experiments were performed according to the relevant guidelines of the Code of Ethics of the World Medical Association for experiments involving humans and the Medical Ethics Committee of Binzhou Medical University (18).

A total of 20 patients (10 males and 10 females), who were pathologically diagnosed with lung adenocarcinoma for the first time and had not yet received chemotherapy, were included in the present study. Fresh lung adenocarcinoma and control tissues were obtained from the patients who underwent surgery. Written informed consent was obtained from all patients prior to the collection of lung tissue samples. The control and lung adenocarcinoma tissues were collected from the same patients, and control samples were normal adjacent control tissues.

Lung adenocarcinoma cells were incubated with let-7a-mimics or let-7a-inhibitor and harvested 48 h after miRNA treatment. Small RNA was isolated using RNAiso for Small RNA reagent (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed as previously described (14). The primers used to amplify let-7a (Shanghai GenePharm Co., Ltd., Shanghai, China) were as follows: Forward, 5′-ACACTCCAGCTGGGTGAGGTAGTAGGTTGT-3′ and reverse, 5′-AACATGTACAGTCCATGGATG-3′. Human 5S rRNA served as the positive control. The primers used to amplify 5S rRNA (GenePharm Co., Ltd.) were as follows: Forward, 5′-GCCATACCACCCTGAACG-3′ and reverse, 5′-AACATGTACAGTCCATGGATG-3′. Total RNA was isolated using TRIzol reagent (Takara Biotechnology Co., Ltd.). The primers used for amplifying cyclin D1 (GenePharm Co., Ltd.) were as follows: Forward, 5′-CTGGCCATGAACTACCTGGA-3′ and reverse, 5′-GTCACACTTGATCACTCTGG-3′. qPCR was performed on an RG3000 system (Qiagen GmbH, Hilden, Germany) under the following reaction conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, annealing at 60°C for 20 sec and extension at 72°C for 30 sec. GAPDH cDNA served as the positive control (18). The primers used for amplifying GAPDH (Shanghai GenePharm, Co., Ltd.) were as follows: Forward, 5′-GTCTTCACCACCATGGAGAAGG-3′ and reverse, 5′-GCCTGCTTCACCACCTTCTTGA-3′.

The sequence of cyclin D1-3′ UTR was obtained from GenBank. The primers were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) and then synthesized by Shanghai GenePharma Co., Ltd. The primers used to amplify cyclin D1-3′ UTR were as follows: forward, 5′-TGCTCTAGATGAATTCTTATCCCCTGCCC-3′ and reverse, 5′-CGCGGATCCAAGAGAAGAGGGACACAGCC-3′. The amplification template was human genomic DNA. Then, cyclin D1-3′ UTR was inserted into the pcDNA3.1-GFP-neo (+) expression vector.

Lung adenocarcinoma cell lines (A549 and H1299) were obtained from the Shanghai Institute of Cell Biology (Shanghai, China). HBE 135E6E7 cells were obtained from the American Type Culture Collection (human bronchus epithelial; ATCC^® CRL-2741; Manassas, VA, USA). The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. Cells (2×10⁵) were seeded into 6-well plates. Let-7a mimics, let-7a inhibitors, negative controls and si-cyclin D1 were synthesized by Shanghai GenePharma Co., Ltd. The target sequence of si-cyclin D1 was as follows: Sense, 5′-CAAACAGAUCAUCCGCAA-3′ and antisense, 5′-UUUGCGGAUGAUCUGUU-3′. Transfection was performed in triplicate at ~60% confluence using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol (19).

Cell proliferation assays were conducted using a modified colorimetric MTT assay as previously described (20). All procedures were repeated three times. Cell colony formation rate was assessed using a plate colony formation assay. The plate was gently washed and stained with crystal violet. Then, the number and size of colonies was analyzed.

An apoptosis assay was performed 48 h after the oligonucleotides were transfected into lung adenocarcinoma cells. The assay was performed using Annexin V-FITC/PI (BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol (19).

A cell cycle assay was performed 48 h after transfection. The cells were collected using a Cell Cycle Detection kit (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China) according to the manufacturer's protocols and counted by flow cytometry (21).

Western blot analysis was performed as previously described (22). The antibodies used were as follows: Rabbit antibodies against GAPDH (1:800; cat. no. AP0063), Rb (1:800; cat. no. BS1311), p-Rb (1:800; cat. no. BS4164), Bcl-2 (1:800; cat. no. BS1511), Bax (1:800; cat. no. BS2538; all from Bioworld Technology, Inc., St. Louis Park, MN, USA) and caspase-9 (1:1,000; cat. no. 9502), caspase-8 (1:1,000; cat. no. 9748) and caspase-3 (1:1,000; cat. no. 9662; all from Cell Signaling Technology, Inc., Danvers, MA, USA). GAPDH was used as an internal reference.

An invasion assay was performed using a modified two-chamber plate with a pore size of 8.0 µm (23) as previously described (14). The Transwell migration assay was conducted according to the same protocol as the invasion assay, with the exception that the cell suspension was added into the upper chamber directly, without Matrigel.

SPSS 22.0 software (IBM Corp., Armonk, NY, USA) was used to analyze the significance of all results. One-way analysis of variance (ANOVA) was used to analyze the differences among three or more groups. A post-hoc test of ANOVA was conducted by performing a Tukey's test. Correlations were calculated with Spearman's rank correlation coefficiant. Group means were compared using an unpaired, two-sided, Student's t-test. Wilcoxen signed-rank test was used to compare the expression of let-7a and cyclin D1 in para-carcinoma and carcinoma tissues. Array data of cyclin D1 were downloaded from data link(s): https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13213. Overall survival was determined using Kaplan-Meier survival analysis by a log-rank test. P<0.05 was considered to indicate a statistically significant difference (11,23). All experiments were performed in triplicate, and all data are expressed as the mean ± standard deviation.

Although let-7a has previously been investigated in other types of cancer (24), the molecular mechanism of let-7a in lung adenocarcinoma remains unclear. In the present study, we first collected the clinicopathological features of patients with lung adenocarcinoma (Table I). Lung cancer tumor-node-metastasis (TNM) staging was based on the 8th edition of the Union for International Cancer Control (UICC) (25). The number of nuclear fission images or proliferation index, necrosis range, invasion status and other parameters were used to determine the lung cancer grade according to the tumor structure and cell atypia in H&E stained sections (26–29). Then, let-7a expression was analyzed in lung adenocarcinoma tissues to investigate the roles of let-7a in lung cancer. The current results indicated that let-7a levels were significantly lower in lung adenocarcinoma tissues (n=20) compared with para-carcinoma tissues (2 cm from the para-carcinoma tissues, n=20, Fig. 1A; P<0.001). The current results indicated that let-7a is involved in the progression of lung adenocarcinoma.

Kaplan-Meier survival analysis was performed to determine the significance of cycin D1 expression in affecting the outcome of lung cancer patients from the GSE13213 dataset. The poor survival time of patients was not significantly correlated with age (P=0.360) or sex (P=0.278). Notably, patients with high cyclin D1 levels exhibited short survival time (P=0.003; Fig. 1B) through a log-rank test analysis, suggesting that higher cyclin D1 levels increase the risk of lung adenocarcinoma. Then, the expression of cyclin D1 was evaluated in lung adenocarcinoma tissues. The results indicated that the expression of cyclin D1 was considerably higher in lung adenocarcinoma tissues (n=10) compared with para-carcinoma tissues (2 cm from the para-carcinoma tissues, n=10, Fig. 1C). Subsequently, we assessed the protein and mRNA expression levels of cyclin D1 in para-carcinoma and carcinoma tissue samples, and our results revealed that cyclin D1 levels were significantly higher in lung adenocarcinoma tissues (n=20) than those in para-carcinoma tissues (Fig. 1D and E; P<0.0001). These results indicated that lower levels of let-7a and higher levels of cyclin D1 may be associated with the development of lung adenocarcinoma.

Let-7a levels were detected in lung adenocarcinoma cell lines (A549 and H1299) and control human bronchus epithelial (HBE) cell line by qPCR. Notably, let-7a levels were reduced in the two lung adenocarcinoma cell lines compared with the HBE cells (Fig. 2A; P<0.001). Let-7a-mimic oligo treatment led to significant upregulation of let-7a compared with the control group (P<0.05). Conversely, the expression of let-7a was significantly reduced in lung adenocarcinoma cell lines following let-7a-inhibitor transfection (P<0.05; Fig. 2B and C).

To elucidate the molecular mechanisms of let-7a in the suppression of the growth of lung adenocarcinoma cells, the putative target gene of let-7a was identified. It was indicated that cyclin D1 was a target gene of let-7a with high probability using TargetScan software online (Fig. 3A; http://www.targetscan.org/vert_71/). Then, a cyclin D1-3′ UTR GFP plasmid was constructed to transfect cells with let-7a. The results indicated that GFP fluorescence intensity and positive rate decreased significantly in let-7a-treated cells compared with the control treatment (P<0.05; Fig. 3B), indicating that cyclin D1-3′ UTR was directly targeted by let-7a. Next, the effects of let-7a on cyclin D1 expression in lung cancer cell lines were investigated by western blot analysis. Notably, the expression of cyclin D1 was significantly reduced in lung adenocarcinoma cell lines following overexpression of let-7a (P<0.05). Conversely, the let-7a inhibitor resulted in the significant upregulation of cyclin D1 compared with the control group (P<0.05; Fig. 3C-F). Cyclin D1 is a positive regulator of cell cycle progression (11), and these results indicated that let-7a suppresses lung cancer cell growth by regulating the pathway of cyclin D1.

To further assess the potential inhibitory roles of let-7a in lung adenocarcinoma, the effects of let-7a on cell proliferation and colony formation were studied. An MTT assay indicated that the proliferation of lung adenocarcinoma cells in the let-7a-treated groups was significantly decreased compared with the scrambled oligo-treated cells (P<0.05). In contrast, the let-7a-inhibitor caused a significant increase in cell proliferation compared with the scrambled oligo treatment (P<0.05; Fig. 4A and B).

A colony formation assay was performed to evaluate the long-term effect of let-7a on cell proliferation. Compared with the scrambled control treatment, a smaller size and a decreased number of clones were observed in the let-7a-treated cells, suggesting that the upregulation of let-7a significantly suppressed the colony formation ability of A549 and H1299 cells (Fig. 4C and D).

Cell apoptosis was detected following transfection of lung adenocarcinoma cells with let-7a mimics and controls. Cell apoptosis was significantly increased in let-7a-treated A549 and H1299 cells, indicating that let-7a overexpression induced lung adenocarcinoma cell apoptosis (P<0.05; Fig. 5A and B). To further characterize the possible mechanisms of let-7a in inducing cell apoptosis, the apoptotic genes, Bcl-2 (as an anti-apoptotic protein) and Bax (as a pro-apoptotic protein), were studied. It was determined that let-7a treatment significantly downregulated Bcl-2 expression and upregulated the expression of Bax and cleaved caspase-3, −8 and −9, compared with the control treatment in A549 and H1299 cells. In contrast, the opposite trends for these proteins were observed in cells transfected with the let-7a-inhibitor (Fig. 5C and D). These findings demonstrated that the Bcl-2/Bax pathways and the activation of caspase-3, −8 and −9 were constitutively downregulated by let-7a overexpression, and that the Bcl-2 family proteins are involved in the apoptosis induced by let-7a.

The effects of let-7a on cell cycle regulation were evaluated by flow cytometric analysis (30). The results indicated that the distribution of the cell cycle in A549 and H1299 cells was notably affected by let-7a overexpression compared with the scrambled group. The cell cycle profile indicated that 77.02% of A549 cells and 79.01% of H1299 cells were arrested at the G₀/G₁ phase at 48 h after let-7a treatment, compared with 68.09% of NC-transduced A549 cells and 68.95% of NC-transduced H1299 cells. However, the let-7a inhibitor-transduced cells exhibited a smaller percentage of arrest at the G₀/G₁ phase and increased cells inhibited in the G₂/M phase compared with let-7a treatment (Fig. 6A and B). These experiments demonstrated that let-7a exerts an inhibitory effect on cell cycle progression. To investigate the potential molecular mechanism of let-7a in cell cycle arrest, the expression of cell cycle molecules, namely cyclin D1, Rb and p-Rb, was detected. Let-7a treatment significantly decreased the expression of cyclin D1 and p-Rb, but increased the Rb protein expression compared with the controls. The opposite trends were observed in let-7a inhibitor-transduced cells (Fig. 6C and D). Therefore, let-7a induces G0/G1 arrest in A549 and H1299 cells, which may be mediated by downregulation of cyclin D1 and upregulation of Rb.

Cyclin D1-associated factors may affect the migration and invasion potential of breast cancer cells (31). In the present study, Transwell experiments were performed to detect whether let-7a inhibits lung cancer cell migration and invasion by influencing cyclin D1-associated factors. The number of migrated cells in let-7a-treated A549 and H1299 cells was significantly reduced compared with the controls (P<0.0001; Fig. 7A and B), while the let-7a inhibitor increased the number of migrated cells. There was no significant increase in the rate of migration in A549 and H1299 cells co-transfected with let-7a-inhibitor and si-cyclin D1 (P<0.001; Fig. 7E and F). Furthermore, an invasion assay demonstrated that invasive ability was significantly decreased in let-7a-transfected A549 and H1299 cells compared with the control groups (P<0.0001; Fig. 7C and D), and the effects of let-7a on invasion could be reversed by knockdown of cyclin D1 using siRNA (P<0.001; Fig. 7G and H). These results indicated that let-7a inhibits cell migration and invasion by targeting cyclin D1.