MicroRNA-346 facilitates cell growth and metastasis, and suppresses cell apoptosis in human non-small cell lung cancer by regulation of XPC/ERK/Snail/E-cadherin pathway

MiR-346 is down-regulated in primary NSCLC tissues and cell lines, and predicts a worse prognosis

miR-346 expression was measured in 114 NSCLC samples and corresponding adjacent normal tissues by qRT-PCR. miR-346 up-regulation was detected in 108/114 (94.74%) of NSCLC tumors (Fig. 1A). Average miR-346 expression was approximately 3.7-fold higher in NSCLC specimens as compared with corresponding adjacent normal tissues (P <0.05, Fig. 1A). Next, we examined miR-346 expression in NSCLC cell lines, and results demonstrated a higher expression of miR-346 in A549, SPC-A-1, H1299, 95-D, SK-MES-1, NCI-H520 and NCI-H460 cell lines, compared with that of in normal lung cells 16HBE (Fig. 1B). Although there was no significant association between miR-346 expression and sex, differentiation, or histological tumor type smoking, up-regulated expression of miR-346 was commonly observed in NSCLC patients with elder age, bigger tumor sizes, smokers, positive lymph node metastasis, and advanced stage (P <0.05, Table 1). Furthermore, multivariate Cox regression analysis revealed that high (>3.7 folds of increase, n=78) miR-346 expression, elder age, and advanced stage are independent predictors of overall survival in NSCLC patients (Table 2). Kaplan-Meier analysis indicated that high miR-346 expression was associated with poorer overall survival (log-rank test, P <0.0001, Fig.1C). Thus, it was concluded that the increased expression of miR-346 might make sense in initiation or development of NSCLC.

Figure 1. MiR-346 is up-regulated in primary human lung cancer and NSCLC cell lines, and predicts a worse prognosis. (A) miR-346 is significantly increased in primary human lung cancer tissues in comparison to adjacent-normal lung cancer tissues. n=114 for each group. (B) The expression level of miR-346 in seven NSCLC cell lines and normal 16HBE cells. Assays were performed in triplicate. (C) Kaplan-Meier survival analysis revealed that up-regulated miR-346 is associated with poor prognosis in patients with non-small cell lung cancer. *P < 0.001, Means ± SEM was shown. Statistical analysis was conducted using student t-test.

Expression of XPC is down-regulated in primary human NSCLC and negatively related to miR-346

XPC is important oncogene that shown strong power of tumorigenesis, by promotion of cell proliferation, metastasis, invasion and epithelial mesenchymal transition (EMT). Thus, we next examined XPC expression in human NSCLC and pair-matched adjacent lung tissues, and our western blot results demonstrated that the expression of XPC protein was significantly decreased in NSCLC tissues compared with lung tissues (Fig. 2A), which were verified by qRT-PCR of XPC mRNA expression (Fig. 2B). Moreover, we evaluated the correlation between XPC mRNA and miR-346 expression in 114 NSCLC tissues, and results revealed expression of XPC mRNA and miR-346 exhibited a significantly inverse correlation as calculated by Pearson correlation (r=-0.51, P <0.0001) (Fig. 2C).

Figure 2. Expression of XPC is up-regulated in primary human lung cancer and negatively expressed related to miR-346. (A-B) Western-blot of XPC protein and qRT-PCR of XPC mRNA in lung cancer tissues and adjacent-normal lung cancers. n=114 for each group. C. Scatter plots showing the inverse association between miR-346 level and XPC mRNA expression. *P < 0.001, Means ± SEM was shown. Statistical analysis was conducted using student t-test.

XPC expression is positively correlated with the outcome of NSCLC patients

A previous analysis of 126 NSCLC patients has shown that median survival of patients with lower XPC mRNA levels was shorter compared with patients with higher XPC mRNA levels [34]. To further explore the critical efficiency of XPC in the survival of NSCLC patients, we analyzed the relationship between the XPC mRNA expression level and the survival of NSCLC patients from 2437 lung tumors using publicly available datasets (2015 version) (http://kmplot.com/analysis/index.php?p=service&cancer=lung). The Kaplan-Meier analyses demonstrated that higher XPC mRNA expression in NSCLC patients is correlated with an improvement of the overall survival (OS), as well as progression-free (FP) survival of patients. These correlations are more pronounced in patients with adenocarcinoma but not squamous cell carcinoma (Fig. 3A-F). These analyses further confirmed the tumor suppressor role of XPC in NSCLC.

Figure 3. Prognostic significance of XPC in lung cancer. (A-B) The effect of XPC mRNA expression level on the overall survival and progression free survival in lung cancer patients was analyzed and the Kaplan-Meier plots were generated by Kaplan-Meier Plotter (http://www.kmplot.com). (C-D) The effect of XPC mRNA expression level on the overall survival and progression free survival in lung adenocarcinoma patients was analyzed and the Kaplan-Meier plots were generated by Kaplan-Meier Plotter (http://www.kmplot.com).

MiR-346 targets human XPC

To clarify the relationship between miR-346 and XPC, basic information about hsa-miR-346 was collected from miRBase. XPC is a putative miR-138-5p target predicted by DIANA (Fig. 4A). To verify XPC targeting by miR-346, reporter constructs in which the XPC 3’-UTR, either wild type or mutated in the miR-346 binding sites, was cloned downstream of the luciferase open reading frame (ORF) (Fig. 4A). miR-346 mimic or mimic NC was transfected into A549 and SPC-A-1 cells. When the XPC 3’-UTR was attached to the luciferase gene, luciferase activity decreased significantly (P <0.05) in A549 and SPC-A-1 cells transfected with miR-346 mimics, demonstrating that XPC was the target of miR-346 (Fig. 4B). Furthermore, expression of mutant XPC 3’-UTR restored luciferase activity (Fig. 4B). To examine the effect of miR-346 on endogenous XPC expression, we treated A549 and SPC-A-1 cells with NC, miR-346, si-XPC or ASO-346 for indicated time. Western blot assay revealed that both miR-346 and si-XPC treatment decreased the protein level of XPC in A549 and SPC-A-1 cells, while ASO-346 treatment showed an increase in the XPC protein expression than NC treated A549 and SPC-A-1 cells (Fig. 4C).

Figure 4. XPC proto-oncogene is a target of miR-346 at specific 3’-UTR sites. (A) The 3’-UTR of XPC harbors one miR-346 cognate site. (B) Relative luciferase activity of reporter plasmids carrying wild-type or mutant XPC 3’-UTR in A549 and SPC-A-1 cells co-transfected with negative control (NC) or miR-346 mimic. (C) Protein expression of XPC in A549 and SPC-A-1 cells after transfected with NC, miR-346, si-XPC and ASO-346. Assays were performed in triplicate. *P < 0.001, Means ± SEM was shown. Statistical analysis was conducted using student t-test.

MiR-346 facilitates cell proliferation and colony formation, and promotes G1/S transition through down-regulation of XPC in NSCLC

miR-346 was ectopically expressed in NSCLC cell lines. We determined the effects of miR-346 overexpression or inhibition, and XPC inhibition on cell proliferation via CCK8 assay. A549 and SPC-A-1 cells (which have high endogenous miR-346 expression) transfected with miR-346 mimics and si-XPC showed increased proliferation (P <0.05), which was rescued by ASO-346 treatment (Fig. 5A-B). However, loss of miR-346 suppressed cell proliferation in A549 and SPC-A-1 cells (Fig. 5A-B). To verify these results, we also did the colony formation assay, and results demonstrated miR-346 mimic treatment caused an increase in the clonogenic survival of A549 and SPC-A-1 cells compared with blank A549 and blank SPC-A-1 cells (Fig. 5C), while miR-346 inhibitor-treated A549 cells showed a significant decrease in the clonogenic survival, when compared with blank A549 and blank SPC-A-1 cells (Fig. 5C). Additionally, in A549 and SPC-A-1 cells transfected with miR-346 mimics or si-XPC, the number of cells in G1 phase of the cell cycle decreased and the number in S phase increased (P <0.05, Fig. 5C-D), and this was again rescued by ASO-346 treatment (P<0.05).

Figure 5. The miR-346 affects cell proliferation and cell cycle. A549 and SPC-A-1 cells were analyzed after transfection. (A-B) CCK8 assays of A549 and SPC-A-1 cells after transfected with NC, miR-346, si-XPC and ASO-346. (C) Shown are representative photomicrographs of colony formation assay after transfected with NC, miR-346, si-XPC and ASO-346 for fourteen days. (D-E) Cell-cycle analysis was performed forty eight hours following the treatment A549 and SPC-A-1 cells with NC, miR-346, si-XPC and ASO-346. The DNA content was quantified by flow cytometric analysis. Assays were performed in triplicate. *P < 0.001, Means ± SEM was shown. Statistical analysis was conducted using student t-test.

MiR-346 promotes NSCLC cell migration and invasion in vitro

Next, we examined the role of miR-346 on A549 and SPC-A-1 cells migration and invasion. We used Transwell migration approache to assess the role of miR-346 on the ability of A549 and SPC-A-1 cells migration. Results revealed that migration of miR-346 mimic or si-XPC treated cells was increased by 0.32-fold or 0.65-fold in A549 and 0.53-fold or 0.96-fold in SPC-A-1 cells, compared with the blank A549 and SPC-A-1 cells (Fig. 6A-B), respectively. However, when treated with ASO-346, migration in miR-346-expression defected A549 and SPC-A-1 cells were significantly decreased by approximately 48% and 31% relative to blank A549 and SPC-A-1 cells (Fig. 6A-B) respectively.

Figure 6. Ectopic expression of miR-346 in A549 and SPC-A-1 cells reduces cell migration and invasion motility. (A-D) A549 and SPC-A-1 cells were loaded onto the top well of a transwell inserts for cell migration or invasion assay. After twenty four hours, cells that migrated to the bottom chamber containing serum-supplemented medium were stained with 0.1% crystal violet, visualized under a phase-contrast microscope, and photographed. Total number of cells in five fields was counted manually. Assays were performed in triplicate. *P < 0.001, Means ± SEM was shown. Statistical analysis was conducted using student t-test.

To investigate the role of miR-346 on A549 and SPC-A-1 cells invasion, we used a transwell invasion assay. As expected, invasion of miR-346 mimic or si-XPC treated cells was increased by 0.46-fold or 0.54-fold in A549 and 1.1-fold or 0.96-fold in SPC-A-1 cells, relative to the blank A549 and SPC-A-1 cells (Fig. 6C-D), respectively. However, when treated with ASO-346, invasion in miR-346-expression defected A549 and SPC-A-1 cells were significantly decreased by approximately 58% and 49% relative to blank A549 and SPC-A-1 cells (Fig. 6C-D), separately. Taken together, these results clearly demonstrated that miR-346 expression markedly promoted the migration and invasion motility of NSCLC cells through targeting XPC.

MiR-346 suppresses NSCLC cell apoptosis

We then explored the efficiency of miR-346 on A549 and SPC-A-1 cells apoptosis. Our results of flow cytometric analysis revealed that miR-346 mimic or si-XPC treatment contributed to a 62% and 70%, or 56% and 67% of decrease in apoptotic cell death of A549 and SPC-A-1 cells (Fig. 7A-B), respectively. However, the percentage of apoptotic cells induced by miR-346 was increased to the basal level when the cells were treated with the specific miR-346 inhibitor (Fig. 7A-B). In addition, we also tested the caspase-3 and caspase-7 activity after treatment of A549 and SPC-A-1 cells with NC, miR-346 mimic, si-XPC or ASO-346, and results showed that miR-346 mimic and si-XPC treatment significantly decreased the caspase-3 and caspase-7 activity in A549 and SPC-A-1 cell lysate, by approximately 74% and 79%, or 54% and 49% of decrease (caspase-3 activity), 83% and 92%, or 47% and 41% of decrease (caspase-7 activity), than that of in blank A549 and SPC-A-1 cells (Fig. 7C-F), respectively. However, loss of miR-346 by transfecting with ASO-346 remarkably reduced the caspase-3 and caspase-7 activity in A549 and SPC-A-1 cell lysate, compared with that of in blank A549 and blank SPC-A-1 cells (Fig. 7C-F), respectively. These results demonstrated that miR-346 indeed promoted apoptosis in A549 and SPC-A-1 cells.

Figure 7. Ectopic expression of miR-346 suppresses apoptosis in A549 and SPC-A-1 cells. (A-B) Shown are statistical analysis of flow cytometric analysis. (C-E) Quantitative representation of caspase-3 and caspase-7 activity in A549 and SPC-A-1 cells transfected with NC, miR-346, si-XPC and ASO-346 for forty eight hours. Assays were performed in triplicate. *P < 0.001, Means ± SEM was shown. Statistical analysis was conducted using student t-test.

MiR-346 suppresses tumor growth in vivo

To validate the oncogenic role of miR-346 in vivo, we established a BALB/c nude mouse xenograft model using A549 cells. After 8 days, NC, miR-346 mimic, si-XPC or ASO-346 was directly injected into the implanted tumor every 4 days for seven times. The tumor volume was measured every 4 days until day 36. The miR-346 expression was examined by qRT-PCR (Fig. 8C). The tumor volume and weight of mice treated with miR-346 mimic or si-XPC were significantly increased (0.56-fold or 0.38-fold of increase in tumor weight, respectively) relative to that of treated with NC (Fig. 8A-B). While ASO-346 treatment significantly decreased the tumor volume and tumor weight of mice. This result demonstrates miR-346 significantly facilitates the tumorigenicity of A549 cells in the nude mouse xenograft model. In addition, our results of western-blot demonstrated that the decreased expression (48% or 55% of decrease) of XPC in tumors developed from miR-346 mimic or si-XPC treated nude mice relative to that of control tumors (Fig. 8D). Thus, miR-346 promotes the growth of established NSCLC xenografts.

Figure 8. Ectopic expression of miR-346 facilitates tumor growth in vivo. (A) Tumor volume in nude mice. (B) Tumor weight in nude mice. Each group contained six mice (n = 6); the data are presented as the mean ± SEM; *p < 0.001, compared with the NC group. (C) The expression of miR-346 in nude mice. (D-F) The expression of XPC protein in nude mice. Assays were performed in triplicate. *P < 0.001, Means ± SEM are shown. Statistical analysis was conducted using student t-test.

miR-346 directly targeting XPC and then activating ERK/Snail pathway, which leads to decrease of E-cadherin

It has been reported that the expression of Snail can be down-regulated through the inhibition of the PI3K/AKT or MAPK/ERK pathway [35,36]. And XPC suppressed Snail expression by inhibition of ERK activation [30] (Fig. 9). To investigate whether these pathways are affected by miR-346 modulation, we determined the phosphorylation of ERK1/2 in A549 and SPC-A-1 cells after being transiently transfected with NC, miR-346 mimic, si-XPC or ASO-346.

Figure 9. miR-346 directly targeting XPC and then activating ERK/Snail pathway, which leads to decrease of E-cadherin. (A-B) Expression of XPC, phospho-ERK1/2, ERK2, Snail, and E-cadherin were detected in A549 and spc-a-1 cells either transiently transfected with NC, miR-346, si-XPC, or ASO-346. The intensity of XPC, phospho-ERK1/2, ERK2, Snail, and E-cadherin bands were quantified using Image J. Assays were performed in triplicate. *P < 0.05. Means ± SEM was shown. Statistical analysis was conducted using ANOVA.

Results demonstrated both miR-346 mimic and si-XPC suppressed the protein expression of XPC, and then resulted in an increased phosphorylation of ERK1/2 in both A549 and SPC-A-1 cell lines (Fig. 10A-B), indicating that XPC is able to inhibit the ERK pathway in NSCLC cells, and miR-346 could activate ERK pathway by directly targeting XPC. In addition, both miR-346 mimic and XPC knockdown -enhanced Snail expression could be blocked by the inhibition of the ERK pathway, accompanied by an increased expression of E-Cadherin (Fig. 10A-B). These data suggest that miR-346 may promote NSCLC cell growth, migration and invasion, and suppress NSCLC cell apoptosis through targeting 3’-UTR of XPC mRNA, leading to inhibition of XPC protein expression, and then contributing to the activation of the ERK pathway. Activated ERK pathway enhances Snail expression, which further suppresses E-Cadherin expression, leading to an accelerated cell proliferation, migration and invasion, and a inhibitory cell apoptosis.

Figure 10. Model of the mechanism by which miR-346 suppresses XPC then activating ERK/Snail pathway, which leads to down-regulation of E-Cadherin expression and facilitates cell proliferation and metastasis, and inhibits cell apoptosis in lung cancer. miR-346 directly targeting 3’-UTR of XPC mRNA, contributes to down-regulation of XPC protein. The deficiency of XPC induces the accumulation of the endogenous DNA damage, which activates the ERK pathway. The activated ERK pathway then enhances the expression of Snail, which further represses the expression of E-Cadherin. Decreased expression of E-Cadherin by XPC silencing leads to a promotion of lung cancer cell proliferation and metastasis, and inhibition of lung cancer cell apoptosis.

Tissue collection

Fresh and formalin-fixed, paraffin-embedded, NSCLC tumor tissue samples were obtained from patients who were diagnosed with primary NSCLC. Elective surgery was carried out on these patients at People's Hospital of Wuhan University (Wuhan, China). In total, 20 cases of fresh NSCLC tissues were freshly frozen in liquid nitrogen and stored at –80°C until further use. 114 cases of archived, formalin-fixed, paraffin-embedded NSCLC tissue samples were collected and used in clinicopathological and prognostic investigation of miR-346. A comprehensive set of clinicopathological data were recorded, including age, gender, size of primary tumor, tumor differentiation, T stage, lymph node metastasis, and distant metastasis. The stage of disease was determined according to the tumor size, lymph node, and metastasis (pTNM) classification system. The use of tissues for this study has been approved by the ethics committee of People's Hospital of Wuhan University. Before using these clinical materials for research purposes, all the patients signed the informed consent. None of these patients received any pre-operative chemotherapy or radiotherapy.

Cell culture and transfection

The human NSCLC cell lines, namely, A549, H1299, SPC-A-1, 95D, SK-MES-1, NCI-H520, NCI-H460 and human normal lung epithelial cell line 16HBE were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10%. fetal bovine serum (FBS). MiR-346 mimic and mimic negative control, miR-346 inhibitor and inhibitor negative control were purchased from GenePharma Co.,Ltd. (Shanghai, China). Complete medium without antibiotics was used to culture the cells at least twenty-four hours prior to transfection. The cells were washed with 1× PBS (pH7.4) and then transiently transfected with 50 nM NC or miR-346, 100 nM ASO-346 or si-XPC, using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Western blot analysis

Western blot was carried out using the protocol described previously [31–33]. The following primary antibodies were used: rabbit anti-XPC (Santa Cruz, USA), rabbit anti-ERK2 (Santa Cruz, USA), rabbit anti-p-ERK1/2 (Santa Cruz, USA), rabbit anti-Snail (Santa Cruz, USA), anti-E-cadherin (Santa Cruz, USA) and rabbit anti-GAPDH (Santa Cruz, USA).

RNA isolation and quantitative reverse transcription poly-merase chain reaction (qRT-PCR)

RNA isolation and qRT-PCR was carried out using the protocol described previously [16,17]. GADPH and U6 were used as endogenous controls. In addition, melting curves were used to evaluate non-specific amplification. The relative expression level was calculated using the 2^-ΔΔCt method. The primer sequences used in this study are as follows: human XPC: sense: 5'- GACAAGCAGGAGAAGGCAAC-3', antisense: 5'- GGTTCGGAATCCTCATCAGA-3'; human E-Cadherin: sense: 5’-TGCCCAGAA AATGAA AAAGG-3’, antisense: 5’-GTGTATGTGGCA ATGCGTTC-3’; human Snail: sense: 5’-CCTCAA GATGCACATCCGAAG-3’, antisense: 5’-ACATGGCCTTGTAGCAGCCA-3’; human GAPDH: sense: 5’-GAAGGTGAAGGTCGGAGT-3’, antisense: 5’-GAAGATGGTGATGGGATTTC-3’. The formula and its derivations were obtained from the ABI Prism 7300 sequence detection system user guide. The relative expression values of XPC, E-Cadherin, and Snail were calculated and normalized to GAPDH in each sample and compared. The experiments were performed in triplicates. human GAPDH: sense: 5'-CTCTGCTCCTCCTGTTCGAC-3', antisense: 5'-ACCAAATCCGTTGACTCCGA -3'.

Colony formation assay

Colony formation assay was carried out using the protocol described previously [16,17].

Luciferase reporter assays

Luciferase reporter assays was carried out using the protocol described previously [16,17].

BrdU immunofluorescence assay

BrdU immunofluorescence assay was carried out using the protocol described previously [16,17].

CCK8 assay

Cell growth was measured using the cell proliferation reagent WST-8 (Roche Biochemicals, Mannheim, Germany). After plating cells in 96-well microtiter plates (Corning Costar, Corning, NY) at 1.0× 10 ³ /well, 10 μL of CCK8 was added to each well at the time of harvest, according to the manufacturer’s instructions. One hour after adding CCK8, cellular viability was determined by measuring the absorbance of the converted dye at 450 nm.

Cell-cycle analysis

Transfected cells were harvested forty-eight hours after transfection. The cells were fixed in 70% ethanol, washed once with PBS, and then labeled with propidium iodide (Sigma-Aldrich) in the presence of RNase A (Sigma-Aldrich) for 30 min in the dark (50 g/mL). Samples were run on a FACSalibur flow cytometer (Becton-Dickinson, FL, NJ, USA), and the percentages of cells within each phase of the cell cycle were analyzed using Cell Quest software.

Statistical analysis

All experiments were repeated 3 times independently. The results are presented as the means ± standard error mean (SEM). Two independent sample t-test or One-Way Analysis of Variance (ANOVA) was performed using SPSS 19.0 software in order to detect significant differences in measured variables among groups. A value of P <0.05 was considered to indicate a statistically significant difference.