Increasing evidence has revealed that aberrant expression of miRNAs contributes to non-small cell lung cancer (NSCLC) development and progression. However, the roles and mechanisms of various miRNAs in NSCLC remain to be determined. In the present study, we confirmed that reduced miR-212 expression was present in NSCLC tissues and cell lines. Our clinical analysis revealed that the reduced miR-212 expression was significantly correlated with poor prognostic features including positive lymph node metastasis and advanced tumor-node-metastasis (TNM) stage. Moreover, we demonstrated that miR-212 is a novel independent prognostic marker for predicting 5-year survival of NSCLC patients. The ectopic overexpression of miR-212 inhibited cell migration, invasion and EMT, while downregulated miR-212 reversed the effect. In addition, miR-212 regulated SOX4 by directly binding to its 3-untranslated region (3′-UTR), leading to suppression of EMT progression. In clinical samples of NSCLC, miR-212 was negatively correlated with SOX4, which was upregulated in NSCLC. Alteration in SOX4 expression reversed the functional effects of miR-212 in regards to migration, invasion and EMT in the NSCLC cells. In conclusion, our data indicated that miR-212 functions as a tumor-suppressor gene by regulating EMT and metastasis of NSCLC by targeting SOX4 signaling, and may represent a novel potential therapeutic target and prognostic marker for NSCLC.
Lung cancer is one of the most common malignancies and the leading cause of cancer-related mortality worldwide. Among all cases of lung cancer, more than 80% of patients present with non-small cell lung cancer (NSCLC) with a dismal 5-year survival (
MicroRNAs (miRNAs) are a family of endogenous, short single-stranded and non-coding RNAs that negatively modulate gene expression by binding to the 3′-untranslated region (3′-UTR) of target mRNAs to cause degradation or inhibition of translation (
In the present study, we investigated the expression level and biological function of miR-212 in NSCLC progression. Our data showed that miR-212 was downregulated in NSCLC and the reduced miR-212 was correlated with poor prognostic characteristics and worse 5-year survival of NSCLC patients. We demonstrated that miR-212 regulated the migration and invasion of NSCLC by targeting SOX4
Clinical specimens were collected from 115 NSCLC patients who received surgical resection in our hospital during 2002 and 2011. All the clinical tissues from the patients were pathologically confirmed as NSCLC before being used for further experiments in the present study. Informed consent was obtained from every patient involved in the present study. Approval for experiments involving patient samples was obtained from the Institutional Research Ethics Committee of The Affiliated Hospital of Hangzhou Normal University.
Cell lines including H292, H1299, A549, SPC-A1 and BEAS-2B were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and the American Type Culture Collection (ATCC; Rockville, MD, USA). All cells were cultured in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco Co., New York, NY, USA). Cell cultures were kept in cell incubators with humidified atmosphere and 5% CO2 at 37°C.
RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) was used to extract RNA from the NSCLC tissues and cells. Transcriptional First Strand cDNA Synthesis kit (Roche, Indianapolis, IN, USA) and SYBR-Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were used for reverse transcription reactions and real-time PCR. Primers for miR-212, U6, SOX4 and GAPDH were purchased from GeneCopoeia (Guangzhou, China). U6 and GAPDH were the internal controls for miR-212 and SOX4, respectively.
Total protein was extracted in RIPA buffer (Beyotime, Shanghai, China) containing a protease and phosphatase inhibitor, and then the protein concentration was determined using the BCA Protein Assay kit (both from Thermo Scientific, Rockford, IL, USA). Equal protein 30 µg was loaded and separated by 10% SDS-PAGE and transferred to polyvinyllidene diflouride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with respective primary antibodies: SOX4 and GAPDH (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight after blocking with 5% non-fat milk in TBS. Then, the membranes were washed three times with Tris-buffered saline with Tween-20 (TBST) and incubated with appropriate peroxidase-conjugated secondary antibody for 2 h at room temperature (ZSGB-BIO, Beijing, China). Protein bands were visualized using an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK).
The tissues were fixed in formalin, embedded with paraffin and sliced into 4-µm sections for immunohistochemical staining. After deparaffinizing, dehydrating and antigen retrieval, the slides were incubated in 3% H2O2 for blocking endogenous peroxidase activity. Then, after incubating with 10% goat serum for 30 min, E-cadherin and vimentin (1:300; Cell Signaling Technology, Inc.) antibodies were applied as the primary antibodies by a streptavidin peroxidase-conjugated (SP-IHC) method. The percentage of positive cells was expressed as: 0 for <10%; 1 for 10–30%; 2 for 31–50%; 3 for >50%.
miR-212 mimic and miR-212 inhibitor were obtained from GeneCopoeia Inc. SOX4 expression vector and SOX4-specific siRNA were purchased from Ruibo Biotechnology Corp. (Guangzhou, China). The tranfection of these vectors into NSCLC cells was performed in 6-well plates using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) based on the instructions from the manufacturer.
The migration and invasion abilities of the NSCLC cells were investigated by Transwell assays without or with Matrigel. Generally, NSCLC suspended in basal DMEM was seeded into the upper chamber and the lower chambers contained 600 µl DMEM with 20% FBS. Twenty-four to 48 h later, NSCLC cells that had migrated or invaded through the membranes and stayed on the lower surface were stained with crystal violet. Cell number for the migrated or invaded cells was counted under a microscope.
Wild-type SOX4 3′-UTR sequence and the mutated SOX4 3′-UTR sequence were constructed into the pGL3 control vector (Promega, Madison, WI, USA) to obtain wt SOX4-3′-UTR and mt SOX4-3′-UTR vectors, respectively. For the luciferase reporter assay, NSCLC cells were co-transfected with the wild-type construct or mutant construct, and, miR-212 mimics or inhibitor or control or negative control vector. Forty-eight hours after transfection, cells were harvested and lysed. The Dual-Luciferase Reporter Assay System (Promega, Shanghai, China) was used to determine firefly and
Data are presented as the mean ± SD and at least three independent replicates were performed. SPSS software 16.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) were used with a two-tailed Students t-test, Pearson's correlation analysis, Kaplan-Meier method and the log-rank test to evaluate the statistical significance. Significant differences were defined as P<0.05.
To determine the expression level of miR-212 in NSCLC, we first evaluated the expression of miR-212 in 115 paired NSCLC and adjacent normal lung tissues. miR-212 was significantly decreased in the NSCLC tissues compared to that noted in the matched tumor-adjacent tissues (P<0.05;
To investigate the clinical significance of reduced miR-212 in NSCLC, we divided the patients into two different miR-212 groups according to the median expression level. As shown in
To investigate the potential role of miR-212 in NSCLC, we performed gain- and loss-of-function experiments based on miR-212 mimics and inhibitors, which were transfected into A549 (P<0.05;
EMT has been identified as having a crucial role in the induction of metastatic progression of cancer (
To explore the molecular mechanism by which miR-212 suppressed NSCLC migration and invasion, we used public bioinformatic algorithms (TargetScan 6.2 and MiRanda) to search for candidate target genes. We selected sex-determining region Y-box 4 (SOX4) for proceeding with experimental confirmation, which was confirmed in other cancers. The 3′-UTR of SOX4 mRNA contains a putative complementary site of miR-212 (
Subsequently, we examined the correlation between miR-212 expression and SOX4 in NSCLC tissues. We found that the expression of SOX4 mRNA and protein in the miR-212 high-expressing tumors were significantly lower than those in the miR-212 low-expressing tumors (P<0.05, respectively;
Alteration in SOX4 expression reverses the functional effects of miR-212 in NSCLC cells. To clarify that SOX4 is a functional target of miR-212, SOX4 was restored by a plasmid vector in miR-212-overexpressing A549 cells (P<0.05;
Increasing evidence has confirmed that aberrant miRNAs play a critical role in NSCLC development and progression. Previous studies indicated that miR-212 is involved in the pathogenic process of diverse human cancers. miR-212 downregulates SMAD2 expression to suppress the G1/S phase transition of the cell cycle and EMT in cervical cancer cells (
Sex-determining region Y-related high-mobility group box 4 (SOX4) has been reported to be upregulated in multiple cancers, including NSCLC (
In summary, we demonstrated that miR-212 was downregulated in NSCLC tissues and cell lines, and its expression was correlated with malignant clinicopathological features. Furthermore, we confirmed that overexpression of miR-212 inhibited cell migration and invasion
Expression of miR-212 in NSCLC tissues and cell lines. (A) Quantification of the data revealed that the mean level of miR-212 expression in NSCLC tissues was significantly lower than that in matched adjacent non-cancer tissues. (B) Comparing differences in the expression levels of miR-212 between NSCLC cell lines compared to the normal gastric epithelial cell line (BEAS-2B). n=6 repeats with similar results. U6 snRNA was used as internal control; *P<0.05, **P<0.01.
The prognostic value of miR-212 for NSCLC patients. NSCLC patients with higher expression of miR-212 had better overall survival; **P<0.01.
miR-212 inhibits NSCLC cell migration and invasion
miR-212 suppresses epithelial-to-mesenchymal transition in NSCLC cells. (A) Overexpression of miR-212 in A549 cells increased the expression of the epithelial cell marker E-cadherin and decreased the expression of the mesenchymal cell markers N-cadherin and vimentin. (B) In contrast, the expression of the antagomiR to miR-212 decreased E-cadherin expression and increased N-cadherin and vimentin expression. Immunohistochemical analysis of E-cadherin and vimentin in NSCLC samples. (C) In cases of high miR-212 expression (a and c); there was strong E-cadherin and no detectable vimentin protein expression in the same tissue section. In contrast, in the case of low miR-212 expression (b and d), there was no detectable E-cadherin and strong vimentin protein expression. Values are depicted as mean ± SEM; *P<0.05 by t-test.
SOX4 is a direct target of miR-212 in NSCLC. (A) miR-212 and its putative binding sequence in the 3′-UTR of SOX4. The mutant binding site was generated in the complementary site for the seed region of miR-212. (B) miR-212 significantly suppressed the luciferase activity that carried the wild-type (WT), but not the mutant (MUT) 3′-UTR of SOX4. (C) qRT-PCR analysis of SOX4 mRNA expression in A549 cells transfected with the miR-212 or miR-control vector and H1299 cells transfected with the anti-miR-212 or anti-miR-NC vector. (D) Overexpression of miR-212 reduced the expression of SOX4 protein in A549 cells and knockdown of miR-212 increased the level of SOX4 protein in H1299 cells. (E) The expression of SOX4 mRNA in miR-212 high-expressing tumors was significantly lower than that in miR-212 low-expressing tumors. (F) The expression of SOX4 protein in miR-212 high-expressing tumors was significantly lower than that in miR-212 low-expressing tumors. n=6 repeats with similar results, *P<0.05; n.s. not significant
Alterations of SOX4 partially reverse miR-212-mediated NSCLC cell migration, invasion and EMT. (A) miR-212-overexpressing A549 cells that were transfected with EV or SOX4 expression plasmid were subjected to western blot analysis for SOX4. (B) Cell migration and invasion of the miR-212-overexpressing A549 cells was increased by SOX4 overexpression. (C) miR-212-silenced H1299 cells that were transfected with scrambled siRNA or SOX4 siRNA were subjected to western blot analysis for SOX4. (D) SOX4 knockdown abrogated the effects of miR-212 knockdown on H1299 cells. (E) Western blot analysis of EMT marker proteins in A549 cells stably expressing miR-212 transduced with SOX4 or control vector. (F) Western blot analysis of indicated proteins in H1299 cells stably expressing miR-212 inhibitor transfected with SOX4 siRNA or control siRNA. n=6 independent experiments. EV, empty vector; *P<0.05.
Correlation between miR-212 expression and clinicopathological features of the NSCLC cases (n=115).
Expression level | ||||
---|---|---|---|---|
Clinical parameters | Cases (n) | miR-212high (n=54) | miR-212low (n=61) | P-value |
Age (years) | 0.716 | |||
<60 | 36 | 16 | 20 | |
≥60 | 79 | 38 | 41 | |
Sex | 0.589 | |||
Male | 89 | 43 | 46 | |
Female | 26 | 11 | 15 | |
Histologic type | 0.724 | |||
Squamous | 68 | 31 | 37 | |
Adenocarcinoma | 47 | 23 | 24 | |
Lymph node metastasis | 0.001 |
|||
Negative | 83 | 47 | 36 | |
Positive | 32 | 7 | 25 | |
TNM stage | 0.003 |
|||
I+II | 78 | 44 | 34 | |
III+IV | 37 | 10 | 27 |
P<0.05. NSCLC, non-small cell lung cancer; TNM, tumor-node-metastasis.
Univariate and multivariate analyses of prognostic factors in NSCLC patients.
Univariate analysis | Multivariate analysis | |||||
---|---|---|---|---|---|---|
Variables | HR | 95% CI | P-value | HR | 95% CI | P-value |
TNM stage | 2.013 | 1.409–2.872 | 0.002 |
1.862 | 1.283–2.708 | 0.003 |
Lymph node metastasis | 2.798 | 1.879–5.682 | 0.001 |
2.214 | 1.387–3.263 | 0.002 |
miR-212 | 3.967 | 1.583–7.382 | 0.001 |
3.223 | 1.237–6.962 | 0.001 |
NSCLC, non-small cell lung cancer; HR, hazard ratio; CI, confidence interval; TNM, tumor-node-metastasis.
Statistically significant.