The Expression of miR-375 Is Associated with Carcinogenesis in Three Subtypes of Lung Cancer

Many studies demonstrated unique microRNA profiles in lung cancer. Nonetheless, the role and related signal pathways of miR-375 in lung cancer are largely unknown. Our study investigated relationships between carcinogenesis and miR-375 in adenocarcinoma, squamous cell carcinoma and small cell lung carcinoma to identify new molecular targets for treatment. We evaluated 723 microRNAs in microdissected cancerous cells and adjacent normal cells from 126 snap-frozen lung specimens using microarrays. We validated the expression profiles of miR-375 and its 22 putative target mRNAs in an independent cohort of 78 snap-frozen lung cancer tissues using quantitative reverse-transcriptase PCR. Moreover, we performed dual luciferase reporter assay and Western blot on 6 targeted genes (FZD8, ITGA10, ITPKB, LRP5, PIAS1 andRUNX1) in small cell lung carcinoma cell line NCI-H82. We also detected the effect of miR-375 on cell proliferation in NCI-H82. We found that miR-375 expression was significantly up-regulated in adenocarcinoma and small cell lung carcinoma but down-regulated in squamous cell carcinoma. Among the 22 putative target genes, 11 showed significantly different expression levels in at least 2 of 3 pair-wise comparisons (adenocarcinoma vs. normal, squamous cell carcinoma vs. normal or small cell lung carcinoma vs. normal). Six targeted genes had strong negative correlation with the expression level of miR-375 in small cell lung carcinoma. Further investigation revealed that miR-375 directly targeted the 3’UTR of ITPKB mRNA and over-expression of miR-375 led to significantly decreased ITPKB protein level and promoted cell growth. Thus, our study demonstrates the differential expression profiles of miR-375 in 3 subtypes of lung carcinomas and finds thatmiR-375 directly targets ITPKB and promoted cell growth in SCLC cell line.


Introduction
Lung cancer has long been the leading cause of cancer-related death in males worldwide [1]. Histologically, lung cancer is classified into 2 major classes, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is a heterogeneous group comprised of 2 most common subtypes, i.e. squamous cell carcinoma (SQ) and adenocarcinoma (AC) [2].Despite the improvements in early diagnosis and recent breakthrough in chemo/targeted therapies, the overall 5-year survival rate of lung cancer remains low and the recurrence rate is high [3].Poor prognosis is due to late disease presentation, heterogeneities, and relatively limited understanding of tumor biology. Therefore, discovery of new molecular markers and targets for the diagnosis and treatment of lung cancer would play pivotal roles in improving prognosis.
MicroRNA (miRNA) is a class of endogenously expressed, noncoding small RNA with around 22 nucleotides. It has been shown that miRNA scan regulate gene expression at the posttranscriptional level through imperfect base pairing with the 3'-untranslated region (3'UTR) of target mRNAs [4]. Growing evidence suggests that deregulation of miRNAs may contribute to certain cancer types including lung cancer. Many studies have demonstrated unique miRNA profiles in lung cancer [5][6][7].In our previous study on the investigation of miRNA biomarkers in 3 subtypes of lung carcinomas, we found significant up-regulation of the microRNA-375(miR-375) expression levels in AC and SCLC but down-regulation of miR-375 in SQ [8]. We hypothesize that miR-375 may be a candidate oncogene in AC and SCLC but a tumor suppressor in SQ. In fact, the phenomena of a single miRNA that plays opposite roles during tumor pathogenesis, either as an oncogene or as a tumor suppressor, has been reported in different cancers [9][10][11].In addition, up-regulation of miR-375 in SCLC was reported recently [12,13]. However, signal pathways regulated by miR-375 and the role of miR-375 in lung cancer is still largely unknown.
We investigated the role of miR-375 in the carcinogenesis of 3 lung carcinoma subtypes to identify new molecular targets for diagnosis and therapy of lung cancer. In this paper, we show successful validation of distinct miR-375 expression profiles in the 3 subtypes. Furthermore, we present expression profiles of 22 putative target mRNAs of miR-375 in 3 lung carcer subtypes. In addition, we demonstrate that miR-375 promotes cell growth in SCLC cell line and inhibits ITPKB expression at the posttranscriptional level by directly targeting the 3'UTR of ITPKB mRNA.

Clinical specimens
The local Ethics Committee approved the study and written informed consent was obtained from all the patients. For sample selection, routine histological classification was used following World Health Organization Classification of lung tumors [2].The diagnosis of AC was confirmed by absence of keratinization or intercellular bridges and positive TTF1 staining. SQ diagnosis was confirmed by intercellular bridges or positive P63 staining (at least 40% of tumor cells). For all the SCLC cases, there was complete agreement on neuroendocrine morphology, small cells and neuroendocrine positive staining. If there was a disagreement, the consensus was reached by the detection of TTF1/P63. All cases were independently reviewed by 2 experienced lung cancer pathologists. Patients who had received pre-operative radiotherapy or chemotherapy were excluded. The clinical characteristics are presented in Table 1. concentration was quantified by NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Waltham, MA). The quality control of RNA was performed by a 2100 Bioanalyzer using the RNA 6000 Pico LabChip kit (Agilent Technologies, Santa Clara, CA). The quality was measured using RNA integrity number (RIN). A RNA sample was discarded if the RIN score was less than 5.0.

qRT-PCR
Quantitative reverse-transcriptase-polymerase-chain-reaction (qRT-PCR) was performed on 78 macrodissected frozen lung tissues to validate the expression profiles of miR-375 and its putative targeted genes in 3 subtypes of lung carcinomas. In the validation of miR-375, qRT-PCR was performed using Taqman microRNA assays (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The U47 small nuclear RNA was used as an endogenous control. All assays were carried out in triplicate. For 22 predicted target genes of miR-375 (ACSL3, CACNG2, CCDC6, CSNK2A1, FZD8, ITGA10, ITPKB, JAK2, JUND, LAMC1, LRP5, MAP3K5, NLK, PAK7, PDGFC, PDPK1, PIAS1, RUNX1, RYR2, SOCS5, SP1andWNT5A), qRT-PCR using SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA) was performed according to the manufacturer's instructions. GAPDH was used as an endogenous control. All assays were carried out in triplicate. Primer sequences are available upon request. Exclusion criteria for statistical analysis are as follows: 1) A target gene that showed cycle threshold (CT) values above 35 cycles in > 20% of the tested samples and 2) A sample that showed CT values above 35 cycles in > 20% of the tested genes.
The KEGG database (http://www.genome.jp/kegg/tool/search_pathway.html) was used to map the predicted targets to lung cancer-associated pathways.

CCK-8 assay
NCI-H82 cells were infected with miR-375-expressing (H82-miR-375) or empty (H82-NC) lentivirus as mentioned above. H82-miR-375 or H82-NC cells were seeded in 96-well plates. The cells were incubated for 24h, 48h, and 72h, respectively. CCK-8 reagent (DOJINDO) was added to each well 3h before the end of incubation. Optical density value (OD) of each sample was measured at a wavelength of 450nm. All experiments were performed in triplicate and independently repeated3 times.

Statistical analysis
For microarray data, one-way analysis of variance (ANOVA) with Benjamini-Hochberg correction (p 0.001) was performed to determine differentially expressed miRNAs among normal, AC, SQ and SCLC tissues. Hierarchical clustering was performed with Pearson correlation using the differentially expressed miRNAs.
For qRT-PCR data, an unpaired, unequal variance t-test with Benjamini-Hochberg correction (p<0.05) was applied to miR-375 and differentially expressed targeted genes in the comparisons of AC vs. normal, SQ vs. normal and SCLC vs. normal tissue. The targeted genes with corrected p< 0.05 and fold expression change > 2 were selected as candidates for further functional studies. All p values were two-sided.

miR-375 Expression in 3 Subtypes of Lung Cancer
Additional methods including laser capture microdissection (LCM), macrodissection and microarray hybridization are described in S2 File.

Results
The miR-375 expression profiles in three subtypes of lung carcinomas In a microarray analysis, expression profiles of 723 human miRNAs were investigated in selected LCM cancerous cell populations and normal cells derived from 36 AC, 30 SQ, 16 SCLC and 44 adjacent normal tissues. Fig 1A illustrates the hierarchical clustering of the differential expression levels of miRNAs in 3 subtypes of lung carcinomas and normal lung tissue. The overall miRNA expression profiles clearly divided the samples into four major groups: AC, SQ, SCLC and normal tissue. Up-regulation of miR-375 expression level was observed slightly in AC and significantly in SCLC, while marked down-regulation of miR-375 expression was observed in SQ ( Fig 1B and Table 2).
The expression profiles of miR-375 in 3 lung carcinoma subtypes were further validated in an independent cohort of 78 snap-frozen surgical lung tissues using qRT-PCR. Similarly, miR-375 expression was significantly up-regulated in AC and SCLC but down-regulated in SQ ( Fig  1C and Table 2).

The association of miR-375 expression with lung carcinogenesis
We identified 191 genes through gateway miRecords to be associated with miR-375. After mapping to the KEGG pathway database, 22 genes were identified to be involved in important signaling pathways in lung cancer (S2 Table.).
The expression levels of the 22 putative target genes were next determined in 78 macrodissected frozen lung tissues. The expression profiles of the putative target mRNAs in 3 subtypes of lung carcinomas are presented in Table 3. Of the 22genes, 11 (ACSL3, CACNG2, CCDC6, FZD8, ITGA10, ITPKB, JUND, LRP5, PIAS1, RUNX1andRYR2) had markedly differential expression levels in at least 2 of 3 pair-wise comparisons of AC vs. normal, SQ vs. normal or SCLC vs. normal. Except ACSL3, all the 11 putative target genes showed significant down-regulation in all the 3 lung carcinomasubtypes. Conversely, ACSL3 showed over 10 fold up-regulation when compareits expression level in SCLC to that in normal tissue (p = 0.011). Two genes (CSNK2A1 and MAP3K5) showed differential expression levels in SQ vs. normal comparison only. Three genes (NLK, PDPK1 and SP1) showed differential expression in SCLC vs. normal control only. Two genes (JAK2 and LAMC1) showed comparable expression levels in the 3 comparisons, while other 4 genes (PAK7, PDGFC, SOCS5 and WNT5A) did not pass the quality control in which the target gene showed CT values above 35 cycles in > 20% of the tested samples.

ITPKB: a direct target of miR-375
Dual luciferase reporter assay was performed on 6 targeted genes (FZD8, ITGA10, ITPKB, LRP5, PIAS1 andRUNX1) that had strong negative correlation with the expression level of miR-375 in SCLC. For the 3 putative target sites at the 3'UTR of ITPKB mRNA, the luciferase activity of the reporter carrying the site 2 and 3 wild-type (ITPKB-Δ2) was significantly suppressed by miR-375, while the luciferase activity of the site 1 wild-type (ITPKB-Δ1) was unaffected. Importantly, the site 2 mutant (ITPKB-Δ2-mut2) was not targeted by miR-375, while the site 3 mutant (ITPKB-Δ2-mut3) was still inhibited by miR-375 ( Fig 3A). Taken together, the results revealed that miR-375 targeted the 3'UTR of the ITPKB mRNA primarily through the target site 2 (seed: 2463-2469).
The effect of miR-375 on the endogenous expression of ITPKB was further examined by Western blot. The ectopic expression of miR-375 significantly suppressed the ITPKB protein in the miR-375-transfected H82 cells (H82-miR-375) compared to the vector control transfected H82 cells (H82-NC) (Fig 3B and 3C). The data indicated that miR-375 inhibited the expression of ITPKB at the posttranscriptional level by directly targeting the 3'UTR (primarily target site2) of ITPKB mRNA.
In addition, we observed ITPKB protein expression was significantly down-regulated in lung cancer tissues compared with adjacent normal tissue, which are in consonance with the expression pattern of ITPKB mRNA mentioned above (Fig 4 and Table 3).

Effect of miR-375 overexpression on SCLC cell growth
The significant up-regulation of miR-375 expression in SCLC samples and its inhibitory effect on ITPKB prompted us to investigate its possible biological role in SCLC cells. After infected, miR-375 expression of both H82-NC and H82-miR-375 was detected using real-time qRT-PCR (Fig 5A). Then we evaluated the cell growth rate by CCK-8 assay. The results shown in Fig 5B suggested that exogenous expression of miR-375 could promote cell growth of H82 cells in a time-dependence manner. After 24h incubation, no difference in cell viability between the two groups was observed(p>0.05).However, significant difference between the two groups  was observed after 48h and 72h incubation, and the promotion efficiencies were 33.1% and 35.9%, respectively(p<0.01).

Discussion
MiR-375 was first identified as a pancreatic islet-specific miRNA regulating insulin secretion [14][15][16]. However, further studies revealed that miR-375 is a multifunctional miRNA participating in pancreatic islet development, glucose homeostasis, mucosal immunity, lung surfactant secretion and more importantly, tumorigenesis. Deregulation of miR-375 has been observed in different types of cancers. For instance, Kong et al reported that miR-375 expression was strongly downregulated in oesophageal squamous cell carcinoma (ESCC), and miR-375 inhibited tumor growth and metastasis through repressing insulin-like growth factor 1 receptor (IGF1R) [17]. Another study convinced that miR-375 was frequently downregulated in ESCC cell lines and tissues, and miR-375 was significantly associated with advanced stage, metastasis and poor outcome of ESCC [18]. Leidner et al found that miR-375 was associated with the progression to invasive carcinoma in Barrett's esophagus [19].Chang et al showed that downregulation of miR-375 in glioma was correlated with unfavorable clinical outcome [20]. Low expression of miR-375 was found in the colorectal cancer [21,22], and also correlated with poor outcome and metastasis in head and neck squamous cell carcinomas [23,24]. He   [25]. In lung cancer, decreased miR-375 expression level in NSCLC tissue samples significantly correlated with advanced stage and lymphatic metastasis [26].Reduced expression of miR-375 was observed in all cancer types mentioned above, while miR-375 upregulation was noticed in breast cancer and prostate carcinoma [27,28]. These findings emphasize a fundamental role of miR-375 in tumorigenesis. Our results were consistent with previous findings. We discovered and validated significant miR-375 up-regulation in AC and SCLC but significant down-regulation in SQ. The current two reports on miR-375 expression in SCLC were consistent with our results [12,13]. Compared to them, our study is unique for the following reasons: First, Zhao et al examined miRNA expression patterns only in cell lines (four SCLC cell lines (H209, H69, H82, and H345), one NSCLC cell line CRL 5908 and one immortalized human lung epithelial cell line Beas-2B), they found that miR-375 was consistently upregulated in all four SCLC cell lines. The other one also drew the conclusion by cultured cells, and used small samples of tissue (7 NSCLC vs. 8SCLC) for verification without normal control. Our study not only included cell lines but also a more comprehensive scanning of clinical samples, enabling us to better identify potential diagnostic miRNA markers. Underscoring the miRNA expression profiles in subtyping SCLC and NSCLC is due to the difficulties in obtaining primary tissue specimens, as most SCLC tumors are not surgically resected. Second, we discovered miRNA biomarkers on LCM selected cancer cells and adjacent normal cells derived from AC, SQ and SCLC. This approach assures the purity of target cells, reducing background signals from non-cancerous cells and improving the reliability of the discovered biomarker candidates [29].And interestingly, we observed that overexpression of miR-375 can drastically promote cell growth in SCLC cell line NCI-H82.
MiRNAs are negative post-transcriptional regulatory elements of gene expression. Single miRNA can regulate multiple mRNAs and are possibly better drug targets. To better understand the molecular mechanism of miR-375 in the 3 subtypes of lung cancer, we further predicted the target genes of miR-375 and filtered the putative target genes through KEGG pathways. We observed that22 target genes are the key members of established signalling pathways in lung cancer including calcium, insulin, Jak-STAT, MAPK, mTOR, PPAR, TGF-beta and Wnt pathways. Among these genes, we found that 6 target genes (FZD8, ITGA10, ITPKB, LRP5, PIAS1 andRUNX1) had strong negative correlation with miR-375 expression level in the comparison of SCLC vs. normal tissue. The association between lung cancer and 4 target genes (FZD8, LRP5, PIAS1 andRUNX1) has been previously reported [30][31][32][33].It has been reported that FZD8is highly expressed in A549 cell line and the dysregulation of FZD8 might play key roles in human cancer through activation of beta-catenin-TCF pathway [30]. Lee et al demonstrated a significant down-expression of LRP5 gene in 6 of 17 lung squamous carcinoma samples [31]. In H1299 cell line, PIAS1 inhibited STAT1-mediated gene activation and the DNA binding activity [32]. In NSCLC patients, RUNX was more frequently methylated in tumor tissues than in noncancerous tissues [33].However, the functions of ITGA10 and ITPKB in lung  cancer are still unknown. In our study, ITPKB mRNA and protein expression was significantly down-regulated in lung cancer tissues. The luciferase assay revealed that ITPKB is the direct target gene of miR-375. The over-expression of miR-375 led to significantly decreased ITPKB protein level. The results confirmed that miR-375 negatively regulated ITPKB at the translation level. For ITGA10gene, we did not observe any effects on miR-375 in the luciferase activity and the suppression of the ITGA10 protein expression by miR-375.
Recently, miR-375 has been found to suppress core hallmarks of cancer by targeting several important genes like AEG-1, YAP1, IGF1R and PDK1 [16].The target genes regulated by miR-375 may function spatiotemporally or in cooperation with each other in different cellular processes. Our identification of ITPKB gene as a direct target of miR-375 provides new insights into the mechanisms underlying tumorigenesis. ITPKB, a member of [InsP3] 3-kinases family, was mapped to the telomeric end of human chromosome 1 and associated with calcium signaling pathway. It has been identified as a candidate gene in the pathogenesis of immune disorders, multiple sclerosis, Alzheimer disease, and malignant melanoma [34,35]. Since little work has been reported on ITPKB function in lung cancer pathogenesis, further studies in vitro and in vivo are needed to explore the potential effect of ITPKB in lung cancer, which will be helpful for understanding the role of miR-375 in lung cancer and if miR-375 upregulation is a cause or effect of cancer pathogenesis. It is well accepted that the expression levels of miRNAs and their direct mRNA targets may be negatively correlated [36,37].Intriguingly, we did observe significant positive correlation in the expression level of miR-375 and its 13 putative targets in SQ and its 4 putative targets in SCLC. It has been demonstrated that a miRNA can function as a switch from repression to activation of its target genes [38,39].Further functional studies are needed to investigate the role of the putative targets showing positive correlation with miR-375 expression level.
In summary, we discovered and validated thatmiR-375 expression level is significantly upregulated in AC and SCLC but down-regulated in SQ. Furthermore, we found that miR-375 directly downregulates ITPKB in SCLC and promotes cell growth in SCLC cell line, which were H82, H82-NC and H82-miR-375 cells were evaluated by western blot. After the transfection of miR-375, the expression level of ITPKB protein was significantly suppressed. C, Quantitative analysis of Western blot. The ITPKB protein intensities were normalized by GAPDH. doi:10.1371/journal.pone.0144187.g003