miR-19 regulates the expression of interferon-induced genes and MHC class I genes in human cancer cells

MicroRNA-19 (miR-19) is identified as the key oncogenic component of the miR-17-92 cluster. When we explored the functions of the dysregulated miR-19 in lung cancer, microarray-based data unexpectedly demonstrated that some immune and inflammatory response genes (i.e., IL32, IFI6 and IFIT1) were generally down-regulated by miR-19 overexpression in A549 cells, which prompted us to fully investigate whether the miR-19 family (i.e., miR-19a and miR-19b-1) was implicated in regulating the expression of immune and inflammatory response genes in cancer cells. In the present study, we observed that miR-19a or miR-19b-1 overexpression by miRNA mimics in the A549, HCC827 and CNE2 cells significantly downregulated the expression of interferon (IFN)-regulated genes (i.e., IRF7, IFI6, IFIT1, IFITM1, IFI27 and IFI44L). Furthermore, the ectopic miR-19a or miR-19b-1 expression in the A549, HCC827, CNE2 and HONE1 cells led to a general downward trend in the expression profile of major histocompatibility complex (MHC) class I genes (such as HLA-B, HLA-E, HLA-F or HLA-G); conversely, miR-19a or miR-19b-1 inhibition by the miRNA inhibitor upregulated the aforementioned MHC Class I gene expression, suggesting that miR-19a or miR-19b-1 negatively modulates MHC Class I gene expression. The miR-19a or miR-19b-1 mimics reduced the expression of interleukin (IL)-related genes (i.e., IL1B, IL11RA and IL6) in the A549, HCC827, CNE2 or HONE1 cells. The ectopic expression of miR-19a or miR-19b-1 downregulated IL32 expression in the A549 and HCC827 cells and upregulated IL32 expression in CNE2 and HONE1 cells. In addition, enforced miR-19a or miR-19b-1 expression suppressed IL-6 production by lung cancer and nasopharyngeal carcinoma (NPC) cells. Taken together, these findings demonstrate, for the first time, that miR-19 can modulate the expression of IFN-induced genes and MHC class I genes in human cancer cells, suggesting a novel role of miR-19 in linking inflammation and cancer, which remains to be fully characterized.


Introduction
MicroRNAs (miRNAs) are implicated in cancer initiation, progression, metastasis and angiogenesis [1]. The miR-17-92 gene cluster is frequently amplified in B-cell lymphomas and in a wide range of solid tumors, including lung and stomach cancers [1,2]. The miR-19 family (including miR-19a and miR-19b-1) is Ivyspring International Publisher considered a key component of the oncogenic miR-17-92 cluster [2].
Our previous study demonstrated that miR-19 triggered epithelial-mesenchymal transition (EMT), accompanied by the reduced proliferation of lung cancer cells [3]. When we explored the roles of dysregulated miR-19 in lung cancer, microarray-based gene expression data unexpectedly demonstrated a significant number of inflammatory and immune response genes up-or down-regulated by miR-19, including interferon(IFN)-stimulated genes such as GBP1, IFRD1, IFI35, IFI6 and PSMB9, and complement components, suggesting a strong relationship between miR-19 and inflammation in cancer. On the other hand, there are several lines of evidence that miR-19 family can act as regulators of inflammatory response in some diseases, including rheumatoid arthritis (RA) [4,5], asthma [6], atherosclerosis [7,8], nasal polyposis [9] and sepsis [10]. miR-17-92 was down-regulated in activated fibroblast-like synoviocytes (FLS) isolated from RA patients, and miR-19a and miR-19b-1 regulate Tolllike receptor 2 (TLR2) expression thereby reducing the inflammatory response induced by bacterial lipoprotein (BLP) in FLS and which is characterized by the secretion of IL-6 and Matrix metalloproteinases (MMP-3) [4,11]. miR-19a upregulation in asthma is an indicator and a cause of increased T H 2 cytokine production in the airways [6]. Moreover, the expression of interferon-regulated genes and MHC class I molecules are modulated by microRNAs, such as miR-9 [12], miR-125a [13], miR-520b [14], let-7f-5p [15], let-7i-5p [15], miR-146b-5p [15] and miR-185-5p [15]. Our findings and published reports strongly indicate a novel role of miR-19 in linking inflammation and cancer, which remains to be fully characterized.
These aforementioned findings prompted us to fully explore whether the miR-19 family was involved in regulating the expression of IFN-related genes, major histocompatibility complex (MHC) class I genes and interleukin (IL)-related genes in cancer cells.

Cell line and cell culture
Human NPC cell lines (i.e., CNE2 and HONE1) were cultured in Roswell Park Memorial Institute 1640 medium (RPMI1640) (Corning) and lung cancer cell lines (i.e., A549 and HCC827) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Corning), respectively, supplemented with 10% fetal bovine serum (FBS) (Biological Industries) in a humidified incubator with 5% CO2 at 37°C. All cells were approved by the Institutional Review Board of Southern Medical University.

miRNA transient transfection
Human miR-19 mimics (including miR-19a mimics and miR-19b-1 mimics), a nonspecific miRNA control (i.e., mimics control), human miR-19 inhibitors (including miR-19a inhibitor and miR-19b-1 inhibitor) and a nonspecific miRNA inhibitor control were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). miRNA inhibitor which is a chemically modified single strand of RNA can sequester miRNAs avoiding their targeting to coding genes of endogenous target miRNA. miRNAs were transiently transfected into cells at a working concentration of 100 nmol/L using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's procedure. Cancer cells (i.e., A549, HCC827, CNE2 or HONE1 cells) were transfected with miR-19 mimics (100 nM) or miR-19 inhibitor (100 nM) for 48h, respectively, followed by evaluating the expression of the indicated IFN-inducible genes, MHC class I genes or IL-related genes via qRT-PCR.

Cytokine production assay
Lung cancer cells (A549 and HCC827) and NPC cells (CNE2 and HONE1) were transfected with 100 nM miR-19 mimics or its mimics control. 24 hours and 48 hours after culture, cell culture supernatants were collected and assayed for supernatant concentrations of IL6 by ELISA kit (MultiSciences, China) according to the manufacturer's instructions.

RNA isolation, reverse transcription and qRT-PCR
For miRNA and mRNA analyses, total RNA from cancer cells was extracted with Trizol Reagent (TaKaRa). Total RNA was reversely transcribed with the PrimeScript RT reagent Kit (TaKaRa). The expression levels of mature miRNA were determined by SYBR Green quantitative PCR amplifications performed on the Stratagene Mx3005P Real-Time PCR system (Agilent Technologies, Inc., USA). U6 was used for internal reference genes for miRNA [16]. Expression of mRNA analysis was performed using SYBR Green Master Mix (TaKaRa), and GAPDH was used for normalization. The primers used for the amplification of the indicated genes were listed in Table 1-4.

mRNA microarray analysis
As described in our paper [3], miR-19-and vector-expressing A549 cells were analyzed by Affymetrix arrays. Expression microarray analysis was carried out with commercially available Affymetrix Human Gene U133 Plus 2.0 array according to the Affymetrix standard protocol, which carries 47,000 transcripts representing 38,500 well-characterized human genes. Data analysis was performed using the Significance Analysis of Microarray software (SAM 3.0, Stanford University, USA; http://www-stat. stanford.edu). Heatmap is plotted using pheatmap R package.

Statistical analysis
Data were presented as mean±SD unless otherwise indicated of at least 3 independent experiments. Statistical analysis was performed using a SPSS 16.0 software (SPSS, North Chicago, IL) software package. Statistical significance was assessed by one-way analysis of variance (ANOVA) (*P<0.5; # P<0.01).

miR-19 overexpression and downregulated endogenous miR-19 expression in cancer cells
To explore the roles of miR-19 dysregulation in the pathogenesis of lung cancer and NPC, two methods were employed to overexpress miR-19 in cancer cells. First, A549 cells expressing both miR-19a and 19b-1 transgenes were simultaneously generated as described in our previous publication [3]. Next, miR-19a or miR-19b-1 mimics were transiently transfected into the indicated cancer cells. As shown in Figure 1, the levels of miR-19a ( Figure 1A) or miR-19b-1 ( Figure 1B) in indicated cells transfected with miR-19a or miR-19b-1 mimics were much higher than those in cancer cells transfected with control mimics. Moreover, the levels of miR-19a ( Figure 1C) or miR-19b-1 ( Figure 1D) in the indicated cancer cells transfected with the miR-19a or miR-19b-1 inhibitor were much lower than those in cells transfected with the control inhibitor.

Global analysis of gene expression changes in miR-19-expressing A549 cells
The global gene expression changes induced by miR-19 overexpression were determined by comparing the gene expression profiles between miR-19-and vector-expressing A549 cells based on microarray data. We found that a total of 352 and 501 genes were significantly downregulated and upregulated, respectively, by miR-19 overexpression [3]. Unexpectedly, among the 853 significantly changed genes, we found that some of the differentially expressed genes were involved in regulating immune and/or inflammatory responses, including IFN-inducible genes ( Figure 2A, and Table  5) and IL-related genes ( Figure 4A). Table 6 lists the GO terms representing biological processes related to immune and inflammatory responses. In summary, the microarray data suggest that miR-19 plays a significant role in regulating the expression of genes involved in immune and inflammatory responses.  Table 2. Primers for qRT-PCR analysis of interferon-regulated genes Table 3. Primers for qRT-PCR analysis of MHC class I genes
To characterize the effects of the individual components of the miR-19 family on the IFN-regulated target genes, cancer cells were transfected with miR-19a ( Figure 1A) and miR-19b-1 mimics ( Figure 1B). As shown in Figure 2C and D, miR-19a and miR-19b-1 mimics significantly downregulated the expression of IFN-regulated genes (i.e., IRF7, IFI6, IFIT1, IFITM1, IFI27 and IFI44L) in both A549 and HCC827 cells. Next, we further explored whether miR-19 overexpression by miR-19a or miR-19b-1 mimics alters the expression of IFN-regulated genes in other cancer cells. qRT-PCR data revealed a general downward trend in the expression of IFN-regulated genes (i.e., IRF7, IFI35, IFI6, IFIT1, IFITM1, IFI27 and IFI44L) in CNE2 cells transfected with miR-19a or miR-19b-1 mimics ( Figure  2E); conversely, endogenous miR-19 inhibition by the miR-19a or miR-19b-1 inhibitor in CNE2 cells correspondingly resulted in the upregulated expression of the aforementioned IFN-regulated genes ( Figure 2F). Overall, miR-19a or miR-19b-1 overexpression leads to a general downward trend in the expression profile of the aforementioned genes involved in IFN induction in cancer cells.

Suppression of MHC class I gene expression by miR-19 in cancer cells
Although MHC class I molecules are constitutively expressed in essentially all nucleated cells, the cumulative evidence shows that tumor cells display downregulated MHC class I antigens, which enables them to evade immune surveillance [17]. Therefore, we further examined the influence of the miR-19 family on MHC class I gene expression in cancer cells. When cancer cells (i.e., A549, HCC827, CNE2 and HONE1 cells) were transiently transfected with miR-19a or miR-19b-1 mimics, qRT-PCR data revealed a general downward trend in the expression profile of the MHC Class I genes (such as HLA-B, HLA-F, HLA-G) ( Figure 3A, C, E, G). Conversely, these tumor cells, when transfected with the miR-19a or miR-19b-1 inhibitor, showed a general upward trend in the indicated MHC Class I genes ( Figure 3B,  D, F, H). Taken together, the most significant change after the enforced miR-19a or miR-19b-1 expression in cancer cells was the generally decreased expression of the abovementioned MHC Class I genes.

miR-19 overexpression resulted in decreased IL6 production by cancer cells
Given that miR-19a or miR-19b-1 mimics suppressed IL6 expression in lung cancer and NPC cells ( Figure 4B, C, D, E), we further investigated the possibility that ectopic miR-19a or miR-19b-1 expression could affect IL6 production by these cancer cells. Strikingly, a marked decrease in the production of IL6 was observed in A549, HCC827, CNE2 and HONE1 cells 24 h ( Figure 5A) and 48 h ( Figure 5B) after mimic transfection. In summary, these findings demonstrate that miR-19 can modulate the IL6 production in cancer cells.

Discussion
Over the last few years, there has been increasing evidence that miR-19a or miR-19b-1 positively regulates cancer cell proliferation and tumorigenesis [18][19][20], stemness [21], and EMT, invasion and metastasis [18][19][20] by targeting different targets, suggesting that miR-19 plays oncogenic roles in cancer progression. Our microarray data unexpectedly revealed immune and inflammatory response genes down-or upregulated by miR-19 overexpression in lung cancer, which encouraged us to investigate the functions of onc-miR-19 in linking inflammation and cancer.
Chronic inflammation is believed to have a crucial role in cancer development, and inflammation not only works as a tumor-promoting agent but also influences other steps of tumorigenesis by inducing DNA damage, angiogenesis, invasion and metastasis [32,33]. There is increasing evidence that miRNAs drive tumor progression by regulating inflammation [34,35]. The inhibition of miR-9, which was overexpressed in Hodgkin lymphoma, decreased the production of cytokines (i.e., TNF-α, CCL-5, IL-6 and IL-5) from L428 and L540 cells of Hodgkin lymphoma, followed by an impaired ability to attract normal inflammatory cells and then impairing tumor outgrowth in vivo [36]. Additionally, our previous study revealed that the most significant change following tumor suppressor miR-9 overexpression in NPC cells was the decreased expression of IL-related genes, including IL1B, IL11, IL1F8, IL1A, IL6 and IL7R [12]. miR-19a promoted colitis and colitis-associated colon cancer by downregulating TNFAIP3 in a targeted manner and constitutively activating NF-κB signaling [27]. miRNA-19a/b-1 exhibits oncogenic activity through negatively regulating SOCS3 via the JAK-STAT pathway together with the increased activation of SOCS3, IL6 and STAT3 [37]. In this study, we revealed that ectopic expression of miR-19a or miR-19b-1 modulated the expression of IL-related genes (including IL1B, IL11RA, IL20RB, IL6 and IL32) and suppressed IL6 production in lung cancer and NPC cells. We found that miR-19a or miR-19b-1 overexpression resulted in decreased IL1B expression in lung cancer and NPC cells, while microglia-derived IL1B triggered p53-mediated cell cycle arrest and apoptosis in neural precursor cells [38], indicating that reduced IL1B expression induced by onco-miR-19 might contribute to cancer progression. In summary, these findings strongly indicate a novel role of miR-19 in linking inflammation and cancer, which remains to be characterized.
IL32 is a novel cytokine involved in inflammation and cancer development [39]. Various published data demonstrated that IL32 promotes or decreases tumor development [39]. For example, IL32 contributes to gastric cancer progression by increasing the metastatic potential resulting from AKT, β-catenin and HIF-1α activation [40]. IL-32 inhibits tumor growth via inhibition of NF-κB and STAT3 signals in colon and prostate cancer [39]. In this study, we found that miR-19a or miR-19b-1 overexpression remarkably downregulated IL32 expression in lung cancer cells and led to an increase in IL32 expression in NPC cells. These findings from this study and other published reports strongly suggest that modulating IL32 expression by miR-19 might contribute to the pathogenesis of lung cancer and NPC, which remains to be examined.
IL6, a multifunctional cytokine, is involved in the host immune defense mechanism as well as the modulation of growth and differentiation in various malignancies [41]. The deregulated overexpression of IL6 is associated with tumor progression through inhibition of cancer cell apoptosis, stimulation of angiogenesis and drug resistance [41]. Clinical studies revealed that increased serum IL6 concentrations in patients are associated with advanced tumor stages of various cancers (e.g., lung and colorectal cancers) and short survival in patients [41]. Thus, blocking IL6 signaling is a potential therapeutic strategy for cancers (i.e., anti-IL6 therapy) characterized by pathological IL6 overproduction. miR-19a and miR-19b-1 repressed the release of the cytokines IL6 and MMP-3 in BLP-activated RAFLS by controlling TLR2 expression, suggesting that miR-19a/b-1 can act as negative regulators of inflammation in humans [11]. Additionally, our present study revealed that miR-19a or miR-19b-1 overexpression reduced IL6 expression and production in cancer cells, which contradicts the pathological IL6 overproduction observed in various cancers described above. In summary, the aforementioned findings encourage us to further explore how the decreased IL6 expression and production resulting from the miR-19 overexpression in lung cancer and NPC cells contributes to cancer progression.
Increasing evidence supports that the IFN-inducible genes that are expressed in tumor cells regulate cancer cell proliferation and tumorigenesis [42] and metastasis [43]. IFITM1 is a negative regulator of cell proliferation and tumorigenesis in hepatocellular carcinoma (HCC), while the anti-proliferative action of interferon-γ is mediated by IFITM1 [42]. The silencing of IRF7 in breast cancer cells promoted bone metastasis through tumor immune escape, while the IRF7-driven suppression of metastasis was reliant on IFN signaling to host immune cells [43]. In this study, we revealed for the first time that the ectopic expression of miR-19a or miR-19b-1 in cancer cells significantly downregulated the expression of PSMB8, PSMB9 and IFN-regulated genes (i.e., IRF7, IFI6, IFIT1, IFITM1, IFI27 and IFI44L), which have never been reported in other physiological and pathological processes. Additionally, the expression of IFNinduced protein family members with tetratricopeptide repeats (IFIT), including IFIT1, IFIT2, IFIT3 and IFIT5, was decreased in HCC tissues [44]. Higher IFIT3 expression in HCC tissues predicts a better response to IFN-a therapy in HCC patients, and IFIT3 promotes IFN-a effector responses and therapeutic effects by strengthening IFN-a effector signaling in HCC [44]. In summary, these findings from this study and other published reports strongly suggest that modulating the expression of IFN-inducible genes in cancer cells by miR-19 might contribute to the pathogenesis of lung cancer and NPC, which remains to be thoroughly investigated.
MHC class I molecules, including the classical MHC class I molecules [i.e., HLA(human leukocyte antigen)-A, HLA-B and HLA-C] and non-classical MHC class I molecules (i.e., HLA-E, HLA-F, HLA-G, HLA-H and HLA-J), are constitutively expressed in essentially all nucleated cells [17]. Many cancer cells display downregulated MHC class I antigen (MHC-I), which enables them to evade immune surveillance, while the downregulation of HLA class I expression contributes to a poor prognosis in cancer patients [17]. The MHC class I promoter is known to be activated by several transcription factors, including CIITA [45][46][47][48] and USF1 [49,50]. CXCR4-mediated cell surface MHC-I downregulation in cancer progression facilitated tumor evasion of immune surveillance [51]. Kinases, including MAP2K1 (MEK1), EGFR and RET, were validated as negative regulators of human HLA expression in multiple cancer types, and activated MAPK signaling in mouse tumors in vivo suppressed components of MHC-I, while the pharmacologic inhibition of MAPK signaling led to improved peptide/MHC target recognition and killing by T cells and TCR-mimic antibodies [52]. EGFR tyrosine kinase inhibitors augmented the expression of MHC-I and MHC-II molecules in primary and malignant human keratinocytes [53]. The inhibition of the MAPK pathway induced the upregulation of HLA-A expression and enhanced the sensitivity of targeted tumors to Ag-specific CTL lysis in esophageal and gastric cancer [54]. Additionally, miR-9, which functions as a tumor suppressor in NPC [55], positively regulated the expression of MHC class I genes (such as HLA-B and HLA-F) in NPC [12]. The previous studies revealed that the non-classical HLA-F and HLA-G act as the important mediators of immune escape [56,57]. This study demonstrated that miR-19a or miR-19b-1 overexpression in cancer cells led to a general downward trend in the expression profile of MHC Class I molecules (such as HLA-B, HLA-E, HLA-F, HLA-G or HLA-J), which has never been reported in other physiological and pathological processes. As described in the discussion section, miR-19a and miR-19b-1 function as oncomiR in various cancer progressions. Taken together, these findings shed new light on the role of the miR-19 family in tumor evasion of immune surveillance by inducing the downregulation of cell surface HLA class I expression, which remains to be fully characterized.

Conclusion
We have demonstrated that miR-19 can regulate IL-related gene expression in lung cancer cells and NPC cells, and we have found for the first time that miR-19 overexpression and/or inhibition alter the expression of IFN-induced genes and MHC class I genes in cancer cells. However, the pathological consequences of the altered expression of immune-or inflammatory-related genes by miR-19 in cancer cells remain to be examined. Future studies are required to clarify the potential contribution of miR-19 to the proliferation, EMT, invasion, metastasis or tumor angiogenesis of cancer cells by playing critical roles in modulating the aforementioned genes involved in immune and inflammation responses. Collectively, we suspect that miR-19 might function as an oncomiR to promote tumor progression by regulating the expression of the above-mentioned genes involved in immunity and inflammation.