CiRS‐7 targeting miR‐7 modulates the progression of non‐small cell lung cancer in a manner dependent on NF‐κB signalling

Abstract The purpose of this study was to figure out the effect of ciRS‐7/miR‐7/NF‐κB axis on the development of non‐small cell lung cancer (NSCLC). In response, the expressions of ciRS‐7, miR‐7 and NF‐κB subunit (ie RELA) within NSCLC tissues and cell lines were determined with real‐time polymerase chain reaction (RT‐PCR) and Western blot. Moreover, the NSCLC cells were transfected with pcDNA3‐ciRS‐7‐ir, pcDNA3‐ciRS‐7, miR‐NC and miR‐7 mimic. Furthermore, the targeted relationships between ciRS‐7 and miR‐7, as well as between miR‐7 and RELA, were confirmed by luciferase reporter assay. The proliferation, migration and apoptosis of NSCLC cells were, successively, measured using CCK‐8 assay, wound‐healing assay and flow cytometry test. Consequently, ciRS‐7, miR‐7, histopathological grade, lymph node metastasis and histopathological stage could independently predict the prognosis of patients with NSCLC (all P < .05). Moreover, remarkably up‐regulated ciRS‐7 and RELA expressions, as along with down‐regulated miR‐7 expressions, were found within NSCLC tissues and cells in comparison with normal ones (P < .05). Besides, overexpressed ciRS‐7 and underexpressed miR‐7 were correlated with increased proliferation, migration and invasion, yet reduced apoptosis rate of NSCLC cells (P < .05). More than that, ciRS‐7 specifically targeted miR‐7 to reduce its expressions (P < .05). Ultimately, the NSCLC cells within miR‐7 + RELA group were observed with superior proliferative, migratory and invasive capabilities than those within miR‐7 group (P < .05), and RELA expression was also significantly modified by both ciRS‐7 and miR‐7 (P < .05). In conclusion, the ciRS‐7/miR‐7/NF‐kB axis could exert pronounced impacts on the proliferation, migration, invasion and apoptosis of NSCLC cells.


| INTRODUCTION
Lung cancer is a common human malignancy with leading prevalence and mortality worldwide, and non-small cell lung cancer (NSCLC) accounted for as high as 80%-85% of all lung cancer cases. 1,2 Furthermore, the treatment efficacy and 5-year survival rate for NSCLC patients were usually undesirable, as NSCLC cases were mostly diagnosed when they have developed into the advanced stage. 3 Thus, it was urgently demanded to seek for a screening technique for earlystage NSCLC that was potentially non-invasive, little traumatized and highly specific.
Notably, exosomes that were shaped as bilayer vesicles were viewed within tumour cells, and their formation was based on a series of regulatory processes, including endocytosis, fusion and efflux. [4][5][6] The exosomes abounded in RNA, protein, microRNA and DNA segments, which participated in regulating the biological behaviour of recipient cells. 7,8 Furthermore, the non-coding regions of DNA segments (eg miRNA, lncRNA and circRNA) accounted for as high as 98% of human genomes, and they were involved with altering the epigenetic inheritance of neoplasms. [9][10][11] It was thus implied that the non-coding regions, especially circRNAs, within exosomes might be advantageous in precisely discriminating tumours at early stage. For instance, plenty of intact and stable circRNAs were discovered within human serum exosomes, and therein the circ-KLHDC10 expression showed evident distinctions between colorectal cancer patients and healthy individuals. 12 In addition, ciRS-7 (also termed as Cdr1as) not merely exhibited a higher expression within hepatocellular carcinoma (HCC) tissues than within para-carcinoma tissues, but also was significantly correlated with hepatic microvascular invasion (MVI) among patients with NSCLC. 13 Therefore, ciRS-7 was speculated as potential biomarker for diagnosing HCC or NSCLC. Interestingly, ciRS-7 expressions displayed an inverse correlation with miR-7 expressions within tumour tissues, and ciRS-7 has also been verified to directly target miR-7 within tumour cells. [13][14][15] Nonetheless, the mechanism underlying the correlation between ciRS-7 and miR-7 within NSCLC cells was still far from clear. Furthermore, miR-7 also participated in the pathogenesis that initiated the development of assorted disorders through acting on the downstream pathways.
To be specific, miR-7 could affect proliferation and migration of NSCLC cells by modulation of ERK/MAPK signalling or mediation of TLR9 signalling. 16,17 The miR-7/NF-jB signalling axis also mattered for its modulating the metastatic conditions of gastric cancer cells, so this axis might also be responsible for the aetiology of additional cancers, such as NSCLC. [18][19][20]

| Cell culture
Human NSCLC cell lines (ie A549, H1299, H1355 and H460) and the normal human embryonic lung fibroblast cell line (ie MRC5) were purchased from Shanghai Institute of Biochemistry and Cell biology, Chinese Academy of Sciences. All the cell strains were inoculated within cell medium that contained 10% bovine foetal serum, 2 lmol/ L glutamine, 100 IU/mL penicillin and 100 lg/mL streptomycin sulphate. Subsequently, the cells were managed to grow in 5% CO 2 and saturated humidity at 37°C.

| Real-time fluorescence quantification polymerase chain reaction (RT-qPCR)
Total RNA was extracted by consulting the operational manual of routine TR1201 reagent (Invitrogen Corporation, USA), and the extracted RNAs were preserved at À80°C. Then, the purity and concentration of RNA were measured by applying spectrophotometry.
Moreover, the expressions of miR-7 and ciRS-7 were measured through qRT-PCR (Invitrogen Corporation), and their primers were designed and composed by Sangon Biotech (Shanghai) Corporation,

| Trypan blue staining
The cells at the exponential phase were digested with pancreatins, and they were processed into cell suspension after low-speed centrifugation. Subsequently, after being mixed with the trypan blue saline solution (percentage: 0.4%), they were dropped upon the blood counting chamber, and cell counting was completed within 3 minutes. Finally, under the microscopic observation, the cells dyed to blue were determined as dead, while the colourless and transparent ones were judged as live.

| MTT test
Each hole of 96-well culture plates was added with 100 lL cell suspension that contained 2 9 10 3 cells, and the cells were cultured in 5% CO 2  Besides, the migration experiment was conducted mostly in accordance with the above procedures, except that Matrigel glue was hardly applied.

| Cell apoptosis
The cells at the logarithmic phase were seeded within the culture vessels at the density of 1. Subsequently, the cells were, respectively, transfected with miR-7 The primers for ciRS-7, miR-7, U6, NFjB and GAPDH investigated in this study

RNAs
Primer sequence ciRS-7 | 3099 mimics and miR-NC, and each group was then transfected with RELA-Wt 3 0 UTR-PGL3 plasmid/RELA-Mut 3 0 UTR-PGL3 plasmid and pRL-TK reporter gene carrier, respectively. The activities of Firefly and Renilla luciferase were evaluated successively by applying dualluciferase reporter assay system (Promega) in 48 hours after transfection. All the assays were repeated for at least 3 times.

| Statistical analysis
All the statistical analyses were conducted with SPSS 20.0 software.
The enumeration data were compared with v 2 test, while measurement data (mean AE SD) were contrasted using t-test. The method of Kaplan-Meier was applied to fit overall survival (OS) curves of the included subjects. It would be considered statistically significant when P value was <.05.  .05). The different trends also rendered a significantly negative correlation between ciRS-7 and miR-7 expressions within NSCLC tissues (P < .05) ( Figure 1B). Analogously, the within NSCLC cell lines (ie A549, H1299, H1355 and H460 cell lines) were associated with strikingly higher ciRS-7 expressions and significantly lower miR-7 expressions, when compared with the normal human embryonic lung fibroblast cell line (ie MRC5 cell line) (P < .05) ( Figure 1C).

| Association of ciRS-7 and miR-7 expressions with NSCLC patients' OS
The incorporated patients with NSCLC were divided into higher ciRS-7 expression (>average ciRS-7 expression) group and lower ciRS-7 expression (<average ciRS-7 expression) group (Table 2). Simultaneously, they were also separated into higher miR-7 expression group (>average miR-7 expression) and lower miR-7 expression (<average miR-7 expression) group. It was derived that highly expressed ciRS-7 and lowly expressed miR-7 both exhibited significant correlations with advanced histopathological grade, larger tumour size and severer lymph node metastasis (P < .05). Moreover, Kaplan-Meier analysis also manifested that NSCLC subjects with highly expressed ciRS-7 or lowly expressed miR-7 possessed inferior OS to ones with lowly expressed ciRS-7 or highly expressed miR-7 (P < .05) ( Figure 1D,E).

| CiRS-7 and miR-7 regulated proliferation, invasion, migration and apoptosis of NSCLC cells
The cell number of pcDNA-ciRS-7 group escalated faster than that of pcDNA group, and miR-7(-) group displayed a better multiplicative capacity than NC group (P < .05) (Figure 2A,B). Meanwhile, the cell viability of pcDNA-ciRS7 group and miR-7(-) group both exceeded that of pcDNA group and NC group (P < .05) ( Figure 2C,D). In addition, pcDNA-ciRS7 group and miR-7(-) group were associated with stronger invasive and migratory abilities of NSCLC cells than pcDNA group and NC group (P < .05) ( Figure 2E,F). Correspondingly, the apoptotic rates of pcDNA-ciRS7 group and miR-7(-) group were largely depressed in comparison with pcDNA group and NC group (P < .05) ( Figure 2G).

| MiR-7 targeted NF-KB subunit (ie RELA) to modify its expression
As Figure 3H-J was indicated, overexpression of miR-7 contributed to attenuated luciferase activity of RELA3 0 -UTR reporter constructs, but this phenomenon could not be observed when the RELA3 0 -UTR was mutated. More than that, miR-7 expression within NSCLC  Interestingly, the optical density (OD) value of miR-7 + RELA group was dramatically higher than that of miR-7 group (P < .05), suggesting that RELA suppressed the lowered effect of miR-7 on NSCLC cells' activity ( Figure 4C). Following a similar trend, miR-7 + RELA group was correlated with more desirable migratory and invasive abilities of NSCLC cells than miR-7 group (P < .05) ( Figure 4D,E).
Finally, the NSCLC cells' apoptosis rate of miR-7 + RELA group was significantly lower than that of miR-7 group (P < .05) (Figure 4F).

| DISCUSSION
It was generally accepted as neoplastic invasion and migration when tumour cells aggressively grew from primary lesions to surrounding tissues, blood circulation and lymphatic circulation. 25 Interestingly, ncRNAs (eg circRNA, lncRNA and miRNA) have been documented to regulate cancers' progression through modulating DNA structures, transcription of RNAs and translation of proteins. 26,27 As a matter of fact, competitive endogenous RNAs (ceRNAs), which mainly incorporated lncRNAs and circRNAs, were revealed to compete with miRNA for the miRNA recognition elements (MREs) of target mRNAs, so as to reduce the control over target genes and to affect the development of diseases. 24,[28][29][30] Distinct from miRNA, cir-cRNA was impregnable to RNA exonuclease, as its special circularclosure structure made itself more stable than linear RNA. [31][32][33][34][35] Hence, circRNAs were more likely to be implicated within the mechanisms that gave rise to dysfunctional bioactivities, such as the onset and aggravation of cancers. For instance, ciRS-7, the natural antisense transcript of cerebellar degeneration-associated protein 1 (CRD1), contained approximately 70 MREs of miR-7. 24,32,36 Its high expression within cytoplasms could effectively lower miR-7 activity through binding to it, thereby down-regulating the expressions of insulin-like growth factor-1 receptor (IGF1R), epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK). [37][38][39] It was also documented that miR-7, which was positioned on chromosome 15, held up the growth of lung cancer cells (ie A549) through down-regulating Bcl-2 expression. 40 Webster et al 41 also speculated that miR-7 might directly restrain the expression of v-raf-1 murine leukemia viral oncogene homologue and the Raf-related signal transmission, finally controlling the proliferation of lung cancer cells. Furthermore, it was miR-7 that hindered the migration of lung cancer through inhibiting phosphatidylinositol-3-kinase (PI3K) and Toll-like receptor 9 (TLR9) expressions. 16 Based on the above-  F I G U R E 3 The relationship between ciRS-7 and miR-7. (A) CiRS-7 was targeted by miR-7 in the specific binding site. The relative luciferase activity of A549 (B) and H1299 (C) cell lines were detected after transfection with pcDNA3-ciRS-7 + miR-7-mut or pcDNA3-ciRS-7 + miR-7. *: P < .05 when compared with NC. (D) The impacts of pcDNA3-ciRS-7 on miR-7 expressions were evaluated within A549 and H1299 cell lines.*: P < .05 when compared with pmirGLO. (E) The impacts of miR-7 mimic on ciRS-7 expressions were evaluated within A549 and H1299 cell lines. The correlation between miR-7 and RELA: (F) CiRS-7 expression was positively correlated with RELA expression within non-small cell lung cancer (NSCLC) tissues, (G) MiR-7 expression was negatively correlated with RELA expression within NSCLC tissues. (H) MiR-7 was targeted by RELA in the specific binding sites. The relative luciferase activity of A549 (I) and H1299 (J) cell lines were determined after transfection with miR-7 mimic + RELA-Mut or miR-7 mimic + RELA-Wt. The RELA expression was determined among groups of pcDNA3-ciRS-7, pcDNA, miR-7 inhibitor, miR-7 mimic and NC within A549 (K) and H1299 (L) cell lines. *: P < .05 when compared with NC; #: P < .05 when compared pcDNA | 3105 mentioned researches, it could be summarized that ciRS-7 combined with miR-7 might modify proliferation, invasion and migration of cancer cells, although hardly any studies have associated ciRS-7 with miR-7 within NSCLC-relevant investigations.
Correspondingly, ciRS-7 and miR-7 expressions were also detected within NSCLC tissues and cells here, validating that they could be widely and stably expressed within biological cells. Also, the apparent differences of ciRS-7 and miR-7 expressions between NSCLC and adjacent normal tissues suggested that they might play a role in the occurrence and development of NSCLC. In addition, the negative association of ciRS-7 with miR-7 further provided hints that ciRS-7 might function as the suppressor of miR-7 or ceRNA that  43 All in all, the current investigation logically linked ciRS-7 with miR-7 and NF-kB signalling in accelerating proliferation, invasion and migration of NSCLC cells, yet several shortcomings were still evident. Firstly, recruiting a larger population of different ethnicities could make the study result be generalized to a wider range of crowds. Secondly, additional molecules, besides NF-kB-p65, should be explored to validate the role of NF-kB signalling in modulating NSCLC development. Finally, animal models should further be established to confirm the study result, so that ciRS-7 might become crucially diagnostic and treatment biomarkers for NSCLC.

CONF LICT OF I NTEREST
The authors declare that no conflict interest existed.