Silencing SIX1 by miR-7160 inhibits non-small cell lung cancer cell growth

The homeoprotein SIX1 is upregulated in non-small cell lung cancer (NSCLC) and associated with NSCLC tumorigenesis and progression. We identified microRNA-7160 (miR-7160) as a SIX1-targeting miRNA. RNA immunoprecipitation results confirmed a direct binding between miR-7160 and SIX1 mRNA in NSCLC cells. In the primary and established NSCLC cells, forced overexpression of miR-7160 downregulated SIX1 and inhibited cancer cell growth, proliferation, migration and invasion. Furthermore, miR-7160 overexpression induced apoptosis activation in NSCLC cells. Conversely, miR-7160 inhibition elevated SIX1 expression and enhanced NSCLC cell progression in vitro. Restoring SIX1 expression, by an untranslated region-depleted SIX1 expression construct, reversed miR-7160-induced anti-NSCLC cell activity. CRISPR/Cas9-inudced knockout of SIX1 mimicked miR-7160-induced actions and produced anti-NSCLC cell activity. In vivo, intratumoral injection of miR-7160-expressing lentivirus downregulated SIX1 mRNA and inhibited NSCLC xenograft growth in severe combined immunodeficient mice. Significantly, miR-7160 expression is downregulated in human NSCLC tissues and is correlated with SIX1 mRNA upregulation. Collectively, miR-7160 silenced SIX1 and inhibited NSCLC cell growth in vitro and in vivo.


INTRODUCTION
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and a major health threat [1,2]. In the United States alone, it is estimated that over 234,000 new cases of NSCLC will be reported each year [1,2]. Recent improvements have been achieved for early diagnosis through emerging technologies and advanced targeted therapies. However, the five-year overall survival of NSCLC patients is still below 15%. The prognosis for the advanced NSCLC patients is extremely poor [2][3][4][5][6]. Considering that the incidence of this devastating disease is rising, particularly in Eastern countries [7][8][9], it is urgent to explore novel therapeutic targets and diagnosis biomarkers for NSCLC [10].

Forced miR-7160 overexpression silences SIX1 and inhibits NSCLC xenograft growth in mice
In order to study the potential effect of miR-7160 on NSCLC cell growth in vivo, we injected pCan-1 cells to the flanks of SCID mice. NSCLC xenografts were established within three weeks, when tumor volumes reached approximately 100 mm 3 ("Day-0"). NSCLC xenografts were than subjected to intratumoral administration of either lv-pre-miR-7160 or lv-miRC. Tumor growth curve results, Figure 6A, demonstrated that NSCLC xenografts with lv-pre-miR-7160 injection grew significantly slower than control NSCLC xenografts with lv-miRC. Tumor volumes in lv-pre-miR-7160 group were significantly lower than lv-miRC tumors ( Figure 6A). On Day-7, one tumor from each group was individually isolated. The qPCR assay results showed that mature miR-7160 levels increased over AGING 25-30 folds in lv-pre-miR-7160-injected NSCLC xenografts ( Figure 6B), with SIX1 mRNA significantly downregulated ( Figure 6C). The animal body weights were not significantly different between the two groups ( Figure 6D). Neither did we notice apparent signs of toxicity. Therefore, forced miR-7160 overexpression silenced SIX1 and inhibited NSCLC xenograft growth in SCID mice.

miR-7160 is downregulated in human NSCLC tissues
At last we tested expression of miR-7160 in human NSCLC tissues. A total of six (6) NSCLC cancer tissues from primary NSCLC patients [29] were tested. As demonstrated, miR-7160 expression in NSCLC cancer tissues ("T") was significantly lower than that in the paired surrounding normal lung epithelial tissues ("N") ( Figure 6E). It correlated with SIX1 mRNA upregulation in cancer tissues ( Figure 6F). Therefore, miR-7160 is downregulated in human NSCLC tissues and SIX1 is upregulated.

DISCUSSION
miRNAs are commonly dysregulated in NSCLC and other lung cancers, which are extremely important for cancer initiation, tumorigenesis, and progression as well as treatment resistance, and prognosis determination [23,24]. Expression and potential functions of miR-7160 in human cancer have not been studied thus far.
Our results here suggest that miR-7160 should be a tumor suppressor in NSCLC. In A549 and primary NSCLC cells, forced overexpression of miR-7160 potently inhibited cell growth, proliferation, migration and invasion. It also provoked cell apoptosis. Conversely, miR-7160 inhibition, by lv-antagomiR-7160, promoted NSCLC cell proliferation, migration and invasion. Intratumoral administration of lv-pre-miR-7160 potently inhibited NSCLC xenograft growth in SCID mice. Importantly, levels of miR-7160 are downregulated in human NSCLC tissues. Therefore, miR-7160 exerted tumor-suppressive activity in NSCLC.

Figure 5. Altering miR-7160 expression is ineffective on the function of SIX1 KO NSCLC cells. The pCan-1 NSCLC cells with the
CRISPR/Cas9-SIX1-KO-GFP construct (SIX1 KO cells) were further infected with pre-miR-7160-expression lentivirus (lv-pre-miR-7160), pre-miR-7160 anti-sense lentivirus (lv-antagomiR-7160) or the control non-sense miRNA lentivirus (lv-miRC), control cells were transduced with CRISPR/Cas9 empty construct (Cas9-C), stable cells were established; Expression of listed proteins was tested by Western blotting assays (A); Cells were further cultured for applied time periods, cellular functions, including cell proliferation (nuclear EdU ratio, B), migration and invasion ("Transwell" assays, C, D), as well as cell apoptosis (nuclear TUNEL staining, E), were tested, with results quantified; Expression of miR-7160 was tested by qPCR (F). The pCan-1 cells bearing the pre-miR-7160-expression lentiviral construct ("lv-pre-miR-7160") were transfected with or without a lentiviral 3'-UTR-null SIX1 expression construct: +SIX1 (UTR-null); Control cells were transfected with the control non-sense miRNA lentivirus (lv-miRC); Expression of listed proteins was tested by Western blotting (G); Cells were further cultured for applied time periods, cell proliferation (H), migration and invasion (I, J), as well as cell apoptosis (K) were tested, with results quantified; Expression of miR-7160 was tested by qPCR (L). Data were presented as mean ± SD (n=5), and results normalized. *P< 0.05 vs. "Cas9-C" cells (B-F). n.s. stands for no statistic difference (B-E, L). Experiments in this figure were repeated five times with similar results obtained.
Our results showed that miR-7160 targeted and silenced SIX1 in NSCLC cells. RNA-IP experiments confirmed a direct binding between miR-7160 and SIX1 mRNA in NSCLC cells. In NSCLC cells forced overexpression of miR-7160 potently inhibited SIX1 3'-UTR luciferase reporter activity and its expression. Conversely, SIX1 expression was increased with miR-7160 inhibition by lv-antagomiR-7160. Importantly, miR-7160 mimics with mutations at the binding sites to SIX1 were unable to alter SIX1 3'-UTR luciferase reporter activity and its expression. In vivo lv-pre-miR-7160 intratumoral administration downregulated SIX1 mRNA in NSCLC xenografts. Importantly, in human NSCLC tissues miR-7160 expression is downregulated and correlated with SIX1 mRNA upregulation. Thus, miR-7160 is a SIX1targeting miRNA in NSCLC.
We here provided strong evidence to support that miR-7160-induced anti-NSCLC activity is due to silencing SIX1. Restoring SIX1 expression, by an UTR-null SIX1 construct, reversed lv-pre-miR-7160-induced anti-NSCLC cell activity. CRISPR/Cas9-inudced SIX1 knockout mimicked miR-7160-induced actions. Importantly, miR-7160-induced anti-cancer activity was were inoculated thorough s.c. injection to SCID mice (n=7 per group). Within three weeks tumor xenografts were established (Day-0), with tumor volume around 100 mm 3 . NSCLC xenografts were intratumorally injected with either lv-pre-miR-7160 or lv-miRC. Tumor volumes (A) and mouse body weights (D) were recorded every 7 days. At Day-7, one NSCLC tumor xenograft per group was isolated, each tumor was randomly cut into five small pieces (n=5, for B, C), and expression miR-7160 (B) and SIX1 mRNA (C) in tumor lysates tested by qPCR. The relative expression miR-7160 (E) and SIX1 mRNA (F) in NSCLC cancer tissues ("T") and paired surrounding normal lung epithelial tissues ("N") was shown. Data were presented as mean ± SD, and results normalized. * p< 0.05 vs. lv-miRC control tumors (A-C) or "N" tissues (D, E).
AGING compromised in SIX1-KO NSCLC cells. In other words, miR-7160 was completely ineffective in SIX1-KO NSCLC cells. These results suggest that SIX1 silencing should be the primary reason of miR-7160-induced anti-NSCLC cell activity.
The current clinical treatment options for NSCLC, including tumor resection, platinum-based chemotherapies, radiation, and newly-developed molecularly-targeted therapies, are not satisfactory [2,4]. There is an urgent need to explore novel and more efficient anti-NSCLC strategies. The results of this study show that miR-7160, a potential tumor suppressor, can inhibit NSCLC cell growth by silencing its target gene SIX1. It may represent a promising therapeutic strategy and diagnosis marker for NSCLC.

Chemicals and reagents
Antibodies were provided by Abcam (Cambridge, MA) and Cell Signaling Tech (Shanghai, China). Fetal bovine serum (FBS) and all other cell culture reagents were purchased from Hyclone (Logan, UT). Puromycin, polybrene, cell-counting kit 8 (CCK-8), and other chemicals were obtained from Sigma-Aldrich Chemicals Co. (St. Louis, Mo). Primers and viral constructs were designed and verified by Genechem Co. (Shanghai, China) unless otherwise specified. Annexin V, propidium iodide (PI), TUNEL dye, and reagents for PCR and transfection assays were obtained from Thermo-Fisher Invitrogen (Shanghai, China).

Cell culture
A549 cell line was provided by Dr. Chen [30] and cultured as described [30]. The primary human NSCLC cells, provided by Dr. Jiang [31,32], were derived from three NSCLC patients, namely pCan-1, pCan-2, pCan-3. The primary cells were cultured as previously described [31,32]. For the established and primary human cells short tandem repeat (STR) profiling, population doubling time, and morphology were routinely checked to verify the genotypes. The protocols for using human cells were approved by the Ethics Committee of Zhengzhou University in accordance to Declaration of Helsinki.

Quantitative real-time PCR (qPCR)
Total RNA was extracted by TRIzol reagents [33], reversely transcripted to complementary DNA (cDNA). qPCR analyses were carried out using a SYBR Premix Ex Taq™ kit [30] under the ABI Prism 7500 Fast Real-Time PCR system (Applied Biosystems). GAPDH was always tested as the reference gene and internal control. A 2 −∆∆Ct method was utilized to quantify targeted mRNAs. miR-7160 expression was examined by a TaqMan microRNA qPCR assay kit, and U6 tested as the internal control. All primers are listed in Table 1.

RNA immunoprecipitation (RNA-IP)
NSCLC cells, transfected with a biotinylated-miR-7160 mimic (wild-type or mutants), were homogenized by cell lysis buffer. RNA-IP was carried out by adding the streptavidin-coated magnetic beads into the cell lysates [35]. The biotinylated-miR-7160-bound beads was purified [30], and SIX1 mRNA expression tested by AGING qPCR. Its expression was always normalized to "Input" controls.

Cell viability
At 3000 cells per well, NSCLC cells were seeded into 96-well tissue-culture plates and cultured for 96h. Ten µL of CCK-8 reagent was added for another two hours, and CCK-8 absorbance tested at 450 nm.

Transwell migration/invasion assay
NSCLC cells (5×10 4 cells per chamber, cultured into serum-free medium) were added to the upper surface of Transwell chambers (BD Biosciences, Shanghai, China). The lower chambers were filled with complete medium (containing 12% FBS). Cells were allowed to migrate for 24h. Migrated cells in the lower chambers were fixed, stained, and counted. For invasion assays, Matrigel (Sigma) was added to chamber surfaces.

BrdU ELISA
As described [36], NSCLC cells with applied genetic modifications were subjected to BrdU incorporation testing, using a BrdU ELISA kit (Roche Diagnostics Basel, Switzerland) according to the manufacturer's protocol. The BrdU ELISA absorbance at 405 nm was recorded.

Cell apoptosis assays
The detailed protocols of routine apoptosis assays, including Annexin V fluorescence activated cell sorting (FACS) and nuclear TUNEL staining, were described in detail in other studies [37,38].

Western blotting
In brief, quantified protein lysates (30 μg per treatment) were separated by 10-12% SDS-PAGE gels [39] and transferred to polyvinylidene difluoride (PVDF) blots (Merck-Millipore). The resulting blots were blocked and incubated with the indicated primary and, subsequently, secondary antibodies. Binding of antibody-antigen was examined and visualized by an enhanced chemiluminescence (ECL) detection kit (Roche, Shanghai, China).

SIX1 knockout
The lenti-CRISPR/Cas9-SIX1-KO-GFP plasmid was constructed by Genechem. The sgRNA sequence was listed in Table 1. NSCLC cells were initially plated into six-well plates (at 8 ×10 4 cells per well) and transfected with the construct. GFP-positive cells were sorted by FACS and the monoclonal NSCLC cells distributed to 96-well plates. Cells were further cultured in puromycincontaining complete medium for four passages to establish stable cells, where SIX1 knockout (KO) was screened by qPCR and Western blotting assays.

UTR-null SIX1
The UTR-null SIX1 cDNA, constructed by Genechem, was sub-cloned into the GV369 lentiviral vector. The vector together with the lentivirus-packing helper plasmids were co-transfected into HEK-293T cells, generating lentivirus. UTR-null SIX1-expressing lentivirus was added to NSCLC cells (cultured in polybrene-containing complete medium). Following selection by puromycin, stable cells were established.

Human tissues
NSCLC cancer tissues and paired surrounding lung epithelial tissues, from six primary NSCLC patients (male, 46 to 64-year old, stage-III), were provided by Dr.

AGING
Li at Wenzhou Medical University [29]. The protocols were approved by the Ethics Committee of Zhengzhou University, in accordance to Declaration of Helsinki.

In vivo tumor growth assay
The pCan-1 NSCLC cells (1×10 7 cells per mouse, cells in Matrigel-containing serum-free medium) were injected subcutaneously (s.c.) into the flanks of severe combined immunodeficient mice (SCID) mice. Mice weighted 18.5-19.2g and were from Soochow University Animal facility (Suzhou, China). When the volume for each tumor reached approximately 100 mm 3 ("Day-0"), mice were randomly assigned into two groups. Group I received intratumoral administration of lv-pre-miR-7160. Group II received lv-miRC administration. Tumor volumes were recorded under a previously described formula [40]. All animal procedures were approved by the Experimental Animal Ethical Committee of Zhengzhou University, in accordance to Declaration of Helsinki.

Statistical analysis
Data were normally distributed and presented as means ± standard deviations (SD). One-way ANOVA and Student-Newman-Keuls post hoc test were performed to determine statistically differences among multiple groups (SPSS 23.0, SPSS Co. Chicago, IL). When comparing the difference between two specific groups, a two-tailed Student's t-test (Excel 2007, Microsoft) was utilized. P<0.05 was considered statistically different.

AUTHOR CONTRIBUTIONS
Huasi Zhao, Xiao-min Tao, Qun Wang and Hong-yu Zhang conducted the cellular experiments. Huasi Zhao, Yuan-yuan Fang and Hua-qi Wang analyzed and interpreted the data. Huasi Zhao and Guo-jun Zhang drafted the manuscript. All the authors read, revised and approved the final manuscript

CONFLICTS OF INTEREST
The listed authors have no conflicts of interest.

FUNDING
This study was supported by Basic Research Funding Support from the first affiliated hospital of Zhengzhou University.