Ninjurin2 overexpression promotes glioma cell growth

Ninjurin2 (Ninj2) is an adhesion protein expressed in neurons and glial cells. The current study tested its expression and potential functions in human glioma. We show that Ninj2 mRNA and protein levels are significantly upregulated in human glioma cells and tissues. In established and primary human glioma cells, Ninj2 shRNA or knockout (by CRISPR/Cas9 gene editing) potently inhibited cell survival, growth, proliferation, cell migration and invasion, while inducing apoptosis activation. Contrarily, ectopic overexpression of Ninj2 promoted glioma cell progression in vitro. In human glioma tissues and cells, Ninj2 co-immunoprecipitated with multiple receptor tyrosine kinases (EGFR, PDGFRβ and FGFR), required for downstream Akt and Erk activation. Akt and Erk activation was potently inhibited by Ninj2 shRNA or knockout, but enhanced with ectopic Ninj2 overexpression in glioma cells. In summary, we show that Ninj2 overexpression promotes glioma cell growth.

Ninjurin2 (Ninj2), a homolog of ninjurin1 (Ninj1) [12], is a novel adhesion protein in neurons and glial cells [12]. Ninj2 and Ninj1 have the same conserved hydrophobic regions in the transmembrane domains. Their adhesion motifs are, however, different [12]. Ninj2 gene is located on chromosome 12p13 [12]. An early genome-wide association study has reported that two Ninj2 single nucleotide polymorphisms (SNPs, rs11833579 and rs12425791) are associated with ischemic stroke in Caucasians [13]. Although inconsistent results have been reported by the following studies [14][15][16][17][18]. Jing et al. have shown that Ninj2 could inhibit oxidative stress-induced injury to neuronal cells [19]. AGING Additionally, Liu et al. demonstrated four-octyl itaconate (4-OI) increased Ninj2 expression and protected neuronal cells from hydrogen peroxide [20]. These results highlighted a key pro-survival activity of Ninj2 in neuronal cells [19,20]. Studies have also found that Ninj2 participates in endothelial inflammation and activation, regulating atherosclerosis progression [21]. The expression and potential functions of Ninj2 in human glioma have not been extensively studied. Here our results will show that overexpression of Ninj2 promotes human glioma cell progression.

Ninj2 is upregulated in human glioma cells and tissues
First, we tested expression of Ninj2 in human glioma cells. As compared to the primary human astrocytes (from Dr. Cao at Soochow University [11]), Ninj2 mRNA levels were significantly elevated in established human glioma cell lines (A172 and U251MG) and primary human glioma cells (derived from two human patients, "P1/P2" [11]) ( Figure 1A). Ninj2 protein levels were upregulated as well in glioma cells ( Figure 1B). Ninj2 protein upregulation was detected as well in human glioma tissues ("T", Figure 1C and 1D), whereas its levels are relatively low in the paired surrounding normal brain tissues ("N", Figure 1C and 1D). In the glioma tissues Ninj2 mRNA upregulation was also detected ( Figure 1E). These results confirm that Ninj2 is upregulated in human glioma cells and tissues, indicating a potential function of Ninj2 in promoting glioma cell progression.

Ninj2 shRNA or KO inhibits human glioma cell survival
To study the potential function of Ninj2 in glioma cells, two lentiviral Ninj2 shRNAs, with non-overlapping and primary human ("P1/P2") glioma cells as well as in the primary human astrocytes ("Astrocytes") were tested by qPCR (A) and Western blotting (B), respectively. A total of ten (10) pairs of human glioma tissues ("T") and paired surrounding normal brain tissues ("N") were homogenized and dissolved in tissue lysis buffer, Ninj2 mRNA and protein expression was tested (C-E). Data were presented as the mean ± SD (same for all Figures).*p<0.05 vs. "Astrocytes" (A). *p<0.05 vs. "N" tissues (D and E). Experiments in this figure were repeated four times, and similar results were obtained. sequences (namely "Seq1/Seq2"), were individually transduced to A172 cells. With selection by the puromycin, the stable cell lines were established ("sh-Ninj2" cells). Furthermore, the lentivirus with the lenti-CRISPR/Cas9 Ninj2 KO construct was transduced to A172 cells, establishing the Ninj2 KO stable A172 cells ("ko-Ninj2" cells). In the stable cells with Ninj2 shRNA and Ninj2 KO construct Ninj2 mRNA levels decreased significantly (over 95% vs. control cells) (Figure 2A). Ninj1 mRNA levels were however unchanged ( Figure  2B). Ninj2 protein levels were also significantly downregulated in "sh-Ninj2" cells and "ko-Ninj2" A172 cells ( Figure 2C), where the Ninj1 protein expression unchanged ( Figure 2C).

Ninj2 shRNA or KO inhibits human glioma cell proliferation
To test the potential effect of Ninj2 on glioma cell proliferation, we utilized the cell counting assay. As shown, the stable A172 cells with Ninj2 shRNA ("Seq1/Seq2") or the KO construct (see Figure 2) grew significantly slower than control cells ( Figure 3A). Furthermore, we show that Ninj2 shRNA/KO potently inhibited BrdU incorporation in A172 cells ( Figure 3B). Additionally, EdU-positive staining in A172 glioma cells was largely decreased after Ninj2 silencing or KO ( Figure 3C). These results clearly show that Ninj2 shRNA or KO inhibited A172 cell proliferation. In U251MG cells and primary human glioma cells ("P1/P2"), Ninj2 shRNA similarly decreased EdU incorporation, suggesting proliferation inhibition ( Figure  3D). PI-FACS assay results demonstrated that Ninj2 silencing or KO disrupted A172 cell cycle progression, causing G1-S arrest ( Figure 3E and 3F). Expression of cell cycle-associated proteins, including cyclin D1 and cdc2, was also downregulated in Ninj2-silenced/-KO A172 cells ( Figure 3G). The control shRNA virus ("sh-C") had no significant effect on proliferation and cell cycle progression of glioma cells ( Figure 3A-3G).

Ninj2 shRNA or KO inhibits human glioma cell migration and invasion
Uncontrolled glioma cell migration and invasion are essential for glioma cell progression [1,22]. Ninj2 is an adhesion protein in neurons. We therefore tested whether Ninj2 was important for glioma cell migration and invasion. Performing the "Transwell" assays, we show that A172 cell in vitro migration was significantly inhibited by Ninj2 shRNA ("Seq1/Seq2") and KO ( Figure 4A). Furthermore, testing cell invasion, by the "Matrigel Transwell" assays, demonstrated that Ninj2 silencing or depletion dramatically attenuated A172 cell invasion as well ( Figure 4B). The control shRNA virus ("sh-C"), as expected, exerted no significant effect on A172 cell migration and invasion ( Figure 4A and 4B). In U251MG cells and the primary human glioma cells ("P1/P2"), "Transwell" and "Matrigel Transwell" assay results show that Ninj2 shRNA significantly decreased the number of migrated ( Figure 4C) and invasive ( Figure 4D) glioma cells. Taken together, Ninj2 shRNA or KO inhibited human glioma cell migration and invasion in vitro.

Ninj2 shRNA or KO induces apoptosis activation in human glioma cells
Apoptosis activation could be one reason of glioma cell death and proliferation inhibition. As shown in Figure  5A, the caspase-3 activity was significantly increased in Ninj2 shRNA-or Ninj2 KO-A172 cells. Western blotting assay results confirmed that Ninj2 shRNA or KO induced cleavages of caspase-3, caspase-9 and ploy ADP ribose polymerase (PARP) in A172 cells ( Figure  5B). The TUNEL-positive nuclei ratio was significantly augmented as well ( Figure 5C). Furthermore, Ninj2 silencing or KO significantly increased Annexin Vpositive staining in A172 cells ( Figure 5D and 5E). These results indicated that Ninj2 shRNA/KO induced apoptosis activation in A172 cells. In U251MG cells and the primary human glioma cells ("P1/P2"), Ninj2 shRNA induced significant apoptosis activation, evidenced by increases of caspase-3 activity ( Figure 5F) and TUNEL-positive nuclei ratio ( Figure 5G).

Figure 5. Ninj2 shRNA or KO induces apoptosis activation in human glioma cells. A172 glioma cells (A-E), U251MG glioma cells
(F and G) or the primary human glioma cells (derived two patients, "P1/P2", F and G) were transduced with lentiviral Ninj2 shRNAs ("sh-Ninj2", two different sequences "Seq1/Seq2"), control shRNA ("sh-C") or the CRISPR/Cas9 Ninj2 KO construct ("ko-Ninj2"), stable cells were established via puromycin selection; The relative caspase-3 activities were tested (A and F); Cell apoptosis was tested by TUNEL staining (C and G) and Annexin V FACS (D and E); Expression of listed proteins was tested by Western blotting (B). Expression of listed proteins was quantified and normalized to the loading control (B). For each assay, n=5. *p<0.05 vs. "sh-C" cells. Experiments in this figure were repeated five times, and similar results were obtained.

DISCUSSION
It has been shown that Ninj2 is an adhesion protein expressed in neurons and glial cells [12,21]. The potential functions of Ninj2 are largely unknown, although its expression is elevated following nerve injuries [12,21], promoting neurite outgrowth [12]. A very recent study has suggested that Ninj2 could be an important pro-survival factor in neuronal cells. Ninj2 knockout by CRISPR/Cas9 gene-editing led to SH-SY5Y neuronal cell death and apoptosis. Jing et al. further show that Ninj2 silencing, by its target mRNA miR-764, inhibited neuronal cell survival [19]. Contrarily, ectopic overexpression of Ninj2 can protect SH-SY5Y cells from hydrogen peroxide (H2O2) [19]. Additionally, Liu et al. have shown that four-octyl itaconate (4-OI), a novel Nrf2 activator, induced Ninj2 expression in human neuronal cells and protected cells against H2O2 [20].
The results of the present study indicate that Ninj2 could be a novel oncogenic protein for human glioma. Ninj2 is upregulated in human glioma cells and tissues. In established and primary human glioma cells, Ninj2 silencing (by targeted shRNAs) or knockout (by CRISPR/Cas9 method) potently inhibited cell survival, proliferation, migration and invasion, while inducing significant apoptosis activation. Contrarily, forced overexpression of Ninj2 by a lentiviral construct efficiently promoted glioma cell progression in vitro.
In human glioma, simultaneous activation of multiple RTKs, including EGFR, VEGFR, FGFR and PDGFR, can constitutively activate key oncogenic downstream cascades, including PI3K-Akt-mTOR and Erk-MAPK signaling [23,25]. Therefore RTKs are important oncotarget proteins for human glioma [25]. Inhibition or silencing of one RTK could only result in partial or cells and primary human glioma cells ("P1/P2") the association between Ninj2 with RTKs (EGFR, PDGFRβ and FGFR) was tested by coimmunoprecipitation ("Co-IP") assays (A); "INPUT" shows expression of the RTKs and Ninj2 in total cell lysates (B). A172 cells were transduced with lentiviral Ninj2 shRNAs ("sh-Ninj2", two different sequences "Seq1/Seq2"), control shRNA ("sh-C") or the CRISPR/Cas9 Ninj2 KO construct ("ko-Ninj2"), stable cells were established via puromycin selection; expression of listed proteins was tested by Western blotting (C and D); Fresh human glioma tissue lysates from three patients ("T1/2/3") were subjected to the same Co-IP assay (E), "INPUT" shows expression of RTKs and Ninj2 in the tumor lysates (F). Experiments in this figure were repeated five times, and similar results were obtained. even no inhibition of these downstream cascades and often little anti-glioma actions [23,25]. Wang et al. have shown that Ninj2 is important for endothelial inflammation and activation, by directly interacting with Toll-like receptor 4 (TLR4) to transduce downstream NF-κB and c-Jun signalings [21]. The results of present study demonstrated that in glioma cells Ninj2 co-immunoprecipitated with multiple RTKs (including EGFR, PDGFRβ and FGFR), required for downstream Akt and Erk activation. In the glioma cells Akt and Erk activation was largely inhibited by Ninj2 shRNA/KO, but augmented with ectopic Ninj2 overexpression.
Our results suggest that fully activation of the oncogenic RTKs (EGFR, PDGFR and FGFR) signaling requires Ninj2 in glioma cells. Ninj2, thought binding directly to the RTKs, should be essential for the downstream signalings (Akt and Erk) transduction. Targeting the Ninj2-RTK signaling will be a potential lentivirus (LV-Ninj2), followed by puromycin selection two stable cell lines ("SL1/SL2") were established ("OE-Ninj2" cells). Control cells were infected with empty vector lentivirus ("Vec"); Expression of listed genes were tested by qPCR and Western blotting (A-D); Cell viability (MTT OD, E) and soft agar colony formation (F) were tested; Cell proliferation was tested by cell counting assay (G) and EdU staining assay (H), with cell migration tested by the Transwell assay (I). Expression of listed proteins was quantified and normalized to the loading control (C and D). For each assay, n=5. *p<0.05 vs. "Vec" cells. Experiments in this figure were repeated three times, and similar results were obtained. valuable novel strategy to inhibit glioma cells. The underlying mechanism of Ninj2-mediated RTK signaling transduction should warrant further investigations.
The identification of novel oncogenic proteins is extremely important for the diagnostic and prognostic determination for human glioma. In the current study, we show that expression of Ninj2 is significantly increased in human glioma tissues, as compared to its levels in the surrounding normal brain tissues. These results further support Ninj2 as a valuable therapeutic target of human glioma. Future studies are certainly needed to further explore the significance of Ninj2 upregulation in the diagnosis and therapeutic of human glioma. Collectively, we show that Ninj2 overexpression promotes glioma cell progression, indicating that it could be a novel and valuable therapeutic target for human glioma.

Cell culture
Established human glioma cell lines, A172 and U251MG, were provided by the Cell Bank of Shanghai Institute of Biological Science (Shanghai, China). A172 cells and U251MG cells were cultured in DMEM with 10% FBS. Primary human glioma cells, derived from two independent informed-consent human patients, were from Dr. Cao at Soochow University [10,11,26]. The primary glioma cells were named as "P1/P2". The primary human astrocytes were provided by Dr. Cao as well. Primary human glioma cells and astrocytes were cultured as described early [10,11,26]. All protocols of the present study, according to Declaration of Helsinki, were approved by the Ethics Board of Nanjing Medical University.

Human tissues
Ten (10) human glioma tissues ("T") together with paired surrounding normal brain tissues ("N") were from Dr. Cao at Soochow University [10,11]. Tissues were immediately stored in liquid nitrogen. For biomedical analyses, tissues were homogenized by using the tissue lysis buffer with proteasome inhibitors (Beyotime Biotechnology, Wuxi, China). Written informed-consent was obtained from each participant.

Quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay
At 1.5×10 5 cells per well human glioma cells or astrocytes were seeded into six-well plates. Following the treatments, TRIzol reagents were added to cultured cells to obtained total RNA. By using an ABI7600 Prism system, qPCR was performed through a SYBR Green PCR kit. Melt curve analysis was always performed to calculate the product melting temperature. The 2 −∆Ct method was utilized for the quantification of targeted mRNA (Ninj1 and Ninj2), with GAPDH mRNA tested as an internal control.

Western blotting and co-immunoprecipitation (Co-IP)
Western blotting was performed through a wellestablished protocol [27]. The same set of lysates were run on separate gels (sister gels) to test different proteins with different molecular weights. Tubulin was always tested as the loading control. The NIH ImageJ software Table 1. Primers utilized in the study.

qPCR primers
was utilized to quantify the intensity of each protein band. For each condition, 1000 μg total cellular lysates of glioma cells were pre-cleared with IgA/G ("Beads", 30 μL of each treatment, Sigma). The endogenous Ninj2 proteins in TCL was captured by an anti-Ninj2 antibody (Santa Cruz Biotech) overnight, followed by incubation with IgA/G "Beads" for another 6-8h. Ninj2-immunoprecipitated proteins were further tested by Western blotting assays.

Ninj2 knockout
At 2×10 5 cells per well A172 glioma cells were seeded into six-well plates. A lenti-CRISPR/Cas9-GFP-puro-Ninj2 KO construct (with targeted DNA sequence, 5′-GCATGGCGTTGGACATGAAC-3′) was provided by Dr. Zhang [19], added to A172 cells. GFP-positive A172 cells were first sorted by FACS, and single cells were cultured for another two weeks. Thestable cells were further selected by puromycin-containing medium for 2-3 passages. In the stable A172 cells Ninj2 knockout was verified by Western blotting and qPCR.

Cell apoptosis analyses
Human glioma cells with applied genetic modifications were seeded into six-well plates at 2×10 5 cells per well. After culture for the applied time periods, cell apoptosis was examined by Annexin V-PI FACS, caspase-3 activity, TUNEL [terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling] staining assays. TUNEL ratio (vs. DAPI) was calculated, recording 500 cells of each treatment from five random views (1:100 magnification). The protocols were described in detail in other studies [28].

Cell proliferation and cell cycle assays
The detailed protocols of proliferation assays, including soft agar colony formation, BrdU ELISA, and EdU staining, as well as the propidium iodide (PI) FACS cell cycle distribution assays were reported early [29,30].

In vitro invasion and migration assays
As described [28], the "Transwell" chambers (Corning Co., New York, NY) with 12 μm pore were pre-coated with or without 1 mg/mL Matrigel (BD Biosciences, Shanghai, China). Human glioma cells were starved overnight, and added to the upper chamber (4 × 10 4 cells of each chamber) in serum-free medium. The lower chamber was filled with completed medium with 10% FBS. After 24h incubation, cells that invaded/migrated to the lower chamber were fixed, stained and counted. Mitomycin (2.5 μg/mL Sigma) was included to exclude the influence of cell proliferation.

Statistical analysis
For all in vitro experiment, n=5. Data of all repeated experiments were pulled together to calculate mean ± standard deviation (SD). Data were analyzed by oneway ANOVA followed by a Scheffe's f-test by using the SPSS 18.0 software (SPSS Inc., Chicago, IL). A two-tailed unpaired T test (Excel 2017) was utilized to examine significance between two treatment groups. Significance was chosen as P < 0.05.

AUTHOR CONTRIBUTIONS
All listed authors designed the study, performed the experiments and the statistical analysis, and wrote the manuscript. All authors have read the manuscript and approved the final version.