LINC01198 promotes proliferation and temozolomide resistance in a NEDD4-1-dependent manner, repressing PTEN expression in glioma

Background: Dysregulation of numerous lncRNAs has been recently confirmed in glioma; however, the majority of their roles and mechanisms involved in this notorious disease remain largely unclear. This study aims to explore the roles and molecular mechanisms of LINC01198 implicated in the proliferation and chemoresistance in glioma. Results: LINC01198 was elevated in glioma, and this predicted a poorer prognosis for patients with glioma. LINC01198 knockdown inhibited, while LINC01198 overexpression promoted, glioma cell proliferation and resistance to temozolomide. Mechanistically, NEDD4-1 (neural precursor cell expressed, developmentally downregulated 4, E3 ubiquitin protein ligase) and phosphatase and tensin homolog (PTEN) were recruited by LINC01198, which functioned as a scaffold. Moreover, we showed that LINC01198 exerted its oncogenic activities by enhancing the NEDD4-1-dependent repression of PTEN. Conclusions: Our study elucidated the role of oncogenic LINC01198 in glioma proliferation and temozolomide resistance, and this role may serve as a promising target for glioma therapy. Methods: LINC01198 expression in glioma tissues and that in paired normal tissues were measured by qRT-PCR. The functional roles of LINC01198 in glioma were demonstrated by a series of in vitro experiments. CCK-8 assay, RNA pulldown, RNA immunoprecipitation and western blotting were used to demonstrate the potential mechanisms of LINC01198.


INTRODUCTION
Glioma is one of the most aggressive and common primary malignant brain tumors, and it accounts for approximately 80% of all primary malignant brain tumor cases [1][2][3]. Even though current advances in therapeutic methods for glioma, the median overall survival of the patients is less than 12-18 months after diagnosis due to there being limited therapeutic options [4][5][6]. Therefore, there is an urgent need to explore the exact molecular mechanisms of glioma progression and developing new and effective treatment strategies to improve patient prognosis.
Long noncoding RNAs (lncRNAs) are a class of RNAs longer than 200 nucleotides in length and without protein-coding capacity. Recently, accumulating studies demonstrate that lncRNAs may be emerging as critical oncogenes in cancer progression [7]. Long intergenic noncoding RNA 1198 (LINC01198), a lncRNA with a gene that is located in chromosome region 13q14. 13, was first identified in glioma, and it shows significantly AGING increased expression in tumor tissues; however, its functions and involvement in glioma cells are still unknown [8].
In this study, we assessed LINC01198 expression in glioma and performed functional studies to explore the effects of LINC01198 on glioma progression. Our results demonstrated that LINC01198 expression was upregulated in glioma tissues. The high level of LINC01198 was associated with disease progression and was a predictor for poor prognosis in glioma patients. LINC01198 knockdown turnover resulted in LINC01198-induced glioma cell proliferation and resistance to temozolomide. Moreover, we showed that LINC01198 exerts its oncogenic activities by enhancing NEDD4-1 (neural precursor cell expressed, developmentally downregulated 4, E3 ubiquitin protein ligase)-dependent repression of PTEN (phosphatase and tensin homolog) expression. Taken together, our findings suggest a role of LINC01198 in promoting glioma progression and provide a potential therapeutic target for glioma.

LINC01198 is upregulated in human glioma and is correlated with poor prognosis
Microarray data from GSE16011, Chinese Glioma Genome Atlas (CGGA) and the Repository for Molecular Brain Neoplasia Data (REMBRANDT) datasets show LINC01198 expression increased with tumor grade in glioma [8]. To examine whether LINC01198 plays an important role in glioma progression, we first analyzed the expression level of LINC01198 in human glioma and adjacent normal tissues using qRT-PCR, and we found that the LINC01198 expression level was significantly increased in glioma tissues compared with adjacent normal tissues (74/90) ( Figure 1A). Next, we explored the relationship between clinicopathological characteristics and the LINC01198 expression in 90 glioma patients, as listed in Table 1. The results showed that glioma patients with LINC01198 High had a bigger tumor size (P = 0.001) and higher WHO grade (P = 0.006) ( Figure 1B). Then, we investigated the prognostic implication of LINC01198 expression. Interestingly, our results showed that patients with LINC01198 High expression had a worse prognosis than those with LINC01198 Low expression ( Figure 1C and 1D). Multivariate analysis identified LINC01198 expression as an independent predictor for prognosis of glioma patients (Tables 2 and 3). These results oracle that LINC01198 is likely participated in the progression of glioma.

LINC01198 enhances the proliferation and temozolomide resistance of glioma cells
The biologic function of LINC01198 in regulating the glioma progression remains unclear. Next, we carried out loss/gain-of-function studies in glioma cells. After detecting the expression levels of LINC01198 in six glioma cell lines, two normal brain tissues, and two glioma tissues, we performed knockdown of LINC01198 in U87 and LN229 cells that had high LINC01198 expression and upregulated its expression in U251 and SNB-19 cells that had low LINC01198 expression ( Figure  2A-2C). CCK-8 assay result showed that knockdown of LINC01198 impaired glioma cell proliferation, whereas LINC01198 overexpression promoted cell proliferation in vitro ( Figure 2D). Furthermore, CCK-8 assay was performed to investigate the role of LINC01198 in the sensitivity of glioma cells to temozolomide. The results showed that knockdown of LINC01198 dramatically increased the sensitivity of glioma cells to temozolomide, while LINC01198 overexpression reduced the sensitivity of glioma cells to temozolomide ( Figure 2E).
To further investigate the potential clinical relevance of LINC01198 in vivo, we subcutaneously injected U251 cells with or without stable LINC01198 overexpression into the dorsal flanks of 4-6 week BALB/c nude mice. Compared to Mock groups, temozolomide slightly inhibit tumor growth which derived from U251 cells overexpressing LINC01198 xenografts ( Figure 2F and 2G). Moreover, the mice bearing subcutaneous xenograft U251 cells overexpressing LINC01198 tumors showed a short survival time compared with Mock groups tumors ( Figure 2H).

LINC01198 functions as a scaffold for NEDD4-1 and PTEN
To investigate the potential mechanisms of LINC01198 in glioma cells, we predicted the interactions between LINC01198 and RNA-binding proteins by an RNAprotein interaction prediction tool (http://pridb.gdcb. iastate.edu/RPISeq/), and we found that LINC01198 probably binds to NEDD4-1 and PTEN (Supplementary Table 3) ( Figure 3A). Then, we carried out RIP assays and found that LINC01198 directly binds NEDD4-1 and PTEN in glioma cells ( Figure 3B). In addition, RNA pulldown assays further confirmed that LINC01198 indeed binds with NEDD4-1 and PTEN in glioma cells ( Figure 3C). Our results showed that LINC01198 could bind to both NEDD4-1 and PTEN, indicating that LINC01198 may function as a scaffold for NEDD4-1 and PTEN in glioma cells. To further confirm this hypothesis, the LINC01198 gene was cut into LINC01198-D1 (1-500 nt) and -D2 (601-955 nt), and their expression plasmids were constructed and AGING transfected into U87 cells ( Figure 3D). Our results showed that LINC01198-D1 mostly bound with PTEN, whereas LINC01198-D2 bound with NEDD4-1 ( Figure  3E and 3F). These data showed that LINC01198 may function as a scaffold and bind with PTEN and NEDD4-1 at its 5′ region and 3′ region in glioma cells, respectively.

LINC01198 increased NEDD4-1-induced PTEN inhibition
Previous studies have demonstrated that NEDD4-1mediated PTEN ubiquitination is crucial in the regulation of PI3K/AKT signaling in neurodegeneration [9]. We thus evaluated the effect of LINC01198 on NEDD4-1induced PTEN inhibition in glioma cells. Overexpression of LINC01198 significantly inhibited the expression of PTEN protein but not mRNA in U257 and SNB-19 cells ( Figure 4A, Supplementary Figure 1A). Conversely, LINC01198 knockdown increased PTEN protein but not mRNA expression in U87 and LN229 cells ( Figure 4B, Supplementary Figure 1B). However, in vitro experiments showed that overexpression or knockdown of LINC01198 did not affect NEDD4-1 expression in glioma cells ( Figure 4C and 4D). Moreover, overexpression or knockdown of LINC01198 did not affect PTEN expression in CRISPR/cas9-induced NEDD4-1-knockout glioma cells ( Figure 4E and 4F; Supplementary Figure 2). Then, to investigate the relationship between LINC01198 and PTEN, we detected the expression of PTEN in 90 cases of glioma tissues using western blotting or qRT-PCR. Scatterplot assays results indicated a negative correlation between LINC01198 and PTEN at the protein level (r = 0.165; P = 0.004; Figure 4G), but this correlation was not shown at the mRNA level (r = 0.003; P > 0.05; Figure 4H). These results indicate that LINC01198 regulates PTEN expression through NEDD4-1.

NEDD4-1 turnover LINC01198-induced glioma progression
To further determine the biological roles of LINC01198 in glioma, CRISPR/cas9-induced NEDD4-1-knockout glioma cells were used in an experiment. A CCK-8 assay showed that forced LINC01198 expression neither increased cell proliferation nor promoted glioma cell resistance to temozolomide in NEDD4-1-knockout glioma cells ( Figure 5A and 5B). Additionally, reduced LINC01198 expression neither inhibited cell proliferation nor increased glioma cell resistance to temozolomide in NEDD4-1 knockout glioma cells ( Figure 5C and 5D). To further understand the mechanism of LINC01198 in glioma, we recovered PTEN expression in LINC01198overexpressing cells ( Figure 5E). The results showed that recovered PTEN expression reversed LINC01198induced tumor growth ( Figure 5F).

LINC01198 enhances AKT activity through regulation of the NEDD4-1/PTEN axis
To explore whether LINC01198 influenced AKT signaling pathway activity, we analyzed the p-AKT expression in glioma cells. As shown in Figure 6A, LINC01198 overexpression increased p-AKT expression. Conversely, knockout of LINC01198 decreased the expression of p-AKT ( Figure 6B). In particular, LINC01198 knockdown inhibited the interplay between NEDD4-1 and PTEN ( Figure 6C;  Supplementary Figure 3). Finally, the LINC01198induced higher AKT activity was abrogated when knockout of NEDD4-1 was performed ( Figure 6D). Collectively, these observations suggest that LINC01198 enhances AKT activity through NEDD4-1induced PTEN ubiquitin degradation in glioma cells.

LINC01198 expression is correlated with poor prognosis in glioma patients treated with temozolomide
We then analyzed retrospective data from 40 recurrent glioma patients after primary tumor resection receiving temozolomide therapy; the clinicopathological characteristics and OS for these patients were recorded in Table 4. Their LINC01198 expression levels were then detected ( Figure 7A), and Kaplan-Meier survival analysis showed that the OS for the LINC01198 high group was significantly lower than LINC01198 low group ( Figure 7B). The median OS was 4.7 months in the   G and H). A negative correlation between LINC01198 and PTEN was observed in tumor tissues at the protein level (r = 0.501; P = 0.013), but this correlation was not observed at the mRNA level (r = 0.296; P > 0.05). AGING LINC01198 high group, and it was 9.6 months in the LINC01198 high group; therefore, we concluded that high levels of LINC01198 led to the temozolomide resistance in glioma patients (Figure 8).

DISCUSSION
Temozolomide is a classic chemotherapeutic drug that is widely used as treatment for glioma. However, the majority of glioma patients exhibit primary or acquired chemotherapy-resistant during temozolomide chemotherapy, which significantly hinders the use of temozolomide. Despite significant advances in the molecular mechanisms of glioma in the last several decades, the exact molecular mechanisms in temozolomide-resistant remain unclearly.
Recently, it has been demonstrated that lncRNAs are dysregulated in many cancers, including in glioma, and they play vital roles in cancer progression through different mechanisms [33,[10][11][12]. Data derived from the GSE16011, CGGA and REMBRANDT datasets verified LINC01198 overexpression in glioma [8]. However, the biological functions of LINC01198 in glioma have not been reported. In this study, we identified the expression level of LINC01198 and investigated its biological functional role in glioma. Consistent with previous reports, our results demonstrated that LINC01198 expression was upregulated in glioma and that this promoted glioma cell proliferation and chemoresistance to temozolomide. To further explore the molecular mechanism by which LINC01198 plays an oncogenic role, RNA pulldown experiments and RIP were used to determine the downstream target genes. Here, we showed that LINC01198 function as a scaffold to regulate the NEDD4-1-induced ubiquitination degradation process of the PTEN protein at post-transcriptional level.
The tumor suppressor PTEN, an antagonist of the PI3K/AKT pathway, has been found to negatively regulate chemotherapy resistance in several tumors, including glioma [13][14][15]. An abundance of PTEN is required for the prevention of several cancers and for AGING carcinogenesis [16]. Interestingly, unlike most observed genomic alterations in other cancers, the loss of PTEN in cancer is mainly due to posttranslational modifications [17,18]. Particularly, E3 ubiquitin ligasemediated proteasomal degradation of PTEN represents a vital posttranslational mechanism for maintaining a higher level of PTEN in physiological conditions [19].
It has been reported in previous studies that NEDD4-1 and WWP2 are the only two well-defined protooncogenic ubiquitin ligases for PTEN degradation; however, PTEN is a stable protein with a long half-life, and only a very robust overexpression of NEDD4-1 can lead to the downregulation of PTEN protein levels in cancer cells [20][21][22].   PTEN acts as a tumor suppressor gene through inhibition of the PI3K/AKT signaling pathway, which participates in the regulation of various biological functions, including cellular growth, metabolism and survival. Recent studies have confirmed the inactivation of PTEN in several malignancy tumors and leukemia [23][24][25][26][27][28]. Notch-1 is required for trastuzumab resistance by repressing PTEN expression to contribute to activation of ERK1/2 signaling in breast cancer cells [29]. PTEN status contributes to acquired resistance to (B-Raf proto-oncogene, serine/threonine kinase) inhibitors in patients who relapse during treatment with BRAF inhibitors [30]. MiR-3142-induced adriamycin resistance occurs through the targeting of PTEN, which leads to downregulation of the PTEN protein and activation of the PI3K/Akt pathway in chronic myeloid leukemia (CML) cells [31]. Here, we showed that PTEN was a RNA-binding protein of LINC01198. Increased of the PTEN protein was detected after inhibition of LINC01198 in glioma cells occurred. Moreover, reduced of PTEN protein expression was detected after upregulation of LINC01198 in glioma cells occurred. We also found that LINC01198 enhances NEDD4-1mediated PTEN ubiquitin, leading to the activation of the PI3K/AKT signaling pathway. Our results also indicate that reversing PTEN expression could reverse many of the biological functions of LINC01198. These results indicate that LINC01198 has a critical role in temozolomide resistance by regulating the PTEN/PI3K/AKT signaling pathway.

CONCLUSIONS
In conclusion, our results indicate that the gliomaassociated lncRNA LINC01198 is an oncogenic lncRNA that promotes glioma progression by serving as a scaffold and recruiting NEDD4-1 enzymes to target specific proteins, such as PTEN. Our findings support the idea that lncRNAs, such as LINC01198, play crucial roles in glioma progression, and they suggest that LINC01198 is potentially an effective target for glioma therapy.
Glioma tissues and adjacent tissues were collected from patients with glioma at Renji Hospital from Jan 2008 to Jan 2012. Forty-eight of the tumor samples were pathologically graded as low-grade tumors (stage I and stage II), and 42 were graded as highgrade tumors (stage III and stage IV) according to the WHO criteria. All tissues were preserved in liquid nitrogen. In this study, patient consent was received for all investigations and experiments, which have been approved by the ethics committee for clinical research of Renji Hospital.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using TRIzol Regentin in accordance with the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Reverse transcription was carried out using the extracted RNA and the M-MLV reverse transcriptase in accordance with the manufacturer's instructions (Invitrogen, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed using SYBR Premix Ex Taq II (Takara) on an ABI StepOnePlus real-time PCR system in accordance with the manufacturers' instructions (Applied Biosystems, Foster City, CA, USA). The quantification of the expression of RNA was normalized to the expression of GAPDH. The 2 -∆∆CT method was used to evaluate the relative expression. The primers in this study used are listed in Supplementary Table 1.

RNA immunoprecipitation
The EZMagna RNA immunoprecipitation (RIP) kit (Millipore) was used according to the manufacturer's protocol. U87 cells were lysed in complete RIP lysis buffer, and the cell extract was incubated overnight at 4°C with magnetic beads conjugated with specific antibodies or control IgG. The beads were washed in PBS three times and incubated with proteinase K to remove proteins. Finally, purified RNA was used to perform the qRT-PCR analysis.

RNA pulldown assay
The RNA pulldown was performed as previously described [32]. RNAs (LINC01198, LINC01198D1, LINC01198D2) were transcribed using T7 RNA polymerase in vitro, purified using the RNeasy Plus mini kit, and treated with RNase-free DNase I. Transcribed RNAs were biotin-labeled with the Biotin RNA labeling mix (Ambio Life). Positive, negative, and biotinylated RNAs were mixed and incubated with U87MG cell lysates. Magnetic beads were added to each binding reaction, and this was followed by incubation of the samples at room temperature for 2 hours. Then, the beads were washed with washing buffer, and the eluted proteins were examined by western blotting.

Western blotting
Western blotting was performed according to standard protocols as described in reference [33]. The antibodies used in this study has added in Supplementary Table 2.

Proliferation and drug sensitivity analyses
For the proliferation analysis, the effect of the test agents on cell viability was assessed with the CCK-8 assay as described in reference [34]. Three independent experiments were performed. For the drug sensitivity analysis, the cells were seeded into 96-well plates at an initial density of 2 × 10 3 cells per well. After 24 hours of incubation, the cells were incubated in fresh culture medium containing different concentrations of temozolomide for 72 hours (4μM, 20μM, 100μM, 500μM, 2,500μM). The CCK-8 assay was performed to analyze cell viability.

Plasmid construction and cell transfection
The full-length complementary cDNA strands of human LINC01198, LINC01198-D1, and LINC01198-D2 were synthesized by Invitrogen and cloned into the expression vector pCDNA3.1. The small hairpin RNA (shRNA) of LINC01198 was provided by Genepharm. Co. Plasmid vectors for transfection were prepared using DNA Midiprep kits and transfected into glioma cells using Lipofectamine 2000 according to the manufacturer's instructions.

Creation of CRISPR/Cas9-mediated knockout cell lines
Knockout of NEDD4-1 was performed by CRISPR/Cas9 technology, using a previously published protocol as reference [35].

Subcutaneous xenograft tumor models
Glioma cells (2 × 10 5 ) were subcutaneously injected into 4-6 weeks old nude mice. When the tumor volumes reached an average of approximately 100 mm 3 , mice were randomly assigned to treatment groups and vehicle solution group. For treatment groups, temozolomide 25 mg/kg i.p. every 2 days for 4 weeks. The tumor volumes were measured twice every week in two dimensions with Vernier calipers. The tumor volumes were calculated using the following formula: length × width 2 × 0.5. The mice are considered dead when the tumor volumes reached 2, 000 mm 3 . Mice were housed according to protocols approved by the Medical Experimental Animal Care Commission of Renji hospital.

Statistical analysis
All analyses were performed using SPSS software (version 16.0). For continuous variables, the results were shown as mean ± SD. Student's t-test was used to AGING compare the difference between two groups. Differentially expressed LINC01198 between glioma and normal tissues was also evaluated by Student's ttest. Kaplan-Meier curve and log-rank test were used to evaluate the effect of LINC01198 on survival of glioma patients. A two-sided P value < 0.05 was considered as statistically significant.

AUTHOR CONTRIBUTIONS
Xiao-Hua Zhang and Jian-Wei Ge designed the study; Wei-Lin Chen, Hong-Jin Chen and Guo-Qiang Hou performed the experiments and analyzed the data; Jian-Wei Ge analyzed the data and drafted the manuscript; Wei-Lin Chen, Hong-Jin Chen and Guo-Qiang Hou collected the clinical data and performed the statistical analysis. All authors reviewed the manuscript before submission. All authors approved the final version of the manuscript.