MicroRNA-136-3p inhibits glioma tumorigenesis in vitro and in vivo by targeting KLF7

Background Malignant brain tumors have been a serious threat to human health worldwide. This study aims to investigate the role of miR-136-3p in glioma development. Methods Hematoxylin-eosin staining (H&E) staining was used to determine the pathologic alterations of glioma tissues. Quantitative real-time PCR (qRT-PCR) analysis and GEO2R analysis was performed to examine the expression of miRNAs and genes. Western blot was applied to detect the protein expression. Cell counting kit-8 (CCK-8) and colony formation were used to analyze the glioma cell growth. Trans-well assay was used to determine the cell migration. Annexin V-FITC/PI staining was conducted to determine the cell apoptosis of transfected glioma cells. The dual-luciferase reporter assay was carried out to confirm the binding sites of miR-136-3p on 3′ untranslated regions (3′ UTR) of Kruppel-like factor 7 (KLF7). Tumor-bearing experiment in nude mice was performed to comprehensively investigate the role of miR-136-3p/KLF7 axis in gliomas. Results Firstly, the results showed that miR-136-3p was decreased in glioma tissues compared with adjacent tissues. Overexpression of miR-136-3p significantly inhibited cell growth of LN-229 and U251 by decreasing expression of Cyclin A1 and PCNA (proliferating cell nuclear antigen), and it suppressed glioma cell migration by downregulating N-cadherin and elevating E-cadherin levels, and it also promotes glioma cell apoptosis by promoting Bcl2-associated X (Bax) expression but suppressing Bcl-2 expression. Furthermore, we observed that KLF7 was a direct target of miR-136-3p, and KLF7 was negatively regulated by miR-136-3p in glioma cells. Finally, overexpression of KLF7 partly blocked miR-136-3p-induced inhibition of tumor growth in vitro and in vivo. Conclusions Targeting miR-136-3p/KLF7 axis might be a novel manner to counter against gliomas.

To explore the potential molecular mechanisms of miR-136-3p in gliomas, we predicted its targets using TargetScan database. Integration of this bioinformatic analysis with the previous studies, we observed that KLF7 was a direct target of miR-136-3p in gliomas. In the previous studies, KLF3, targeted by miR-450b-3p and miR-185, often severs as an oncogene in gastric cancer [18] and non-small cell lung cancer respectively [19]. And KLF7, regulated by Linc00669/miR-193a axis, may also promote progression of non-small cell lung cancer [20]. Importantly, KLF7 can enhance glioma progression by transcriptionally activating argininosuccinate lyase [21]. Consequently, we can conclude that KLF7 is an oncogene in cancers, such as glioma. Herein, we investigated the role of miR-136-3p/KLF7 axis in glioma cell proliferation, migration, and apoptosis, and we also explored the potential molecular mechanisms. The findings showed that overexpression of miR-136-3p might provide a novel therapeutic target for glioma development.

Collection of cancer tissues
In this study, adjacent tissues and glioma tissues were collected from 41 patients in Shandong Provincial Lanling People's Hospital. The clinical processes were approved by the ethics committee of this Hospital. None of the patients had received treatment before surgery. These collected tissues were put in liquid nitrogen.

Cell culture
The U251 and LN-229 were bought from ATCC (American Type Culture Collection, Manassas, VA, USA), and they were cultured in Dulbecco's Modified Eagle Medium (DMEM) basic media (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and the media contained 10% fetal bovine serum, and 100 mg/ mL streptomycin, and 100 U/mL penicillin (Thermo Fisher Scientific). They were cultured in a 37°C sterile incubator with 5% CO 2 .

Western blot
The supernatant, from lysed cancer cells or tissues, was used for determining the protein expression in this study using radio-immunoprecipitation assay (RIPA) lysate (Thermo Fisher Scientific). The lysates were boiled at 98°C for 10 min. Then, 60 μg proteins were added on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) (Sangon). Afterward, these gels were transferred to 0.22 nm polyvinylidene difluoride (PVDF) membranes for 60 min (Bio-Rad, Hercules, CA, USA). Then, PVDF membranes were blocked with 2% phosphate buffer saline (PBS)-diluted bovine serum albumin (BSA) were incubated with the primary antibody at 4°C for 8 h. The protein bands were captured by using Tanon 4600SF system (Tanon, Shanghai, China) after coating with horseradish peroxidase (HRP)-conjugated goat-anti-mouse or goat anti-rabbit secondary antibody (Thermo Fisher Scientific). The anti-KLF7 and anti-β-actin were bought from Bioworld Technology (Nanjing, China). The experiments were carried out for three times independently.

Cell counting kit-8 (CCK-8) assay
In our study, the cell proliferation of LN-229 or U251 was assessed by performing CCK-8 assay (Sigma). Firstly, 3 × 10 3 cells/well was prepared in 96-well plates (Corning, Shanghai, China). After 48 h transfection with miR-NAs or plasmids, the OD value was measured in the microplate reader (Thermo Fisher Scientific). The experiments were conducted for three times independently, and 5-well was prepared for each group.

Colony formation assay
Firstly, 2000 cells/well was plated into a 6-well plate (Corning). After 48 h transfection with miRNAs or plasmids, the cells (LN-229 or U251) were maintained in normal fresh media. Next, the media was replaced every 3 days. Post 14 days incubation, the visible colonies were stained with 0.1% crystal violet (Sigma) after fixation with methanol for 1 h, and they were captured using a microscopy (Leica, WETZLAR, German). The experiments were conducted for three times independently, and 5-well was prepared for each group.

Trans-well assay
Here, we put 1.0 × 10 5 cells per well into the topchambers of multi-well plates (Corning). Then, we added 10% FBS-contained DMEM into the down chambers. Post 16 h incubation, the migrated cells were stained with crystal violet after fixation with 4% paraformaldehyde. In the end, we observed the migrated cells with a microscopy under × 200 magnification (Leica). The experiments were carried out for three times independently, and 5-well was prepared for each group.

Annexin-V/fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining
The cancer cells were seeded in 24-well plates, and they were harvested at 80 g for 5 min post 48 h transfection. Afterward, the LN-229 or U251 was stained with an Annexin V-FITC/PI kit (Solaribio, Beijing, China) in the dark following the manufacturer's instruction. Finally, the cell apoptosis rate of cancer cells was determined on Bio-Rad S3e flow cytometry (Hercules, CA, USA). The experiments were conducted for three times independently, and 3-well was prepared for each sample.

Animal experiments
The LN-229 cells were collected, and 6 × 10 6 cells were planted into 5-week-old BALB/c nude mice. The mice were bought from the Model Animal Research Center of Nanjing University (Nanjing, China). At the initiation of tumor growth (on the third day), the lenti-viruses were injected into tumors, and this injection was repeated every 6 days. These animal experiments were approved by the ethics committee of Shandong Provincial Lanling People's Hospital. Afterward, the tumor volume was measured using the formula (length × width 2 )/2 at the indicated time. Finally, the tumors were captured and weighted.

Data analysis
The data here were indicated as the mean ± standard deviation (S.D.). A p value < 0.05 was statistically significant. All the showed data were analyzed statistically using GraphPad Prism 6 (GraphPad Software, CA, USA). The statistical significance was examined by Two-tailed Student's t test for two groups comparisons and one-way analysis of variance (ANOVA) test with post hoc analysis contrasts for multi-groups comparisons.

MiR-136-3p is elevated in glioma tissues
To investigate the role of miR-136-3p in glioma development, we collected tumors from patients with glioma in the hospital. The clinical characteristics of 41 patients were showed in Table 1. The H&E staining showed that glioma tissues showed significant pathologic alterations compared with adjacent tissues (Fig. 1a). GEO2R analysis and qRT-PCR analysis revealed that miR-136-3p was significantly decreased in glioma tissues compared with adjacent tissues (Fig. 1b, c).   Overexpression of miR-136-3p suppresses glioma cell growth and migration Here, we overexpressed miR-136-3p in LN-229 and U251 to detect the glioma cell growth and migration. The data showed that miR-136-3p mimics efficiently overexpressed miR-136-3p in LN-229 and U251 cells (Fig. 2a). The CCK-8 assay told that overexpression of miR-136-3p significantly inhibited glioma cell proliferation (Fig. 2b). Soft-agar colony formation assay analysis demonstrated that overexpression of miR-136-3p obviously repressed glioma cell colony formation of LN-229 and U251 cells (Fig. 2c, d). Meanwhile, overexpression of miR-136-3p significantly downregulated cell growthassociated gene expression, including Cyclin A1 and PCNA in LN-229 and U251 cells (Fig. 2e, f). Finally, trans-well assay demonstrated that overexpression of miR-136-3p markedly decreased the migration of LN-229 and U251 (Fig. 2g, h). Finally, we found that overexpression of miR-136-3p elevated expression of migration-associated gene E-cadherin, and it reduced Ncadherin levels in LN-229 and U251.

KLF7 is a direct target of miR-136-3p in glioma cells
To investigate the potential molecular mechanism of miR-136-3p in gliomas, we predicted the downstream targets of miR-136-3p using TargetScan tools. Base on comprehensive consideration of both the bioinformatic prediction and the previous studies [19][20][21][22], we can conclude that KLF7 is an oncogene in glioma or other cancers. Thus, we selected KLF as the target of miR-136-3p in glioma (Fig. 4a). Luciferase reporter assay expressed that overexpression of miR-136-3p significantly inhibited luciferase activity of KLF7 3′ UTR, which was upregulated by knocking miR-136-3p down in LN-229 and U251 (Fig. 4c, d). Then, qRT-PCR analysis and western blot analysis revealed that miR-136-3p negatively modulated KLF7 expression (Fig. 4e, f, g). Moreover, the miR-136-3p expression is negatively associated with KLF7 levels in clinical samples (Fig. 4h). Additionally, KLF7 was increased in glioma tissues (Fig. 4i).
MiR-136-3p has been reported to inhibit progression of thyroid cancer [16], implying that miR-136-3p may serve as a tumor suppressor in tumors. However, little is known about the functions of miR-136-3p in other cancers, including gliomas. In this study, miR-136-3p was decreased in glioma tissues compared with the adjacent tissues. Importantly, the ability of cell growth and migration was inhibited in miR-136-3p-overexpressed cancer cells (LN-229 and U251), and the cell apoptosis was promoted in miR-136-3poverexpressed cancer cells, suggesting that miR-136-3p may function as a tumor suppressor in gliomas. Consistently, overexpression of miR-136-3p significantly inhibited tumor growth in nude mice, conforming that miR-136-3p can serve as a cancer suppressor and it can inhibit the tumorigenesis of glioma.
MiRNAs show its function mainly by targeting 3′ UTR of mRNA in many diseases [7][8][9]. We predicted the target genes of miR-136-3p via using TargetScan database. Our results demonstrated that KLF7 was a direct target of miR-136-3p. Previous studies have revealed that KLF7 can promote polyamine biosynthesis and glioma development by activating argininosuccinate lyase [21], and meanwhile, KLF7 can be modulated by Linc00669/miR-193a axis and it can advance non-small cell lung cancer [20], suggesting that KLF7 is an oncogene. In this study, we observed that KLF7 expression was inhibited by overexpression of miR-136-3p, and KLF7 levels was increased by knockdown of miR-136-3p in cancer cells, indicating that KLF7 was a direct target of miR-136-3p in glioma. Then, in vitro and in vivo experiments were performed to determine whether KLF7 was required for miR-136-3p exerting its functions in glioma. The data showed that overexpression of KLF7 can partly block overexpression of miR-136-3p-mediated suppression of glioma progression by regulating cell growth, migration, and apoptosis of cancer cells.

Conclusion
These findings demonstrated that miR-136-3p is significantly decreased in cancer tissues and it serves as a tumor suppressor by directly targeting KLF7 in gliomas. The results also encourage that miR-136-3p and KLF7 might be promising novel biomarkers and therapeutic targets for glioma patients' treatment.