Inhibiting mirna-30a-3p has anti-tumor effects in glioma cancer via PTEN

Inhibiting mirna-30a-3p has anti-tumor effects in glioma cancer via PTEN Liexiang Zhang 1,3,*, Wei Jin 2, Jing Zheng 3, Yuxiang Dai 2,Yue Song 1, Hongbin Ni 2, 1. Department of Neurosurgery, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjng210008, Jingsu, China 2. Department of Neurosurgery, The Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing210008, Jingsu, China 3. Department of neurosurgery, Suqian People s Hospital, Nanjing Drum Tower Hospital Group, Suqian223800, Jingsu, China *: Corresponding Author E-mail: zhangliejun0921@126.com Abstract: The aim of this study is to explain the relationship and mechanism of mirna-30a-3p in glioma cancer development. Collecting the 30 glioma patients who were treated in our hospital, the adjacent and cancer tissues were took to evaluate the pathological morphology and PTEN protein expression by H&E staining and immunohistochemistry (IHC). The correlation between mirna-30a-3p and PTEN were confirmed by dual luciferase assay. The LN229 was divided into 3 groups. The LN229 cell proliferations of 3 groups were measured by MTT assay. The apoptosis rates and cell cycles of difference groups were evaluated by flow cytometry. The cell invasion of difference groups were measured by transwell and the cell migration of difference groups were evaluated by wound healing assay. The relative proteins expression (PTEN, MAPK, P53, MMP-2 and MMP-9) were measured by WB assay. Depending on H&E staining, The cancer cell s invasion and migration ability and PTEN protein expression was increased with stage enhancement. The mirna-30a-3p was certain target with PTEN by dual luciferase assay. Compared with NC group, the cell proliferation of si-mirna group was significantly down-regulation; however, the 3

cell apoptosis rate of si-mirna group was significantly up-regulation. By flow cytometry, The G1 phase of si-mirna was significantly higher than that of NC group. The cell invasion and migration abilities of si-mirna group were significantly decreased compared with NC group. With mirna-30a-3p inhibiting, The PTEN and P53 protein expression were significantly enhanced and MAPK, MMP-2 and MMP-9 proteins expression were down-regulation in si-mirna group compared with those in NC group. In conclusion, mirna-30a-3p suppressor had anti-tumor abilities via PTEN. Key words: mirna-30a-3p; LN-229; Glioma; PTEN; MAPK; P53; MMP-2; MMP-9 4

Introduction PTEN (Phosphatase and Tensin homologue deleted on chromosome ten), also called MMAcl or TEP1, was a tumor suppressor gene, Li et al (1) found in 1997, it has dual specificity phosphatase activity. PTEN can promote a group of proteins that regulate the growth of cells by regulating the phosphorylation of proteins (2, 3). microrna (mirna) is a length of about 20 to the highly conserved non encoding single stranded molecule of 22 nucleotides regulate RNA, which can play an important role by regulating target genes in cell proliferation, differentiation, growth and apoptosis. At present, there are 3 main mechanisms of mirna regulated target genes: one is the incomplete complementary binding to the target gene mrna 3'untranslated region (3'UTR) to inhibit protein translation; two is binding to the target gene mrna 3'UTR complementary target genes similar to mrna degradation mediated by sirna; three is the integration of the above two (4). In recent years, the role of mirnas in the tumor has attracted great attention. A number of studies have confirmed that mirnas are closely related to the occurrence, development, diagnosis, treatment and prognosis of cancer (5, 6). In our present study, we firstly discuss the correlation between PTEN protein expression and glioma pathological staging and studied the mirna-30a-3p inhibitor had anti-tumor effects in glioma cancer cell line LN-229. Material and Methods Clinical data The giloma patients who were treated in our hospital were took the adjacent and cancer tissues, and the tissues were stored in -80 until used. The tissues were paraffin embedded and sliced. Evaluating the pathology of tissues by H&E staining and measuring the PTEN protein expression of these tissues by IHC assay. Material 5

Human glioma cancer cell lines LN229 (ATCC, USA) were cultured in RPMI 1640 contained 10% FBS; lipofecter were purchased from Nanjing Kingsy Biological Technology; mirna-30a-3p was 5 -GTTCGTCCCTTTCCAGCTTTACA-3 (Shang hai Biological Engineering Technology Service Co., Ltd, Shanghai). Rabbit anti human PTEN, MAPK, P53, MMP-2 and MMP-9 anti-body were purchased from Abcam (USA). Cell culture and transfection The LN229 cells were cultured in RPMI 1640 contained 10% FBS in incubator which contained 5% CO 2 and 37. After the cells were filled with culture bottles, they were digested, centrifuged and counted. After that, the cells were inoculated in 96 holes. The LN229 cell were divided into 3 groups: NC group: The LN229 cell were treated with nothing; mirna group: The LN229 cell were transfected with mirna-30a-3p and si-mirna group: The LN229 cell were transfected with si-mirna-30a-3p. Dual luciferase reporter gene assay Luciferase reporter gene plasmid (PTEN-WT) containing wild type PTEN 3'-UTR sequence and luciferase reporter gene plasmid (PTEN-Mut) containing mutant PTEN 3'-UTR sequence were constructed. Collect the logarithmic growth phase LN229, adjusting the cell density to 2 * 105 cells/well, and inoculated in 24 Kong Banzhong, PTEN-WT PTEN-Mut and mir-30a-3p mimics plasmid, plasmid or control oligonucleotide was transfected into LN229, with 3 wells in each group, the cells were placed in a constant temperature incubator (37, 5% CO 2 ) to culture 48 h; the blank plasmid fluorescence value as reference, to detect the expression of luciferase activity. Cell proliferation by MTT assay After transfection the cells were placed in an incubator (37, 5% CO 2 ) in cultured 4 h after culture, 800 r/min centrifugation 10 min, supernatant per hole adding MTT solution (5 mg/ml) 20 L on Incubator (37, 5% CO 2 ) cultured for 4 h, after absorbing abandoned hole Culture Raising supernatant, adding DMSO (100 μl/, 6

10 min hole) to the crystal oscillation, fully dissolved, the automatic ELISA test OD 490nm absorption value. Cell cycle and apoptosis by flow cytometry The next step was detected after transfection of 36 h. The detection of cell cycle, with 75% frozen ethanol suspension cells, as far as possible gently dispersed into a single cell, Fixed -20 for 1h; Suspension of cells with 200~500 μl 4 pre cooled PBS buffer. Adding 400 μl Prolidium Iodide (PI), 4 light stain for 15 min, using BDLSR II flow cytometry to measure. The cell invasion ability by Transwell assay 60 μl Matrigel glue were added on the polycarbonate film on the Transwell room at 37 for 30 min. The cells were inoculated on Transwell cells by 10 4 cells / holes, and the DMEM medium containing 600 l of bovine serum was added to the lower chamber. 48 h after taking out the room, wet cotton swab wipe membrane surface through the membrane of cells, hematoxylin mounting. Count the number of cells through the membrane under inverted microscope. The cell migration ability by wound healing test The cells of difference groups were inoculated in 24 well plates as 2 10 4 cell/hole. Scratching the cultured cells with sterile 250 μl gun head, After the scratch, the plates were carefully cleaned with the culture medium for 2 times to remove the exfoliated cells, and then the fresh medium was added to the culture medium. In the scratch 0 h, 48 h, respectively, to select a different field of view to take photographs. Each group was set up with 4 replicates, each of which was selected for 5 field views. Measuring the cell would heal rates. The relative protein expression by WB assay The cells of difference groups were washed by PBS for 3 times after treated by difference methods. Adding the RIPA RIPA lysis solution was mixed with PMSF (100: 1) for cell lysis of 15 min, and the supernatant was collected after centrifugation. The protein was quantified by BCA method. The total protein was obtained by SDS-PAGE gel electrophoresis (concentration of 80V, 120 V), and the protein was transferred to 7

NC membrane (V) by semi dry transfer method, and then closed at room temperature with 5% skimmed milk powder at 1 h. Adding PTEN (1:500), MAPK (1:500), P53 (1:500), MMP-2 (1:500), MMP-9(1:500) and GAPDH (1:1000) first anti-body., culturing overnight at 4, After washing by PBS for 3 times, The corresponding two resist (1: 10000) was incubated at room temperature for 1 h, and the membrane was washed for 4 times. Odyssey infrared laser imaging system was used to scan, and the ratio of /GAPDH was the relative expression of the protein. Statistical analysis The data were analysis by SPSS 19.0 software, the data was shown as mean ± standard deviation ( s), Variance analysis was used to compare between groups, Enumeration data were compared using x 2 test, P<0.05 was statistically significant. Results Clinical analysis The invasion and migration abilities of glioma cancer were increased with degree increasing by H&E staining (Figure 1A); Meanwhile, PTEN protein expression of glioma cancer tissues were decreased with degree increasing by IHC (Figure 1B). Figure 1. The glioma cancer tissues of difference degree by H&E staining and PTEN protein 8

expression by IHC 1A. The pathological morphology of difference glioma cancer tissues by H&E staining 1B. The PTEN protein expression of difference glioma cancer tissues by IHC Dual luciferase Report MicroRNA target gene prediction of Targetscan predicted that mir-30a-3p was associated with the presence of a 3 'UTR binding site of tumor suppressor gene PTEN. It is proved that PTEN may be the target gene of mir-30a-3p by dual luciferase target. The data was shown in Figure 2. Figure 2. Double luciferase assay **: P<0.05, Compared with Control The cell proliferation rate by MTT assay The cell apoptosis rate of si-mirna group was significantly down-regulation compared with NC group (P<0.05, Figure 3). 9

Figure 3. The cell proliferation of difference groups **: P<0.05, Compared with NC group The cell apoptosis of difference group by flow cytometry The cell apoptosis of si-mirna group was significantly increased compared with NC group (P<0.05, Figure 4). Figure 4. The cell apoptosis rates of difference groups 10

**: P<0.05, Compared with NC group The cell cycle of difference group by flow cytometry The G1 phase of si-mirna group was significantly increased compared with NC group (P<0.05, Figure 5). Figure 5. The cell cycle of difference groups **: P<0.05, Compared with NC group The cell invasion ability of difference groups by transwell The invasion cell number of si-mirna group was significantly decreased compared with NC group (P<0.05, Figure 6). 11

Figure 6. The cell invasion abilities of difference groups **: P<0.05, Compared with NC group The cell migration ability of difference groups by wound healing assay The LN229 cell wound healing rate of si-mirna group was significantly increased compared with NC group (P<0.05, Figure 7). 12

Figure 7. The cell migration abilities of difference groups by wound healing assay **: P<0.05, Compared with NC group The relative protein expression by WB assay Compared with NC group, The PTEN and p53 protein expression were significantly up-regulation and MAPK, MMP-2 and MMP-9 protein expression were significantly down-regulation (P<0.05,respectively), The data was shown in Figure 8. 13

Figure 8. The relative proteins expressions by WB assay **: P<0.05, Compared with NC group Discussion mirna is a highly conserved non coding small molecule single stranded RNA, and is widely found in eukaryotes. Under normal physiological conditions, the expression of strict organization and sequence specific mirna in the body; but in the tumor environment, different mirna showed different biological activity, and is similar to that of oncogenes or tumor suppressor genes (7). PTEN/MAPK signaling pathway is an important pathway for many biological processes, such as apoptosis, metabolism, cell proliferation and cell growth regulation (8). PTEN gene, as an effective tumor suppressor gene, has been found to be mutated or deleted in a variety of human cancers, including hepatocellular carcinoma. As a tumor suppressor gene PTEN and protein phosphatase with lipid phosphatase activity can influence the nucleus, and can affect the cell surface, can stabilize and enhance tumor cell adhesion, cell proliferation and inhibit migration of tumor and non anchorage dependent growth, and induce apoptosis, and inhibit the degradation of extracellular matrix and tumor angiogenesis, thus the inhibition of tumor invasion and metastasis, tumor suppressor role (9). In our study, we found that PTEN protein expression was reduced with glioma cancer degree increasing. Further, we found mirna-30a-3p was the target gene to PTEN by bioinformatics. The mirna-30a-3p suppressor (si-mirna) had effects to inhibit mirna-30a-3p expression and improve PTEN expression and to suppress the glioma cancer cell lines LN-229 cell biological activities (proliferation, invasion and migration) in vitro experiments. MAPK signaling pathway, including ERK, p38 and JNK3 pathway, plays an important role in cell proliferation, apoptosis and differentiation (10, 11). p53 is a downstream gene of MAPK. The p53 gene plays an important role in the regulation of cell cycle and growth, and its mutation or deletion is directly related to the occurrence of a variety of tumors. About 50% of lung cancer has the mutation or deletion of this 14

gene (12). p53 can regulate cell cycle, apoptosis and senescence by activating p21waf /cip1, regulating Bax, Fas and Smad4 (13-15). In our study, we found that mirna-30a-3p suppressor (si-mirna) had effects to inhibit cell proliferation and stimulate cell apoptosis by regulation MAPK/p53 pathway. Matrix metalloproteinases are a large family, because it requires Ca 2 +, Zn 2 + and other metal ions as cofactors. MMPs can degrade almost all kinds of protein components in extracellular matrix, destroy the histological barrier of tumor cell invasion, and play a key role in tumor invasion and metastasis (16, 17). Among the MMPs proteins, MMP-2 and MMP-9 were involved in angiogenesis and degradation of the basement membrane is directly related with tumor metastasis and prognosis (18, 19). In our present study, The LN-229 cell invasion and migration abilities were inhibited by mirna-30a-3p inhibitor (si-mirna-30a-3p) transfection by regulation MMP-2/9 proteins expression. References: 1. Li J, Yea C, Liaw D, et al: PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275:1943-1947, 1997. 2. Wullachleger S, Loewith R, Hall MN: TOR signaling in growth and metabolism. Cell 124:471-484, 2006. 3. Tsang CK, Zheng XF: TOR-in (g) the nucleus. Cell Cycle 6:25-29, 2007. 4. Bi Y, Liu G, Yang R: MicroRNAs: novel regulators during the immune response. J Cell Physiol 218:467-472, 2009. 5. Yang S, Li Y: MicroRNAs: novel factors in clinical diagnosis and prognosis for nasopharyngeal carcinonia. Acta Pharmacol Sin 33: 981-982, 2012. 6. Wang D, Qiu C, Zhang H, et al: Human microrna oncogenes and tumor suppressors show significantly different biological patterns: from functions to gargets. PLoS One 5: pii: e13067, 2010. 7. Xu X, Yang X, Xing C, et al: mirna: The nemesis of gastric cancer (Review). Oncol Lett 6: 631-641, 2013. 8. Song G, Ouyang G, Bao S: The activation of AKT/PKB signaling pathway and cell survival. J 15

Cel Mol Mel 9:59-71, 2005. 9. Di Cristofano A, Pandolfi PP: The multiple roles of PTEN in tumor suppression. Cell 100: 387-390, 2000. 10. Fecher LA, Amaravadi RK, Flaherty KT: The MAPK pathway in melanoma. Curr Opin Oncol, 20: 183-189, 2008. 11. Junttila MR, Li SP, Westermarck J: Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival. FASEB J 22: 954-965, 2008. 12. Fisher MD: Strategies to restore p53 function in patients with lung cancer (J). Clin Lung Cancer 3: 99-101, 2001. 13. Moll UM, Wolff S, Speidel D, et al: Transcription-independent pro-apoptotie functions of P53. Curr Opin Cell Biol 17: 631-636, 2005. 14. Levine AJ, Finlay CA, Hinds PW: P53 is a tumor suppressor gene. Cell 116: 67-69, 2004. 15. Kalo E, Buganim Y, Shapira KE, et al: Mutant p53 attenuates the SMAD-dependent transforming growth factor β1 (TGF-β1) signaling pathway by repressing the expression of TGF-β receptor type II. Mol Cell Biol 27: 8228-8242, 2007. 16. Curran S, Murray GI: Matrix metalloproteinases: Molecular aspects of their roles in tumour invasion and metastasis. Eur J Cancer 36: 1621-1630, 2000. 17. Dong Z, Bonfil RD, Chinni S, et al: Matrix metalloproteinase activity and osteolasts in experimental prostate cancer bone metastasis tissue. Am J Pathol 166: 1173-1186, 2005. 18. Chang HR, Chen PN, Yang SF, et al: Silibinin inhibits the invasion and migration of renal carcinoma 786-O cells in vitro, inhibits the growth of xenografts in vivo and enhances chemosensitivity to 5-fluorouracil and paclitaxel. Mol Carcinog 50: 811-823, 2011. 19. Rink M, Chun FK, Robinson B, et al: Tissue-based molecular markers for renal cell carcinoma. Minerva Urol Nefrol 63: 293-308, 2011. 16