RGS16 regulated by let-7c-5p promotes glioma progression by activating PI3K-AKT pathway

doi:10.1007/s11684-022-0929-y

Front. Med.

2023, Vol. 17

Issue (1) : 143-155 https://doi.org/10.1007/s11684-022-0929-y

RESEARCH ARTICLE

RGS16 regulated by let-7c-5p promotes glioma progression by activating PI3K-AKT pathway

Chaochao Wang^1,², Hao Xue^1,², Rongrong Zhao^1,², Zhongzheng Sun^2,³, Xiao Gao^1,², Yanhua Qi^1,², Huizhi Wang^1,², Jianye Xu^1,², Lin Deng^1,²(

), Gang Li^1,²(

)

¹. Department of Neurosurgery, Qilu Hospital, Cheeloo College of Medicine and Institute of Brain and Brain-Inspired Science, Shandong University, Jinan 250012, China
². Shandong Key Laboratory of Brain Function Remodeling, Jinan 250012, China
³. The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250033, China

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Abstract

Gliomas are the most common central nervous system tumours; they are highly aggressive and have a poor prognosis. RGS16 belongs to the regulator of G-protein signalling (RGS) protein family, which plays an important role in promoting various cancers, such as breast cancer, pancreatic cancer, and colorectal cancer. Moreover, previous studies confirmed that let-7c-5p, a well-known microRNA, can act as a tumour suppressor to regulate the progression of various tumours by inhibiting the expression of its target genes. However, whether RGS16 can promote the progression of glioma and whether it is regulated by miR let-7c-5p are still unknown. Here, we confirmed that RGS16 is upregulated in glioma tissues and that high expression of RGS16 is associated with poor survival. Ectopic deletion of RGS16 significantly suppressed glioma cell proliferation and migration both in vitro and in vivo. Moreover, RGS16 was validated as a direct target gene of miR let-7c-5p. The overexpression of miR let-7c-5p obviously downregulated the expression of RGS16, and knocking down miR let-7c-5p had the opposite effect. Thus, we suggest that the suppression of RGS16 by miR let-7c-5p can promote glioma progression and may serve as a potential prognostic biomarker and therapeutic target in glioma.

Keywords RGS16 let-7c-5p glioma proliferation migration

Corresponding Author(s): Lin Deng,Gang Li

Just Accepted Date: 30 August 2022 Online First Date: 23 November 2022 Issue Date: 15 March 2023

Cite this article:

Chaochao Wang,Hao Xue,Rongrong Zhao, et al. RGS16 regulated by let-7c-5p promotes glioma progression by activating PI3K-AKT pathway[J]. Front. Med., 2023, 17(1): 143-155.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-022-0929-y
https://academic.hep.com.cn/fmd/EN/Y2023/V17/I1/143

Fig.1 Expression of RGS16 is upregulated in gliomas and is associated with tumor grade. (A) Quantification of RGS16 mRNA expression levels in LGG and GB tissues compared with normal brain tissues in TCGA. (B) IHC staining images for RGS16 in gliomas of various grades and normal brain specimens. Magnification, 200×, upper; 400×, lower. (C) Quantitative analysis of RGS16 staining scores in gliomas of various grades and normal brain specimens. Scores were presented as the median + interquartile range, significance was determined using one-way ANOVA of non-parametric test (Kruskal-Wallis). RGS, regulator of G-protein signaling; TCGA, The Cancer Genome Atlas; LGG, low grade glioma; GB, glioblastoma; WHO, World Health Organization.

Fig.2 Knockdown of RGS16 inhibits glioma cell proliferation. U87 and U251 cells transfected with RGS16 siRNA or NC were characterized. (A, B) Expression of RGS16 was downregulated in glioma cells at the mRNA and protein level. (C, D) CCK-8 assays revealed that knockdown of RGS16 reduced the proliferation of U87 and U251 glioma cells compared to transfection with NC. (E, F) Colony-formation assays showed that knockdown of RGS16 reduced colony numbers. (G, H) EdU staining performed 48 h after transfection showed fewer proliferating cells (DAPI staining) in cells with RGS16 knockdown compared with the NC-transfected cells. (I) Variations in protein expression levels were detected via Western blotting. GAPDH was used as the loading control. RGS, regulator of G-protein signaling; siRNA, small interfering RNA; NC, negative control; CCK-8, Cell Counting Kit-8.

Fig.3 Knockdown of RGS16 reduces migration and invasion in glioma cells in vitro. (A–D) Cell migration/invasion assays were performed after RGS16 knockdown in glioma U87 and U251 cells, scale bar = 200 μm. Data are shown as the mean ± standard deviation. (E) Changes in protein expression levels were assessed by Western blotting and showed that MMP2 and MMP9 levels were lower in the si-RGS16 group compared with the NC group. (F) Western blotting assay showed that AKT2 and p-AKT levels were lower in the si-RGS16 group compared with the NC group. GAPDH was used as the internal control. (G, H) Wound-healing assays were performed on monolayers of U87 and U251 glioma cells after 48 h of transfection. Images were taken 0 and 24 h after the wounds were made, scale bar = 200 μm. RGS, regulator of G-protein signaling; siRNA, small interfering RNA; NC, negative control; p-, phospho.

Fig.4 Overexpression of miR-let-7c-5p inhibits glioma cell proliferation/migration/invasion ability. (A) Target site of let-7c-5p in the 3′-UTR of RGS16 was predicted by TargetScan. The mutant sequence used was identical to the wild-type sequence except for mutations at the 3′ end of the target site. (B) Relative expression of miR let-7c-5p in NBTs and GBM tissues. (C) U87 and U251 cells were transfected with let-7c-5p mimics or inhibitor; two independent scrambled sequence was used as the negative control. RT-qPCR assays proved that the expression of let-7c-5p was upregulated when transfected with mimics, while downregulated with inhibitor. (D) EdU staining showed fewer proliferating cells (DAPI staining) in the mimics group compared with the NC group and inhibitor group. (E) Cell migration/invasion assays were performed, and the mimics group exhibited the lowest amount of migrating/invading cells. (F) A bioluminescence imaging system was used to evaluate tumor development in each group as indicated. Representative bioluminescent images are shown. (G) Survival analysis for mice implanted with ov-let-7c-5p or NC. n = 5. (H,I) mRNA and protein levels of RGS16 were downregulated in glioma cells overexpressing let-7c-5p, while upregulated after let-7c-5p inhibitor was transfected. (J) Reporter constructs, including wild-type and mutant, were co-transfected with let-7c-5p mimics or NC for 48 h and luciferase activity was detected. (K) AGO2-dependent RNA immunoprecipitation (RIP) assay in U87 and U251 cells, vs. anti-IgG. The results are presented as the mean of the normalized luciferase intensity. RGS, regulator of G-protein signaling; UTR, untranslated region.

Fig.5 RGS16 is a target of let-7c-5p. (A) CCK8 assays showing the proliferation ability of GBM cells co-transfected with NC, mimics and ov-RGS16 as indicated, n = 3. (B) Representative EdU assays showing the proliferation ability of GBM cells co-transfected with NC, mimics and ov-RGS16 as indicated. Quantification histogram represented relative cell numbers, n = 3. (C) Wound-healing assays were performed on monolayers of GBM cells in each group. Images were taken 0 and 24 h after the wounds were made. (D) Representative Transwell migration and invasion assays showing the migration and invasion ability of GBM cells co-transfected with NC, mimics and ov-RGS16 as indicated. Quantification histogram represented relative cell numbers.

Fig.6 Knockdown of RGS16 inhibits tumorigenesis in vivo. Luciferase-labeled U87 cells were infected with sh-RGS16 or control. (A) Expression of RGS16 was downregulated after transfected with sh-RGS16. (B) A bioluminescence imaging system was used to evaluate tumor development in each group weekly. Representative bioluminescent images are shown. (C) Relative radiance is represented as the total flux value. (D) Survival analysis for mice implanted with sh-RGS16 or control. n = 5. (E) Relative mRNA levels of RGS16 were detected when mice died, tumors were taken out and RNA was extracted for RT-qPCR. (F) Hematoxylin and eosin staining of tumor and peripheral tissue border in the two groups of xenografts. (G–J) Immunohistochemical analysis of RGS16 and Ki-67 expression in sections from sacrificed mice. Magnification, 200×, upper; 400×, lower. Arrows indicate the tumor margin. sh, short hairpin; T, tumor, NBT, normal brain tissue.

1	GP Dunn, ML Rinne, J Wykosky, G Genovese, SN Quayle, IF Dunn, PK Agarwalla, MG Chheda, B Campos, A Wang, C Brennan, KL Ligon, F Furnari, WK Cavenee, RA Depinho, L Chin, WC Hahn. Emerging insights into the molecular and cellular basis of glioblastoma. Genes Dev 2012; 26(8): 756–784 https://doi.org/10.1101/gad.187922.112 pmid: 22508724
2	C Villa, C Miquel, D Mosses, M Bernier, AL Di Stefano. The 2016 World Health Organization classification of tumours of the central nervous system. Presse Med 2018; 47(11–12): e187–e200 https://doi.org/10.1016/j.lpm.2018.04.015 pmid: 30449638
3	F Bray, J Ferlay, I Soerjomataram, RL Siegel, LA Torre, A Jemal. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68(6): 394–424 https://doi.org/10.3322/caac.21492 pmid: 30207593
4	J Chen, RM McKay, LF Parada. Malignant glioma: lessons from genomics, mouse models, and stem cells. Cell 2012; 149(1): 36–47 https://doi.org/10.1016/j.cell.2012.03.009 pmid: 22464322
5	EM Ross, TM Wilkie. GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu Rev Biochem 2000; 69(1): 795–827 https://doi.org/10.1146/annurev.biochem.69.1.795 pmid: 10966476
6	KM Druey, KJ Blumer, VH Kang, JH Kehrl. Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family. Nature 1996; 379(6567): 742–746 https://doi.org/10.1038/379742a0 pmid: 8602223
7	G Liang, G Bansal, Z Xie, KM Druey. RGS16 inhibits breast cancer cell growth by mitigating phosphatidylinositol 3-kinase signaling. J Biol Chem 2009; 284(32): 21719–21727 https://doi.org/10.1074/jbc.M109.028407 pmid: 19509421
8	EN Johnson, TM Seasholtz, AA Waheed, B Kreutz, N Suzuki, T Kozasa, TL Jones, JH Brown, KM Druey. RGS16 inhibits signalling through the G alpha 13-Rho axis. Nat Cell Biol 2003; 5(12): 1095–1103 https://doi.org/10.1038/ncb1065 pmid: 14634662
9	M Berthebaud, C Rivière, P Jarrier, A Foudi, Y Zhang, D Compagno, A Galy, W Vainchenker, F Louache. RGS16 is a negative regulator of SDF-1-CXCR4 signaling in megakaryocytes. Blood 2005; 106(9): 2962–2968 https://doi.org/10.1182/blood-2005-02-0526 pmid: 15998835
10	MB Carper, J Denvir, G Boskovic, DA Primerano, PP Claudio. RGS16, a novel p53 and pRb cross-talk candidate inhibits migration and invasion of pancreatic cancer cells. Genes Cancer 2014; 5(11–12): 420–435 https://doi.org/10.18632/genesandcancer.43 pmid: 25568667
11	N Miyoshi, H Ishii, M Sekimoto, Y Doki, M Mori. RGS16 is a marker for prognosis in colorectal cancer. Ann Surg Oncol 2009; 16(12): 3507–3514 https://doi.org/10.1245/s10434-009-0690-3 pmid: 19760045
12	X Sun, C Charbonneau, L Wei, Q Chen, RM Terek. miR-181a targets RGS16 to promote chondrosarcoma growth, angiogenesis, and metastasis. Mol Cancer Res 2015; 13(9): 1347–1357 https://doi.org/10.1158/1541-7786.MCR-14-0697 pmid: 26013170
13	D Sayed, M Abdellatif. MicroRNAs in development and disease. Physiol Rev 2011; 91(3): 827–887 https://doi.org/10.1152/physrev.00006.2010 pmid: 21742789
14	GA Calin, CM Croce. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; 6(11): 857–866 https://doi.org/10.1038/nrc1997 pmid: 17060945
15	X Fu, X Mao, Y Wang, X Ding, Y Li. Let-7c-5p inhibits cell proliferation and induces cell apoptosis by targeting ERCC6 in breast cancer. Oncol Rep 2017; 38(3): 1851–1856 https://doi.org/10.3892/or.2017.5839 pmid: 28731186
16	M Huang, X Gong. Let-7c inhibits the proliferation, invasion, and migration of glioma cells via targeting E2F5. Oncol Res 2018; 26(7): 1103–1111 https://doi.org/10.3727/096504018X15164123839400 pmid: 29362021
17	PY Wen, S Kesari. Malignant gliomas in adults. N Engl J Med 2008; 359(5): 492–507 https://doi.org/10.1056/NEJMra0708126 pmid: 18669428
18	R Huang, G Li, Z Zhao, F Zeng, K Zhang, Y Liu, K Wang, H Hu. RGS16 promotes glioma progression and serves as a prognostic factor. CNS Neurosci Ther 2020; 26(8): 791–803 https://doi.org/10.1111/cns.13382 pmid: 32319728
19	I Chu, J Sun, A Arnaout, H Kahn, W Hanna, S Narod, P Sun, CK Tan, L Hengst, J Slingerland. p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2. Cell 2007; 128(2): 281–294 https://doi.org/10.1016/j.cell.2006.11.049 pmid: 17254967
20	G Choe, S Horvath, TF Cloughesy, K Crosby, D Seligson, A Palotie, L Inge, BL Smith, CL Sawyers, PS Mischel. Analysis of the phosphatidylinositol 3′-kinase signaling pathway in glioblastoma patients in vivo. Cancer Res 2003; 63(11): 2742–2746 pmid: 12782577
21	L Wang, ZG Zhang, RL Zhang, SR Gregg, A Hozeska-Solgot, Y LeTourneau, Y Wang, M Chopp. Matrix metalloproteinase 2 (MMP2) and MMP9 secreted by erythropoietin-activated endothelial cells promote neural progenitor cell migration. J Neurosci 2006; 26(22): 5996–6003 https://doi.org/10.1523/JNEUROSCI.5380-05.2006 pmid: 16738242
22	QW Fan, C Cheng, C Hackett, M Feldman, BT Houseman, T Nicolaides, D Haas-Kogan, CD James, SA Oakes, J Debnath, KM Shokat, WA Weiss. Akt and autophagy cooperate to promote survival of drug-resistant glioma. Sci Signal 2010; 3(147): ra81 https://doi.org/10.1126/scisignal.2001017 pmid: 21062993
23	Y Suzuki, K Shirai, K Oka, A Mobaraki, Y Yoshida, SE Noda, M Okamoto, Y Suzuki, J Itoh, H Itoh, S Ishiuchi, T Nakano. Higher pAkt expression predicts a significant worse prognosis in glioblastomas. J Radiat Res (Tokyo) 2010; 51(3): 343–348 https://doi.org/10.1269/jrr.09109 pmid: 20410674
24	G Choe, S Horvath, TF Cloughesy, K Crosby, D Seligson, A Palotie, L Inge, BL Smith, CL Sawyers, PS Mischel. Analysis of the phosphatidylinositol 3′-kinase signaling pathway in glioblastoma patients in vivo. Cancer Res 2003; 63(11): 2742–2746 pmid: 12782577
25	H Mure, K Matsuzaki, KT Kitazato, Y Mizobuchi, K Kuwayama, T Kageji, S Nagahiro. Akt2 and Akt3 play a pivotal role in malignant gliomas. Neuro-oncol 2010; 12(3): 221–232 https://doi.org/10.1093/neuonc/nop026 pmid: 20167810
26	DF Quail, JA Joyce. The microenvironmental landscape of brain tumors. Cancer Cell 2017; 31(3): 326–341 https://doi.org/10.1016/j.ccell.2017.02.009 pmid: 28292436
27	MD Jansson, AH Lund. MicroRNA and cancer. Mol Oncol 2012; 6(6): 590–610 https://doi.org/10.1016/j.molonc.2012.09.006 pmid: 23102669
28	A Esquela-Kerscher, FJ Slack. Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 2006; 6(4): 259–269 https://doi.org/10.1038/nrc1840 pmid: 16557279
29	M Piwecka, K Rolle, A Belter, AM Barciszewska, M Żywicki, M Michalak, S Nowak, MZ Naskręt-Barciszewska, J Barciszewski. Comprehensive analysis of microRNA expression profile in malignant glioma tissues. Mol Oncol 2015; 9(7): 1324–1340 https://doi.org/10.1016/j.molonc.2015.03.007 pmid: 25864039

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