In the present study, lncRNA SNHG1 was highly expressed in human glioma tissues and cell lines. There existed a regulatory relationship between lncRNA SNHG1 and miR-128-3p, as well as between miR-128-3p and RRAS2. Compared with normal brain tissues, miR-128-3p was down-regulated, whereas RRAS2 was up-regulated in glioma tissues. Overexpression of RRAS2 promoted the proliferation and metastatic potential of glioblastoma cells, whereas the miR-128-3p mimic targeted and inhibited the expression of RRAS2. Furthermore, both SNHG1 and RRAS2 facilitated the EMTprocess in GBM cells. Finally, we showed that lncRNA SNHG1 acted as a sponge of miR-128-3p and promoted the EMT process in GBM by regulating RRAS2, which in turn enhanced the malignant progression of GBM.
In recent years, increasing evidence has shown that lncRNAs play a critical role in the development of different tumors, as well as being prognostic markers for tumor patients (Shuai et al. 2020; Marín-Béjar et al. 2017). Small nucleolar RNA host genes (SNHGs) are newly recognized lncRNAs, which have become the focus of current oncology studies. For example, lncRNA SNHG6 is overexpressed in primary high grade ductal breast cancers, and it may promote the proliferation, migration and EMT of breast cancer cells (Jafari-Oliayi and Asadi 2019). Moreover, lncRNA SNHG1 promotes sorafenib resistance in hepatocellular carcinoma (HCC) by activating the Akt pathway, its nuclear expression is promoted by miR-21, and its nuclear translocation is induced by sorafenib, suggesting that SNHG1 may be a valuable target for overcoming sorafenib resistance to HCC (Li et al. 2019). In addition, Wang et al. reported that SNHG1 was upregulated in gliomas, which predicted poor prognosis (Wang et al. 2017). However, the role and mechanism of SNHG1 in glioma still needs to be further characterized. In the present study, we found that SNHG1 expression was upregulated in both glioma tissues and cell lines, which is consistent with the Wang findings, which indicated that SNHG1 may play an important role in glioma genesis and development.
GBM is the most common and serious malignancy among intracranial primary gliomas, which is incurable owing to its highly invasive and metastatic potentials (Shergalis et al. 2018). There is therefore an urgent need to find ways to control the invasive and metastatic ability of GBM. In this study, we performed functional experiments in two GBM cell lines, A172 and U251. Our results showed that the proliferation, migration, and invasive abilities of GBM cells were effectively inhibited after SNHG1 knockdown, suggesting that SNHG1 functioned as an oncogene in GBM.
MiR-128-3p is also known as an miR-128 and its precursor is miR-128-1 (Hu et al. 2014). MiR-128-3p plays an important role in tumor development (Chen et al. 2019). For example, Zhao et al (Zhao et al. 2019) found that overexpression of miR-128-3p suppressed the expression of LIMK1, thereby inhibiting breast cancer progression. Simultaneously, they also found that patients with high expression levels of miR-128-3p may have a better prognosis. In addition, Shi et al (Shi et al. 2012) found that overexpression miR-128 suppressed the expression of p70S6K1, thereby attenuating glioma development and progression. This result was similar to the findings of our present study, in which miR-128-3p overexpression suppressed SNHG1 expression and also regulated RRAS2 expression, thereby affecting GBM development and progression.
Previous studies had shown that many lncRNAs bound and interacted with miRNAs as ceRNAs, which in turn regulated downstream target genes (Wang et al. 2021; Liu et al. 2019). For example, SNHG1 activated the oncogene HOXA1 in breast cancer cells by repressing the expression of miR-193a-5p, thereby promoting malignant progression of breast cancer (Li et al. 2020). Based on these results, we conducted an in-depth mining of the regulatory mechanism of SNHG1 as ceRNA in gliomas. Bioinformatics prediction helped us to identify six down-regulated miRNAs and 17 up-regulated mRNAs associated with SNHG1. KEGG pathway analysis was used to analyze the significant pathway of 17 mRNAs. From this analysis, we found that RRAS2 played a key role in tumor progression. RRAS2 is a member of the RAS superfamily of GTPases (Drivas et al. 1990). RRAS2 controls a variety of cellular processes, including proliferation, survival, and migration, and its dysfunction had been shown to contribute to tumorigenesis (Capri et al. 2019). For example, RRAS2 partially attenuates the effect of the anticancer drug JQ1 in NMC tumor cells by maintaining ERK pathway function during oncogene BRD4 inhibition, thereby promoting the progression of midline cancers in the NUT (Liao et al. 2018). In our research, we confirmed that the expression of RRAS2 was also decreased after SNHG1 knockdown in GBM cells. This suggested the potential regulatory effect of SNHG1 on RRAS2. Furthermore, knockdown of RRAS2 also effectively inhibited the proliferation, migration, and invasion ability of GBM cells, indicating that RRAS2 may function as an oncogenic gene in GBM.
EMT is a transformation process that causes cells to lose their epithelial properties and acquire mesenchymal cell characteristics, which plays an important role in primary tumor formation and metastasis (Bakir et al. 2020). E-cadherin, N-cadherin, and vimentin proteins play key roles in the EMT. E-cadherin is a tumor suppressor protein whose major effect in normal cells involves inhibiting cancer cell metastasis, and the loss of E-cadherin expression has been correlated with the EMT in the progression of cancer metastasis (Na et al. 2020). The expression of N-cadherin is increased, which promotes tumor cell shedding, leading to tumor cell invasion and metastasis (Cao et al. 2019). Cancer cells expressing the intermediate filament protein, vimentin, are more motile and invasive in vitro, and also more metastatic in vivo (Thompson et al. 1992). Furthermore, it was found that EMT-related induction factors played important roles in the malignant progression of glioma (Tao et al. 2020). In the present study, the expression of the EMT marker proteins E-cadherin, N-cadherin, and vimentin were lowered after SNHG1 and RRAS2 knockdown in GBM cells, suggesting that SNHG1 and RRAS2 regulated the EMT. To our knowledge, this is the first report that SNHG1 regulated the EMT process in GBM.
Nevertheless, there were several limitations of this study. To better explain the regulatory mechanism of lncRNA SNHG1 in glioblastomas, in vitro experiments in nude mice need to be further investigated. In addition, the regulatory mechanism of the SNHG1/miR-128-3p/RRAS2 network in other biological functions of glioblastoma cells remains to be further investigated.
In the present study, we found miR-128-3p up-regulation after SNHG1 knockdown in GBM. Both SNHG1 and RRAS2 expressions were down-regulated after overexpression of miR-128-3p in GBM cells. These results suggested that SNHG1 and miR-128-3p had antagonistic effects in the pathological process of GBM, and that miR-128-3p also had a dampening effect on the expression of its downstream target RRAS2. In addition, luciferase reporter gene experiments further confirmed the close relationship between SNHG1 and miR-128-3p. Taken together, our results suggested that SNHG1 regulated the EMT process through the SNHG1/miR-128-3p/RRAS2 axis, which in turn promoted the malignant progression of GBM.