ABSTRACT
Background and purpose: Long non-coding RNA small nucleolar RNA host gene 5 (lncRNA SNHG5) plays a cancer-promoting role in many cancers, however its effect on colorectal cancer (CRC) and its regulatory mechanism are not clear. This study aimed to explore the mechanism of lncRNA SNHG5/miR-26a-5p/metadherin (MTDH) signal axis promoting metastasis of CRC. Methods: The data of The Cancer Genome Atlas (TCGA) database was analyzed, the abnormal expression of lncRNA in CRC was explored and analyzed the survival. Samples of CRC, paracancerous tissues and complete clinical data of patients who underwent surgical resection from October 2020 to October 2021 were collected. The expression levels of SNHG5 and miR-26a-5p in lncRNA were detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR), and the expression level of MTDH was detected by immunohistochemistry. The relationship between the relative expression level of lncRNA SNHG5 in CRC and clinicopathological features and survival time was analyzed. The effects of lncRNA SNHG5 on the proliferation, migration and invasion of CRC cells were detected by cell counting kit-8 (CCK-8), clone formation, scratching assays, transwell test and in vivo xenotransplantation. The relationship between CRC cell metastasis, the expression level of epithelial-mesenchymal transition related molecules and lncRNA SNHG5 expression level by Western blot and immunohistochemical detection were explored. The physical interaction between SNHG5 and miR-26a-5p, MTDH and miR-26a-5p was studied by RNA pull-down test, double luciferase reporter gene detection and RNA co-immunoprecipitation. The functional relationship among the three was verified by CCK-8, EdU and transwell experiments. The effect of SNHG5, miR-26a-5p and MTDH expression on migration and invasion related molecules was analyzed by Western blot. Results: The results of TCGA database analysis showed that lncRNA SNHG5 was significantly upregulated in CRC. The results of RTFQ-PCR and immunohistochemistry showed that the levels of lncRNA SNHG5 and MTDH in CRC tissues were significantly upregulated (P<0.05), the level of miR-26a-5p was decreased (P<0.05), and the level of MTDH in samples with high expression of SNHG5 was also increased. The expression of lncRNA SNHG5 in CRC tissues with serosa and extraserosal invasion, distant metastasis, lymph node metastasis and TNM stage Ⅲ was significantly higher compared with subserosal invasion, no distant metastasis and lymph node metastasis and TNM stage Ⅰ-Ⅱ (P<0.05). The results of survival analysis showed that the high expression of lncRNA SNHG5 was significantly correlated with overall survival rate (P<0.05). Overexpression of lncRNA SNHG5 could enhance the proliferation, clone formation, migration and invasion of CRC cells, promote the growth and lung metastasis of transplanted tumor, increase the relative expression level of Ki-67 proliferation index and vimentin (P<0.05), and decrease the relative expression level of E-cadherin (P<0.05). However, the development of CRC cells was inhibited after inhibition of lncRNA SNHG5 expression. RNA pull-down test, double luciferase reporter gene detection and RNA co-immunoprecipitation confirmed the physical interaction between SNHG5 and miR-26a-5p, MTDH and miR-26a-5p. Upregulation of miR-26a-5p or downregulation of MTDH expression in lncRNA SNHG5 overexpressed cells partially reversed the effects of lncRNA SNHG5 on proliferation, migration, invasion and expression of related molecules in CRC cells. Conclusion: LncRNA SNHG5 is upregulated in CRC tissues and cells, and its high expression is related to tumor progression and poor survival. It can be used as a molecular sponge of miR-26a-5p to regulate the expression of MTDH to promote the proliferation and metastasis of SW620 cells.
ABSTRACT
Objective:To explore the effect of lncRNA SNHG5 on injury of astrocytes induced by hypoxia/reoxygenation (H/R).Methods:(1) Astrocytes were cultured in vitro. The H/R cell model was established by hypoxia culture for 6 hours and then reoxygenaion culture for 18 hours. Lipofectamine? 2000 liposome method was used to transfect lncRNA SNHG5 into astrocytes. RT-qPCR was used to detect the expression of lncRNA SNHG5 in H/R cells and after transfection. (2) Astrocytes were divided into normal control group, model group, transfection control group (pcDNA-NC was transfected first, then H/R cell model was established) and transfection group (pcDNA-lncRNA SNHG5 was transfected first, then H/R cell model was established). Then the effect of overexpression of lncRNA SNHG5 on astrocytes was observed. (3)The astrocytes transfected with lncRNA SNHG5 and H/R intervention were divided into transfection+ vehicle group (0.1% DMSO incubation) and transfection+ inhibitor group (20 μmol/L LY294002 incubation), and then observe the effect of the inhibitor of PI3K/Akt signaling pathway LY294002 on H/R astrocytes was observed. (4) CCK-8 was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Western blot was used to detect the expression of cell proliferation proteins (Cyclin D1 and Cyclin E), apoptotic proteins (Caspase-3 and Bax), p-PI3K and p-AKT protein. ELISA was used to detect the levels of IL-1β and TNF-α. The colorimetric method was used to detect the level of lactate dehydrogenase(LDH) in cell culture supernatants and the level of malondialdehyde(MDA) and superoxide dismutase(SOD) in cells. SPSS 22.0 software was used for independent sample t-test and one-way ANOVA, and LSD- t test was used for further pairwise comparisons. Results:(1) RT-qPCR results showed that the level of lncRNA SNHG5 in astrocytes induced by H/R was lower than that in the normal cultured cells ( t=33.28, P<0.05). (2) lncRNA SNHG5 overexpression experiment: The cell proliferation activity of the model group was lower than that in the normal control group (CCK-8 OD value: (0.64±0.02), (1.23±0.02), t=62.58, P<0.05). The levels of proliferation proteins Cyclin D1 and Cyclin E in the model group were lower than those of the normal control group ( t=33.54, 32.20, both P<0.05). The cell proliferation activity of the transfection group was higher than that of the transfection control group (CCK-8 OD value: (1.49±0.02), (0.65±0.03), t=69.89, P<0.05), the levels of cell proliferation proteins Cyclin D1 and Cyclin E in the transfection group were lower than those in the transfection control group ( t=24.96, 28.46, both P<0.05). The apoptosis rate of the model group was higher than that of the control group (flow cytometry results: (25.33±1.13)%, (9.06±0.21)%, t=42.47, P<0.05), and the levels of apoptotic proteins Caspase-3 and Bax were also higher than those of the control group ( t=57.41, 41.60, both P<0.05). The Caspase-3 rate of the transfection group was lower than that of the transfection control group((16.56±0.60)%, (25.89±1.18)%, t=21.14, P<0.05), and the levels of apoptotic proteins Caspase-3 and Bax were also higher than those of the transfection control group( t=77.79, 58.34, both P<0.05). The levels of p-PI3K and p-AKT proteins in the model group were lower than those in the control group ( t=56.35, 33.94, both P<0.05), and the levels of p-PI3K and p-AKT proteins in the transfection group were higher than those in the transfection control group ( t=130.14, 76.37, both P<0.05). The results of ELISA showed that the levels of IL-1β and TNF-α in the model group were higher than those in the control group ( t=58.04, 30.63, both P<0.05), but the levels of IL-1β and TNF-α in the transfection group were lower than those in the transfection control group ( t=33.63, 39.01, both P<0.05). The colorimetric method showed that the levels of LDH and MDA in the model group were higher than those in the control group ( t=65.51, 41.85, both P<0.05), but the level of SOD was lower than that in the control group ( t=48.82, P<0.05). The levels of LDH and MDA in the transfection group were lower than those in the transfection control group ( t=37.93, 30.72, both P<0.05), but the level of SOD was higher than that in the transfection control group ( t=30.32, P<0.05). (3) PI3K/Akt signaling pathway inhibition experiment: the cell proliferation activity of the transfection+ inhibitor group was lower than that of the transfection+ vehicle group (CCK-8 OD value: (0.97±0.02), (1.46±0.03), t=15.24, P<0.05), and the related proliferation proteins Cyclin D1 and Cyclin E were also lower ( t=11.41, 13.15, both P<0.05). The apoptosis rate of the transfection+ inhibitor group was higher than that of the transfection+ vehicle group (Flow cytometry: (26.11±0.86)%, (16.06±0.44)%, t=10.45, P<0.05). The apoptosis rate of the transfection+ inhibitor group was higher than that of the transfection+ vehicle group (Flow cytometry: (26.11±0.86)%, (16.06±0.44)%, t=10.45, P<0.05), and the related apoptosis protein Caspase-3 and Bax were also higher ( t=19.06, 13.54, both P<0.05). The expression levels of p-PI3K and p-AKT protein in the transfection+ inhibitor group were lower than those in the transfection+ vehicle group ( t=36.67, 27.34, both P<0.05). ELISA results showed that the levels of IL-1β and TNF-α in the transfection+ inhibitor group were higher than those in the transfection+ vehicle group ( t=15.17, 9.44, both P<0.05). The colorimetric method results showed that the levels of LDH and MDA in the transfection+ inhibitor group were the same as those in the transfection+ vehicle group ( t=15.33, 9.05, both P<0.05), but the level of SOD was lower than the transfection+ vehicle group ( t=11.04, P<0.05). Conclusion:Overexpression of lncRNA SNHG5 may promote the proliferation of astrocytes induced by hypoxia/reoxygenation, and inhibit cell apoptosis, inflammation and oxidative stress.