ABSTRACT
ObjectiveTo investigate the mechanism of Biejiajian Wan in the intervention of primary liver cancer based on long non-coding RNA SNHG5 （lncRNA SNHG5）/micro RNA-26a-5p （miRNA-26a-5p）/glycogen synthase kinase-3β （GSK-3β） signal axis. MethodDouble luciferase reporting assay was used to verify the targeted interaction between lncRNA SNHG5 and miRNA-26a-5p， miRNA-26a-5p， and GSK-3β in HepG2 cells. Nude-mouse transplanted tumor model of human HepG2 were established and randomly divided into model group， Biejiajian Wan low-dose group （0.5 g·kg-1）， medium-dose group （1.0 g·kg-1）， and high-dose group （2.0 g·kg-1）， and sorafenib group （100 mg·kg-1）， with 10 mice in each group. The mice were given intragastric administration of normal saline or drug for 28 days， and the tumor volume was measured at different time. Hematoxylin-eosin （HE） staining was used to observe the histological changes of tumors. The nucleic acid levels of lncRNA SNHG5， miRNA-26a-5p， GSK-3β， and β-catenin mPNA in tumor tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of GSK-3β and β-catenin in tumor tissue were detected by western blot. ResultCompared with the SNHG5-WT （wild type） + miRNA NC （negative control） group， the relative luciferase activities of the SNHG5-WT + miRNA-26a-5p mimic group were decreased （P<0.05）. Compared with the GSK-3β-WT + miRNA NC group， the relative luciferase activity of the GSK-3β-WT + miRNA-26a-5p mimic group was decreased （P<0.05）. Compared with the model group， the tumor volume of Biejiajian Wan low-dose， medium-dose， and high-dose groups was significantly decreased （P<0.05， P<0.01）. Compared with the model group， the cells in the tumor tissue of nude mice in each dose group of Biejiajian Wan were sparsely arranged with necrocytosis， which showed concentration-dependent changes. Compared with the model group， the expression levels of lncRNA SNHG5， GSK-3β， and β-catenin were decreased （P<0.05， P<0.01）， while the expression of miRNA-26a-5p was increased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. Compared with the model group， the protein expression levels of GSK-3β and β-catenin were decreased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. ConclusionBiejiajian Wan may affect the necrosis of liver cancer cells through lncRNA SNHG5/miRNA-26a-5p/GSK-3β signal axis and thus play an anti-tumor role. This research will provide more theoretical basis for the clinical application of Biejiajian Wan.
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Objective To investigate the molecular mechanism of SNHG5 regulating the proliferation, invasion and apoptosis of glioblastoma multiforme (GBM) cells by targeting miR-421. Methods Real-time quantitative PCR test was performed to detect the expression levels of SNHG5 and miR-421 in 31 cases of GBM tissue samples and 32 cases of normal brain tissue samples. After increasing or decreasing SNHG5 expression in U87 cell lines by lentivirus or plasmid transfection, the changes of miR-421 expression were measured by real-time quantitative PCR, to explore the correlation between SNHG5 and miR-421 in GBM. The dual-luciferase reporter test was performed to explore the target interaction of SNHG5 and miR-421. The plasmids with low expression of SNHG5 and miR-421 were cotransfected into U87 cells for the rescue experiment. CCK-8 test, Transwell test, flow cytometry and tumor cell xenograft in nude mice were used to verify molecular mechanism of SNHG5 regulating the proliferation, invasion and apoptosis of GBM in vitro and vivo. Results The expression level of miR-421 was decreased in U87 cell line after SNHG5 upregulation. In addition, the expression level of miR-421 was increased in U87 cell line after SNHG5 downregulation (P < 0.05). The expression level of SNHG5 was correlated negatively with the expression of miR-421 in GBM and U87 cell line. The result of luciferase reporter tests indicated SNHG5 targetedly interacted with miR-421. Rescue experiment results showed that compared with si-SNHG5+miR-421-inhibitor group, the proliferation, invasion and anti-apoptosis ability of U87 cells were significantly inhibited in the si-SNHG5+control-inhibitor group, the expression levels of BAX and p21 were significantly higher, the expression levels of CyclinD1 and Bcl-2 were lower remarkably (P < 0.05). Conclusion SNHG5 promotes the proliferation, invasion and anti-apoptosis of GBM by targeting miR-421 and regulating the expression of CyclinD1, p21, BAX and Bcl-2. Downregulation of miR-421 is related to SNHG5 overexpression in GBM.
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@#[Abstract] Objective: To explore the regulatory effect of long non-coding RNA (lncRNA) SNHG5 on invasion and migration of hypoxia-induced hepatocellular carcinoma (HCC) cells. Methods: A total of 20 pairs of cancer and para-cancerous tissue specimens resected from HCC patients in the First Affiliated Hospital of Xi'an Jiaotong University from January 2017 to June 2018, and human HCC cell lines (HepG2, MHCC-97L, MHCC-97H , Huh7) as well as immortalized human liver LO2 cells were collected for this study. Bioinformatics methods were used to analyze the binding sites between hypoxia-inducible factor 1α (HIF-1α) and SNHG5. pCMVHIF-1α and shRNA-SNHG5 (sh-SNHG5) plasmids were transfected into HCC cells, respectively. qPCR was used to detect the expres‐ sion level of SNHG5 in HCC tissues and hypoxia-induced HCC cells. Western botting was used to detect the expression level of HIF-1α protein in HCC cells, and Transwell chamber method was used to detect the migration and invasion ability of HCC cells after SNHG5 si‐ lence under normoxia and hypoxia condition. Results: Compared with para-cancerous tissues and immortalized human liver LO2 cells, the expression of SNHG5 was significantly up-regulated in HCC tissues and cell lines (all P<0.01). Hypoxia promoted the expression level of SNHG5 in HCC cells, and its mechanism might be related to the combination of hypoxia-activated HIF-1α and SNHG5 promoter to promote its transcription. Hypoxia promoted the invasion and migration ability of HepG2 and MHCC-97L cells (all P< 0.01), but knockdown of SNHG5 significantly inhibited the invasion and migration ability of HepG2 and MHCC-97L cells under hy‐ poxic conditions (all P<0.01). Conclusion: SNHG5 is highly expressed in HCC tissues and cell lines and plays an important role in the invasion and migration of HCC cells induced by hypoxia.
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Objective: To investigate the expression of small nucleolar RNA host gene 5 (SNHG5) in hepatocellular carcinoma (HCC) and its effect on epithelial-mesenchymal transition (EMT) and tumor stem cell proliferation. Methods: The expression of SNHG5 gene was detected by qRT-PCR in 48 cases of HCC tissues and corresponding adjacent tissues. The results and clinical pathological parameters were analyzed. The SNHG5 knockdown lentivirus was constructed and transfected into HCC cell lines, and the efficiency was verified by qRT-PCR. The cell invasion and migration abilities were detected by Transwell test; the formation and proliferation of HCC stem cells were detected by stem cell glomeration test. The expressions of EMT and stem cell-related genes mRNA and protein were detected by qRT-PCR and Western blot. Results: In 48 HCC patients, the expression of SNHG5 was significantly higher in HCC than in adjacent normal tissue. The high expression of SNHG5 was associated with tumor size, HBV infection, pathological differentiation type and clinical stage of HCC (P<0.05). At the cellular level, knockdown of SNHG5 inhibited the invasion and migration of HCC lines HepG2 and Huh7 and the number and diameter of cancer stem cells (CSCs). The expression of E-cadherin was significantly increased in the SNHG5 knockdown group while the expressions of N-cadherin and stem cell-associated proteins OCT4 and SOX2 were significantly reduced (P<0.05). Conclusion: SNHG5 is highly expressed in HCC. Knockdown of SNHG5 inhibits the invasion and migration of HCC cells and the proliferation of CSCs. The mechanism may be related to the knockdown of SNHG5 to promote the reversal of HCC cell EMT and reduce the characteristics of HCC cancer stem cells.