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[Abstract] Objective To investigate whether levosimendan (Lev) affects hypoxia / reoxygenation (H / R) - induced cardiomyocyte proliferation, apoptosis and fibrosis by regulating the molecular axis of long chain noncoding RNA (LncRNA) eosinophil granule ontogeny transcript (EGOT) / microRNA (miR) -641. Methods Rat cardiomyocytes H9C2 were cultured in vitro, and H / R-treated cells were used to establish cell damage models, which were randomly divided into control group, H / R group, H / R + Lev 1 μmol / L (H / R + Lev-L) group, H / R + Lev 5 μmol / L (H / R + Lev-M) group, and H / R + Lev 10 μmol / L (H / R + Lev-H) group, 9 samples per group. MTT method was used to detect cell proliferation. Flow cytometry was used to detect the apoptosis rate. Real-time P CR was used to detect the expression levels of EGOT and miR-641 mRNA. P cDNA-EGOT and EGOT small interfering RNA (si-EGOT) were transfected into H9 C2 cells respectively, and the cell proliferation and apoptosis rates were detected by the above method. The dual luciferase report experiment verified the targeting relationship between EGOT and miR-641. Western blotting was used to detect the expression levels of Bax, Bcl-2, collagen I (colI), collagen Ⅲ (col Ⅲ), tissue inhibitor of matrix metalloproteinase 2 (TIMP 2), matrix metalloproteinase-2 (MMP -2) . Results Compared with the control group, the cell survival rate of the H / R group reduced significantly (P < 0. 05), the apoptosis rate increased significantly (P < 0. 05), and the protein levels of Bax, c I, col Ⅲ, TIMP 2, and MMP -2 increased significantly (P < 0. 05), the level of Bcl-2 protein reduced significantly (P < 0. 05), the expression level of EGOT reduced significantly (P < 0. 05), the expression level of miR-641 increased significantly (P < 0. 05) . Compared with the H / R group, the cell survival rate of the H / R + Lev-L group, H / R + Lev-M group, and H / R + Lev-H group increased significantly (P < 0. 05), and the apoptosis rate decreased significant (P < 0. 05), the protein levels of Bax, colI, colⅢ, TIMP 2, MMP -2 reduced significantly (P < 0. 05), the level of Bcl-2 protein increased significantly (P < 0. 05), the expression level of EGOT increased significantly (P < 0. 05), the expression level of miR-641 reduced significantly (P < 0. 05), and each index of H / R + Lev-L group, H / R + Lev-M group, H / R + Lev-H group, the difference was statistically significant (P < 0. 05) . The dual luciferase report experiment confirmed that EGOT ccould target and bind to miR-641. The effect of transfecting pcDNA-EGOT and Lev was similar. Transfection of si-EGOT could reduce the effect of Lev on H / R-induced proliferation, apoptosis and fibrosis of H9 C2 cells. Conclusion Levosimendan may promote H / R-induced H9 C2 cell proliferation and inhibit apoptosis and fibrosis by up-regulating EGOT expression and down-regulating miR-641 expression.
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Objective: To investigate the miR-488-3p-mediated effects of the long-chain noncoding RNA (lncRNA) family with sequence similarity 201-member A (FAM201A) on the biological behavior and radiosensitivity of gastric cancer cells. Methods: Sixty-three pairs of gastric carcinoma tissues and paracancerous tissues were resected from gastric carcinoma patients, who underwent surgery (no radiotherapy or chemotherapy treatment before surgery) at Laiyang Central Hospital of Yantai City during January 2014 and January 2017. The expression levels of FAM201A and miR-488-3p in the 63 gastric cancer tissue samples and their paracancerous tissues were detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The gastric cancer cell line MGC803 with inhibited FAM201A expression was constructed. The proliferation and viability of MGC803 cells were assessed by the Methyl Thiazolyl Tetrazolium (MTT) assay; their apoptosis was measured by flow cytometry; and their migration and invasion abilities were tested by the Transwell assay. Further, the MGC803 cells were irradiated with different radiation intensities (0, 2, 4, 6, and 8 Gy). Cell survival fractions were measured by colony-formation assay, and cell survival curve was stimulated by the single-hit multi-target model. The dual-luciferase reporter gene assay and qRT-PCR were used to verify whether FAM201A targets miR-488-3p. Results: The expression level of FAM201A was significantly higher, while that of miR-488-3p was significantly lower in gastric cancer tissues than in the paracancerous tissues. Inhibiting FAM201A expression significantly suppressed the proliferation, migration, and invasion abilities of MGC803 cells, promoted their apoptosis, and increased their radiosensitivity. FAM201A negatively regulated miR-488-3p expression. Inhibiting miR-488-3p expression reversed the effects of the inhibition of FAM201A expression on the proliferation, apoptosis, migration, and radiosensitivity of MGC803 cells. Conclusions: Inhibiting the expression of the lncRNA FAM201A suppressed the proliferation, migration, and invasion abilities of gastric cancer cells, promoted their apoptosis, and increased their radiosensitivity by targeting miR-488-3p. Thus, FAM201A may serve as a novel molecular target for gastric cancer treatment.
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@#[Abstract] Objective ： ：To investigate the long-chain noncoding RNA (Lnc RNA) PCGEM1 regulating the lung cancer (LC) cell invasion and metastasis through the TGF-β/Smad signaling pathways. Methods： ：From March 2016 to May 2018, total 62 cases of LC patients receiving surgical treatment in our hospital were collected, including cancer tissues and normal tissues more than 2 cm away from the cancer tissues. qRT-PCR was used to detect the expression of lncRNA PCGEM1 and miR-148a in LC, corresponding para-cancer tissues and different LC cell strains. LncRNA PCGEM1 silenced cell line A549-siPCGEM1 and negative control A549-NC were constructed, and A549 was used as blank control. MTT and plate cloning assay were used to detect the effect of PCGEM1 on the proliferation of A549 cells. Transwell and scratch assay were used to detect the effect of PCGEM1 on the invasion and migration of A549 cells. The bioinformatics website StarBase was used to predict the complementary binding miRNAof PCGEM1. Furthermore, according to the website Targetscan, the genes that the corresponding miRNAs could target and bind were predicted. Results： ：qRT-PCR results showed that the expression of PCGEM1 in LC tissues and lung cancer cell lines was higher than that in normal tissues, and the expression level of miR-148a was lower than that in normal tissues (all P<0.05). The expression level of PCGEM1 in A549 cells was the highest, and the difference was statistically significant compared with other cell lines (P<0.05). After successful construction of PCGEM1 silenced cells, compared with the blank control group and A549-NC group, the cell OD492nm value of A549-siPCGEM1 group was significantly decreased, the number of cell clones and the number of matrigel matrix gels was significantly reduced, the cell migration rate was significantly reduced, the differences were statistically significant (P<0.05). According to the prediction results of StarBase website, PCGEM1 could be complementary to miR-148a, and the prediction analysis on microRNA.org website shows that miR-148a had a targeted binding site with TGF-β2. qRT-PCR and Western blotting results showed that the expression of miR-148a was significantly increased in the A549-siPCGEM1 group compared with the blank control group and A549-NC group, and the expression of TGF-β2 and p-Smad 2 was significantly decreased (P<0.05), while the expression of the above indicators in the blank control group and A549-NC group was not statistically significant (P>0.05). Conclusion： ：Lnc RNA PCGEM1 is highly expressed in lung cancer. High expression of PCGEM1 may enhance the TGF-β2/Smad2 signaling pathway by downregulation of miR-148a, thus promoting the development of LC and the malignant biological behavior.
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Objective To investigate the differential expression profile of epithelial mesenchymal transition (EMT) related long chain noncoding RNA (LncRNA) in hepatocellular carcinoma (HCC) cells after incomplete radiofrequency ablation (RFA) in vitro.Methods Incomplete RFA was simnulated in vitro by using Huh7 cells in water bath at 47℃.EMT change was detected by microscopy and Western blot.Cell invasion and migration were detected by transwell assays.Cell proliferation was determined by cell counting kit-8 (CCK-8) assay.Differential expression profile of EMT-related LncRNAs between Huh7-H and Huh7 were analyzed by LncPath human EMT array,and it was validated by RT-PCR.Results Under microscopy,Huh7-H presented characteristic EMT morphological changes.Western blot analysis showed that the expression of E-cadherin in Huh7-H cells was decreased,while the expressions of N-cadherin and Vimentin were increased.Transwell assay test indicated that cell migration and invasion were increased in Huh7-H cell when compared with Huh7 cell (61.0±5.2 vs 138.0±11.8 and 33.3±7.8 vs 82.7±39.4,respectively),the abilities of Huh7-h cell in migration and invasion were evidently strengthened (P＜0.05).CCK-8 assay shawed that the proliferation ability of Huh 7-H was obviously higher than that Huh 7 (P＜0.05).LncPath human EMT array screened out 3 differential expressed LncRNAs (P≤0.05 and fold changes ≥ 1.5),including two down-regulated LncRNAs (FUNDC2P4,RPL27P7) and one up-regulated LncRNA (MTND4LP14).RT-PCR validation revealed that the expressions of FUNDC2P4,RPL27P7 and MTND4LP14 in Huh7-H cells were 0.137,0.869 and 1.037times of that in Huh7 cells,respectively.Clinical specimen validation showed that the expression of LncRNA FUNDC2P4 in peficancerous tissue was higher than that in HCC tissue,and the expression of LncRNA FUNDC2P4 in HCC tissue was higher than that in the residual HCC tissue after RFA.Conclusion EMTrelated LncRNA FUNDC2P4 in HCC cells with low expression after incomplete RFA is successfully screened out,which provides basis for the further investigation of the function and molecular mechanism of this gene.