Journal: Virologica Sinica

Article Title: Human cytomegalovirus RNA2.7 inhibits RNA polymerase II (Pol II) Serine-2 phosphorylation by reducing the interaction between Pol II and phosphorylated cyclin-dependent kinase 9 (pCDK9)

doi: 10.1016/j.virs.2022.02.011

Figure Lengend Snippet: Regulation of host cell cycle by HCMV RNA2.7. A Block of host cells entry into S phase by RNA2.7. HELF cells were infected with HAN or HANΔRNA2.7 (MOI ​= ​1.0). DRB was added 4 ​h before infection with a final concentration of 200 ​nmol/L and was used as a positive control. Cells were stained and analyzed by flow cytometry. Percentages of cells in the G0/G1 phases of the cell cycle were calculated and presented as mean ​± ​SEM. B Model of pre-RC formation for cellular DNA replication. Components involved in pre-RC formation were up-regulated in HANΔRNA2.7 infected cells and were facilitated cellular DNA replication. Gene that mRNA fold change more than 2 is indicated with red color. Gene that mRNA fold change less than −2 is indicated with green color. C Effects of RNA2.7 on genes facilitating pre-RC formation. Transcriptions of MCM2, MCM4, MCM5, Cdt1 and Cdc6 were increased in HANΔRNA2.7 infected cells, and could be decreased by inhibiting Pol II S2 phosphorylation. Data are presented as mean ​± ​SEM. D Effects of RNA2.7 on expressions of Cdt1 and Cdc6. Protein levels of Cdt1 and Cdc6 were increased in HANΔRNA2.7 infected cells, and could be decreased by inhibiting Pol II S2 phosphorylation. Data are presented as mean ​± ​SEM. Statistical analysis was performed by Student's t- test. ∗ P ​< ​0.05; ∗∗ P ​< ​0.01; ∗∗∗ P ​< ​0.0001. DRB, 5,6-dichloro-1-b-D-ribofuranosyl benzimidazole; pre-RC, pre-replication complex.

Article Snippet: siRNAs specific to Cdt1 and Cdc6 (RiboBio, Guangzhou, China) were diluted with RNase free water to a final concentration of 20 μmol/L.

Techniques: Blocking Assay, Infection, Concentration Assay, Positive Control, Staining, Flow Cytometry, Phospho-proteomics

Journal: Virologica Sinica

Article Title: Human cytomegalovirus RNA2.7 inhibits RNA polymerase II (Pol II) Serine-2 phosphorylation by reducing the interaction between Pol II and phosphorylated cyclin-dependent kinase 9 (pCDK9)

doi: 10.1016/j.virs.2022.02.011

Figure Lengend Snippet: Effects of RNA2.7 on viral DNA replication. A Growth curves of HAN and HANΔRNA2.7 in HELF cells. Growth curves were performed in quiescent HELF cells infected with HAN or HANΔRNA2.7 ​at an MOI of 0.1 and 3. Supernatant samples were collected at 1, 3, 6 and 9 dpi. Titers were determined by TCID 50 . HAN is indicated with solid lines, and HANΔRNA2.7 is indicated with dashed lines. Data are averages from three independent experiments, and standard errors are indicated. B Up-regulation of viral replication by Cdt1 and Cdc6 knockdown. Evaluated siRNA specific to Cdt1 and Cdc6 were transfected into HELF cells. Cells transfected with siRNAs specific to Cdt1 or Cdc6 were infected with HANΔRNA2.7 (MOI ​= ​1.0). Cells transfected with siRNA negative control were infected with HAN or HANΔRNA2.7. Viral DNA levels were quantified at 2, 24 and 96 hpi. Relative viral DNA levels are presented as mean ​± ​SEM. Statistical analysis was performed by Student's t- test. ∗ P ​< ​0.05; ∗∗ P ​< ​0.01. TCID 50 , standard median tissue culture infective dose.

Article Snippet: siRNAs specific to Cdt1 and Cdc6 (RiboBio, Guangzhou, China) were diluted with RNase free water to a final concentration of 20 μmol/L.

Techniques: Infection, Knockdown, Transfection, Negative Control

Journal: Virologica Sinica

Article Title: Human cytomegalovirus RNA2.7 inhibits RNA polymerase II (Pol II) Serine-2 phosphorylation by reducing the interaction between Pol II and phosphorylated cyclin-dependent kinase 9 (pCDK9)

doi: 10.1016/j.virs.2022.02.011

Figure Lengend Snippet: Model for the mechanism of HCMV RNA2.7 in cell cycle control. RNA2.7 binds to Pol II and inhibits Pol II S2 phosphorylation by blocking interactions between pCDK9 and Pol II. The inhibition of Pol II S2 phosphorylation decreases the levels of MCMs, Cdt1 and Cdc6, and disturbs the formation of pre-RC which leads to host cell cycle arrest at G0/G1 phase and facilitates viral DNA replication.

Article Snippet: siRNAs specific to Cdt1 and Cdc6 (RiboBio, Guangzhou, China) were diluted with RNase free water to a final concentration of 20 μmol/L.

Techniques: Control, Phospho-proteomics, Blocking Assay, Inhibition

Journal: Acta Pharmaceutica Sinica. B

Article Title: Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

doi: 10.1016/j.apsb.2019.01.002

Figure Lengend Snippet: MiR-489-3p and miR-630 directly target OCT2. (A) Summary of miRNAs potentially target OCT2 3′-UTR. (B) Dual-luciferase reporter gene assay in HEK293 cells cotransfected with miRNA mimics and wild-type pGL3-OCT2 3′-UTR plasmid together with the Renilla luciferase plasmid. (C) MiR-489-3p, miR-630 and their putative binding sequences in the 3′-UTR of OCT2. A 6-bp deletion mutation was generated in the complementary site that binds to the seed region. (D) and (E) Dual-luciferase reporter gene assay in HEK293 cells cotransfected with miRNA mimics and mutant pGL3-OCT2 3′-UTR plasmid (seed deletion of miR-489-3p and miR-630 separately). (F) Dual-luciferase reporter gene assay in HEK293 cells separately or synchronally transfected with 10 μmol/L miR-489-3p and 5 μmol/L miR-630 together with pGL3-OCT2 3′-UTR and Renilla luciferase plasmid. Student׳s t -test (two-tailed) was used. Data are the mean±S.E.M ( n =6). * P <0.05, *** P <0.001.

Article Snippet: Biotin-labeled OCT2 mRNA probes, synthesized and purchased from RiboBio Co., Ltd. (Guangzhou, China), were transfected into 786-O-OCT2-489 and 786-O-OCT2-630 to hybridize OCT2 mRNA for 48 h. Biotech and the miRIP assay were performed as previously described with minor modification.

Techniques: Luciferase, Reporter Gene Assay, Plasmid Preparation, Binding Assay, Mutagenesis, Generated, Transfection, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

doi: 10.1016/j.apsb.2019.01.002

Figure Lengend Snippet: Construction of 786-O cell line, stably overexpressing of OCT2, together with miR-489-3p or miR-630. (A) The fluorescence intensity in 786-O-OCT2-489 and 786-O-OCT2-630. Green fluorescence indicates overexpression of OCT2 while red fluorescence indicates miRNAs overexpression. (B)–(D) Upregulation of OCT2 mRNA and protein expression level and the uptake of MPP + in 786-O-OCT2 compared with 786-O-pCDH. (E) Detection of miR-489-3p and miR-630 expression level by qRT-PCR, with U6 as an endogenous control, in 786-O-OCT2-489, 786-O-OCT2-630 and 786-O-OCT2-NC.

Article Snippet: Biotin-labeled OCT2 mRNA probes, synthesized and purchased from RiboBio Co., Ltd. (Guangzhou, China), were transfected into 786-O-OCT2-489 and 786-O-OCT2-630 to hybridize OCT2 mRNA for 48 h. Biotech and the miRIP assay were performed as previously described with minor modification.

Techniques: Stable Transfection, Fluorescence, Over Expression, Expressing, Quantitative RT-PCR

Journal: Acta Pharmaceutica Sinica. B

Article Title: Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

doi: 10.1016/j.apsb.2019.01.002

Figure Lengend Snippet: MiR-489-3p and miR-630 suppress OCT2 expression and activity. (A)–(C) Downregulation of OCT2 mRNA, protein expression levels and the accumulation of MPP + in 786-O-OCT2-489, 786-O-OCT2-630 compared with 786-O-OCT2-NC. 100 nmol/L of microRNA inhibitor NC, miR-489-3p or miR-630 inhibitor were transiently transfected into 786-O cells for 48 h: (D) Quantitative analysis of endogenous mature miR-489-3p and miR-630 expression levels were measured by qRT-PCR with U6 as an endogenous control. (E)–(F) The mRNA expression level of OCT2 and uptake of MPP + were measured by qRT-PCR and LC–MS. (G) OCT2 mRNA and probe binding sites. The green bar, 5′-UTR; the blue arrow, coding sequence; the yellow bar, 3′-UTR; the black bars and the numbers at the bottom denote regions the probe binding sites; the red balls indicate where biotin binded. (H)–(I) OCT2, miR-489-3p and miR-630 in 786-O-OCT2-489 and 786-O-OCT2-630 cell lysis was pulled down and enriched with OCT2 specific probes and then detected by qPCR.

Article Snippet: Biotin-labeled OCT2 mRNA probes, synthesized and purchased from RiboBio Co., Ltd. (Guangzhou, China), were transfected into 786-O-OCT2-489 and 786-O-OCT2-630 to hybridize OCT2 mRNA for 48 h. Biotech and the miRIP assay were performed as previously described with minor modification.

Techniques: Expressing, Activity Assay, Transfection, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Sequencing, Lysis

Journal: Acta Pharmaceutica Sinica. B

Article Title: Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

doi: 10.1016/j.apsb.2019.01.002

Figure Lengend Snippet: MiR-489-3p and miR-630 promote chemoresistance to oxaliplatin in RCC cells. (A) Overexpression of OCT2 increased oxaliplatin cellular accumulation which can be suppressed by miR-489-3p and miR-630. (B) The cell viability of 786-O-pCDH, 786-O-OCT2-NC, 786-O-OCT2-489 and 786-O-OCT2-630 treated with different concentrations (0–250 μmol/L) of oxaliplatin for 48 h. C) Oxaliplatin administration timeline and experiment schedule for xenograft models. The in vivo effect of miR-489-3p and miR-630 was evaluated in xenograft models bearing tumours originating from 786-O-pCDH, 786-O-OCT2-NC, 786-O-OCT2-489 and 786-O-OCT2-630 cell lines, n =5/group. In the middle of administration timeline, a mouse randomly selected from each group ( n =1) was sacrificed to detect the expression of OCT2 , miR-489-3p and miR-630. (D) Representative images of mice bearing xenografts and tumours after resection from each group at day 26. In 786-O-OCT2-630 model, the mouse drawn in dashed line represents one which was euthanized when tumour size reached 1500 mm 3 at day 19. In 786-O-OCT2-NC model, “×” indicates that the tumour in one mouse eventually disappeared. (E) Tumour volume was periodically measured for each mouse and tumour growth curves were plotted. Data are means±S.E.M. ( n =4), except for the one in 786-O-OCT2–630 model, euthanized ahead of the end point for bearing oversized tumour. (F) and (G) OCT2 protein expression level and platinum accumulation in tumours resected from each group ( n =3).

Article Snippet: Biotin-labeled OCT2 mRNA probes, synthesized and purchased from RiboBio Co., Ltd. (Guangzhou, China), were transfected into 786-O-OCT2-489 and 786-O-OCT2-630 to hybridize OCT2 mRNA for 48 h. Biotech and the miRIP assay were performed as previously described with minor modification.

Techniques: Over Expression, In Vivo, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

doi: 10.1016/j.apsb.2019.01.002

Figure Lengend Snippet: MiR-489-3p and miR-630 are overexpressed in RCC. (A) Comparison of OCT2 , miR-489-3p and miR-630 expression separately in human matched renal normal-tumour samples ( n =33, for the expression of miR-630 in some tissues is below the detection limit, n =15 for miR-630 analysis). Wilcoxon signed rank test was used, * P <0.05, ** P <0.01, *** P <0.001. (B) Association between the expression level of OCT2 , miR-489-3p, miR-630 and clinic pathological characteristics of RCC patients. (C) MiR-489-3p, miR-630 expression in renal cancer cells 786-O, 769-P and CAKI compared to normal renal epithelial cells HK-2. U6 was used as an internal control.

Article Snippet: Biotin-labeled OCT2 mRNA probes, synthesized and purchased from RiboBio Co., Ltd. (Guangzhou, China), were transfected into 786-O-OCT2-489 and 786-O-OCT2-630 to hybridize OCT2 mRNA for 48 h. Biotech and the miRIP assay were performed as previously described with minor modification.

Techniques: Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

doi: 10.1016/j.apsb.2019.01.002

Figure Lengend Snippet: C-Myc upregulates miR-630 expression by binding to its promoter region. (A) and (B) Comparison of C-Myc mRNA and protein expression in human matched renal normal-tumour samples. (C) The expression of C-Myc , pri-miR-630, pre-miR-630 and OCT2 in c-Myc knockdown 786-O cells. (D) The uptake of MPP + in c-Myc knockdown 786-O cells. (E) Chip-qPCR analysis of c-Myc at the promoter of miR-630 in c-Myc knockdown cell model and 786-O cells that transiently transfected with c-Myc expression plasmid or empty expression vector (pENTER). IgG, immunoglobulin G. (F) The expression of C-Myc , pri-miR-630 and abundance of c-Myc occupied around E2 in human matched renal normal-tumor samples. (G) Relative light unit (RLU) was detected in HEK293 cells transfected with wild pri-miR-630 promoter (gray) or E-Box element (CACGTG) mutated (white) reporter plasmid along with either c-Myc or empty expression vector (pENTER).

Article Snippet: Biotin-labeled OCT2 mRNA probes, synthesized and purchased from RiboBio Co., Ltd. (Guangzhou, China), were transfected into 786-O-OCT2-489 and 786-O-OCT2-630 to hybridize OCT2 mRNA for 48 h. Biotech and the miRIP assay were performed as previously described with minor modification.

Techniques: Expressing, Binding Assay, Transfection, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

doi: 10.1016/j.apsb.2019.01.002

Figure Lengend Snippet: Potential of miR-489-3p and miR-630 as candidate biomarkers. (A) Representative electron microscopy images of exosomes secreted by 786-O-OCT2 cells. Scale bar, 100 nm. (B) ZetaView particle tracking analysis of the size distributions and number of exosomes. (C) Western blot analysis of protein markers in cells and exosomes. GRP94 (ab3674, Abcam) was rarely expressed in exosomes but rich in cells. ALIX (ab117600, Abcam), TSG101 (sc-7694, Santa Cruz) and CD63 (ab59479, Abcam) were all often used as identification protein marker of exosomes. (D) MiRNAs expression in 786-O-OCT2-NC, 786-O-OCT2-489 and 786-O-OCT2-630 cell lines and respective exosomes.

Article Snippet: Biotin-labeled OCT2 mRNA probes, synthesized and purchased from RiboBio Co., Ltd. (Guangzhou, China), were transfected into 786-O-OCT2-489 and 786-O-OCT2-630 to hybridize OCT2 mRNA for 48 h. Biotech and the miRIP assay were performed as previously described with minor modification.

Techniques: Electron Microscopy, Western Blot, Marker, Expressing

Journal: Cancers

Article Title: LncRNA LINC01094 Promotes Cells Proliferation and Metastasis through the PTEN/AKT Pathway by Targeting AZGP1 in Gastric Cancer

doi: 10.3390/cancers15041261

Figure Lengend Snippet: Circos plot displayed screening of metastasis-related lncRNAs in FUSCC RNA sequence data. Circos plot displayed the distribution and expression of lncRNAs on human chromosomes. The outermost layer was a chromosome map of the human genome. The inner scatter diagrams corresponded to the distribution and expression of detected lncRNAs on the chromosomes; the red represents tumor tissue, while the blue represents normal tissue. The next inner histogram corresponded to the p -value for the differential expression analysis between tumor tissue and normal tissue, and the first 200 p -values were marked red. The next inner heat map from outside to inside corresponded to the expression of the first 200 lncRNAs in tumor tissue of HMG, normal tissue of HMG, tumor tissue of LMG, and normal tissue of LMG. The innermost histogram corresponded the expression ratio between HMG and LMG; ratios > 2 were marked red; and LINC01094 was represented by the red column, pointing to chromosome 4.

Article Snippet: LINC01094-fluorescence conjugated probes were designed and purchased from RiboBio (Guangzhou, China).

Techniques: Sequencing, Expressing

Journal: Cancers

Article Title: LncRNA LINC01094 Promotes Cells Proliferation and Metastasis through the PTEN/AKT Pathway by Targeting AZGP1 in Gastric Cancer

doi: 10.3390/cancers15041261

Figure Lengend Snippet: Survival and GSEA analysis in TCGA and GEO data sets. Kaplan–Meier survival plot showed that LINC01094 high-expression gastric cancer (separated by cutoff expression of LINC01094 calculated by ROC analyses) conferred a worse prognosis in the TCGA gastric cancer ( A ), GSE15459 ( B ), and GSE62254 ( C ) cohorts. Upregulated pathways analyzed by GSEA in TCGA gastric cancer cohort ( D ), GSE15459 ( E ), and GSE62254 ( F ).

Article Snippet: LINC01094-fluorescence conjugated probes were designed and purchased from RiboBio (Guangzhou, China).

Techniques: Expressing

Journal: Cancers

Article Title: LncRNA LINC01094 Promotes Cells Proliferation and Metastasis through the PTEN/AKT Pathway by Targeting AZGP1 in Gastric Cancer

doi: 10.3390/cancers15041261

Figure Lengend Snippet: Analysis in FUSCC RNA sequence and PCR data. ( A ) Upregulated pathways analyzed by GSEA in FUSCC RNA sequence data. ( B ) LINC01094 expression was evaluated in 106 paired cancerous and noncancerous tissues from FUSCC. ( C ) For the whole cohort, the Kaplan–Meier curve showed that LINC01094 high expression was associated with an unfavorable prognosis. Patients in the LINC01094 high-expression group had higher T ( D ) and N ( E ) stages. ( F ) For the stage I–II subgroup, the Kaplan–Meier curve showed that LINC01094 high expression was associated with an unfavorable prognosis. ( G ) For the stage III–IV subgroup, the Kaplan–Meier curve showed that LINC01094 high expression was associated with an unfavorable prognosis. *: p < 0.01, ***: p < 0.001.

Article Snippet: LINC01094-fluorescence conjugated probes were designed and purchased from RiboBio (Guangzhou, China).

Techniques: Sequencing, Expressing

Journal: Cancers

Article Title: LncRNA LINC01094 Promotes Cells Proliferation and Metastasis through the PTEN/AKT Pathway by Targeting AZGP1 in Gastric Cancer

doi: 10.3390/cancers15041261

Figure Lengend Snippet: Clinical-pathological characteristics of the 106 enrolled patients from FUSCC.

Article Snippet: LINC01094-fluorescence conjugated probes were designed and purchased from RiboBio (Guangzhou, China).

Techniques:

Journal: Cancers

Article Title: LncRNA LINC01094 Promotes Cells Proliferation and Metastasis through the PTEN/AKT Pathway by Targeting AZGP1 in Gastric Cancer

doi: 10.3390/cancers15041261

Figure Lengend Snippet: The characteristics of LINC01094 in GC. ( A ) LINC01094 was predicted as a noncoding RNA by PhyloCSF, CPAT, CPC, and the ORF finder. QRT-PCR for the separated nuclear and cytoplasmic fraction of HGC-27 ( B ) and MGC-803 ( C ) cell lines showed that LINC01094 was in both the nucleus and the cytoplasm. FISH assay showed that LINC01094 was in both the nucleus and the cytoplasm in the HGC-27 ( D ) and MGC-803 ( E ) cell lines (objective: 63×). The scale bar equals 50 μm, as indicated in each graph.

Article Snippet: LINC01094-fluorescence conjugated probes were designed and purchased from RiboBio (Guangzhou, China).

Techniques: Quantitative RT-PCR

Journal: Cancers

Article Title: LncRNA LINC01094 Promotes Cells Proliferation and Metastasis through the PTEN/AKT Pathway by Targeting AZGP1 in Gastric Cancer

doi: 10.3390/cancers15041261

Figure Lengend Snippet: LINC01094 promotes the malignant phenotype of GC in vitro. ( A ) LINC01094 expression levels in the normal human gastric mucosal cell line (GES-1) and human gastric cancer cell lines. ( B ) QRT-PCR results showed the knockdown efficacy of ASOs and overexpression efficacy of lentivirus. The migration abilities were weakened after the knockdown of LINC01094 ( C ) and were strengthened after the overexpression of LINC01094 ( D ) (objective: 10×). Western blotting was applied to assess the expression levels of EMT and stemness-associated proteins after silencing ( E ) and overexpressing ( F ) LINC01094 in HGC-27 and MGC-803 cells, respectively. The colony-formation results showed that the proliferation of HGC-27 and MGC-803 cells was inhibited and strengthened after silencing ( G ) and overexpressing ( H ) LINC01094, respectively. ( I ) The CCK-8 assay showed that silencing LINC01094 weakened the proliferation of HGC-27 and MGC-803 cells. ( J ) The CCK-8 assay showed that overexpressing LINC01094 strengthened the proliferation of HGC-27 and MGC-803 cells. ( K ) The EdU assay showed that silencing LINC01094 weakened the proliferation of HGC-27 and MGC-803 cells (objective: 4×). ( L ) The EdU assay showed that overexpressing LINC01094 strengthened the proliferation of HGC-27 and MGC-803 cells (objective: 4×). ( M ) Sphere-forming assay showed that LINC01094 overexpression cell lines formed more spheres (objective: 4×). *: p < 0.01, **: p < 0.01, ***: p < 0.001. The scale bar equals 100 or 200 μm, as indicated in each graph.

Article Snippet: LINC01094-fluorescence conjugated probes were designed and purchased from RiboBio (Guangzhou, China).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Over Expression, Migration, Western Blot, CCK-8 Assay, EdU Assay

Journal: Cancers

Article Title: LncRNA LINC01094 Promotes Cells Proliferation and Metastasis through the PTEN/AKT Pathway by Targeting AZGP1 in Gastric Cancer

doi: 10.3390/cancers15041261

Figure Lengend Snippet: LINC01094 promotes the malignant phenotype of GC in vivo. ( A ) After overexpressing LINC01094, the proliferation of MGC-803 cells was strengthened in vivo. ( B ) Ki-67 IHC staining of tumor tissues dissected from subcutaneous xenograft models (objective: 20×). ( C ) The representative H- and E-stained slice pictures for the negative control and LINC01094 overexpression groups (objective: left 10×, right 20×). *: p < 0.01, **: p < 0.01, ***: p < 0.001. The scale bar equals 20, 50, or 100 μm, as indicated in each graph.

Article Snippet: LINC01094-fluorescence conjugated probes were designed and purchased from RiboBio (Guangzhou, China).

Techniques: In Vivo, Immunohistochemistry, Staining, Negative Control, Over Expression

Journal: Cancers

Article Title: LncRNA LINC01094 Promotes Cells Proliferation and Metastasis through the PTEN/AKT Pathway by Targeting AZGP1 in Gastric Cancer

doi: 10.3390/cancers15041261

Figure Lengend Snippet: LINC01094 promotes migration of tumor cells through the PTEN/AKT pathway by targeting AZGP1 in GC. ( A ) The western blot results showed that only AZGP1 specifically combined LINC01094. ( B ) The qRT-PCR results showed that LINC01094 was significantly more enriched with the anti-AZGP1 antibody than with the rabbit IgG. ( C ) The qRT-PCR results showed that the RNA level of PTEN underwent no significant change after overexpression of AZGP1 in HGC-27 and MGC-803 cells. ( D ) Western blotting was applied to assess the expression levels of PTEN, pAKT, and total AKT after overexpressing AZGP1 in HGC-27 and MGC-803 cells. ( E ) The qRT-PCR results showed that the RNA level of PTEN underwent no significant change after overexpression of LINC01094 in HGC-27 and MGC-803 cells. ( F ) Western blotting was applied to assess the expression levels of AZGP1, PTEN, pAKT, and total AKT after overexpressing LINC01094 in HGC-27 and MGC-803 cells. ( G ) Rescue experiments showed that the migration-promotion effect caused by overexpression of LINC01094 could be rescued by overexpression of AZGP1 (objective: 10×). ( H ) The western blot results showed that the downregulation of PTEN and upregulation of pAKT effects caused by overexpression of LINC01094 could be rescued by overexpression of AZGP1. ( I ) Rescue experiments showed that the migration-promotion effect caused by overexpression of LINC01094 could be rescued by perifosine (objective: 10×). ( J ) The western blot results showed that perifosine successfully downregulated the expression of pAKT. *: p < 0.01, **: p < 0.01, ***: p < 0.001, ns: no statistical significance. The scale bar equals 100 μm, as indicated in each graph.

Article Snippet: LINC01094-fluorescence conjugated probes were designed and purchased from RiboBio (Guangzhou, China).

Techniques: Migration, Western Blot, Quantitative RT-PCR, Over Expression, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: circ‐AKT3 aggravates renal ischaemia‐reperfusion injury via regulating miR‐144‐5p /Wnt/β‐catenin pathway and oxidative stress

doi: 10.1111/jcmm.16072

Figure Lengend Snippet: Circ‐AKT3 sponges to miR‐144‐5p A, binding sites between circ‐AKT3 and miR‐144‐5p. B, The luciferase activity in 293T cells was measured by Dual‐luciferase assay. C, The correlation between miR‐144‐5p and circ‐AKT3 in 293T cells was tested by using RIP assay, anti‐Ago2 and anti‐IgG antibody were used to immunoprecipitated cellular lysates. D, The relationship between miR‐144‐5p and circ‐AKT3 in 293T cells was tested by using RNA pull‐down experiment. E, relative expression of miR‐144‐5p in pcDNA‐circ‐AKT3 and its NC plasmids treated rat models were measured by qRT‐PCR. F, circ‐AKT3 expression in RI/R rat and sham rat kidney tissues at postoperative time 4, 12, 24, 48 h were measured by qRT‐PCR. G, the representative images of FISH of circ‐AKT3 expression in HK‐2 and NRK‐52E cells. Three independent experiments were carried out. * P < .05, ** P < .01, *** P < .001

Article Snippet: The biotin‐labelled circ‐AKT3 probe was purchased from Genepharma.

Techniques: Binding Assay, Luciferase, Activity Assay, Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: circ‐AKT3 aggravates renal ischaemia‐reperfusion injury via regulating miR‐144‐5p /Wnt/β‐catenin pathway and oxidative stress

doi: 10.1111/jcmm.16072

Figure Lengend Snippet: Circ‐AKT3 promotes RI/R injury progression by sponging to miR‐144‐5p A and B: Tail vein injection was conducted to inject pcDNA‐circ‐AKT3, pcDNA‐circ‐AKT3 + miR‐144‐5p mimic plasmids into the RI/R injury rats, and the level of BUN and Serum Cr were measured by qRT‐PCR. C, Observation of the effect of pcDNA‐circ‐AKT3, pcDNA‐circ‐AKT3 + miR‐144‐5p mimic on RI/R injury rats presented by HE staining. D, Observation of cellular ultrastructure changes of pcDNA‐circ‐AKT3, pcDNA‐circ‐AKT3 + miR‐144‐5p mimic on RI/R injury rat by TEM. E, Detection of the effect of pcDNA‐circ‐AKT3, pcDNA‐circ‐AKT3 + miR‐144‐5p mimic on apoptotic ratio in renal I/R injury tissues by TUNEL method. F, the expression of Bax, Bcl‐2 and c‐caspase3 in pcDNA‐circ‐AKT3, pcDNA‐circ‐AKT3 + miR‐144‐5p mimic transfected hypoxia‐treated HK‐2 cells were evaluated by Western blot. G, Bax/Bcl‐2 ratio was shown. H, Apoptosis rate of HK‐2 cells (con), hypoxia‐treated HK‐2 cells (circ‐AKT3), and pcDNA‐circ‐AKT3 + miR‐144‐5p mimic transfected hypoxia‐treated HK‐2 cells (circ‐AKT3 + mimic) was measured by flow cytometry. G, the level of β‐catenin, c‐myc and cyclinD1 in pcDNA‐circ‐AKT3, pcDNA‐circ‐AKT3 + miR‐144‐5p mimic transfected RI/R rat kidney tissues was measured by Western blot (I) and qRT‐PCR (J). Each experiment was repeated three times, * P < .05, # P < .05, ** P < .01

Article Snippet: The biotin‐labelled circ‐AKT3 probe was purchased from Genepharma.

Techniques: Injection, Quantitative RT-PCR, Staining, TUNEL Assay, Expressing, Transfection, Western Blot, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: circ‐AKT3 aggravates renal ischaemia‐reperfusion injury via regulating miR‐144‐5p /Wnt/β‐catenin pathway and oxidative stress

doi: 10.1111/jcmm.16072

Figure Lengend Snippet: Circ‐AKT3 enhances RI/R injury‐induced oxidative stress pcDNA‐circ‐AKT3 (AAV) and its NC were injected into rat by performing tail vein injection surgery. After 24 h, tissues were collected for MDA (A), O 2 ‐ (B), SOD (C), CAT (D) measurement. N = 6 in each experiment. ** P < .01

Article Snippet: The biotin‐labelled circ‐AKT3 probe was purchased from Genepharma.

Techniques: Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: circ‐AKT3 aggravates renal ischaemia‐reperfusion injury via regulating miR‐144‐5p /Wnt/β‐catenin pathway and oxidative stress

doi: 10.1111/jcmm.16072

Figure Lengend Snippet:

Article Snippet: The biotin‐labelled circ‐AKT3 probe was purchased from Genepharma.

Techniques: