Journal: Acta Pharmaceutica Sinica. B

Article Title: Reprogrammed siTNF α /neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy

doi: 10.1016/j.apsb.2022.08.012

Figure Lengend Snippet: Schematic illustration of the reprogrammed siRNA/neutrophil cytopharmaceuticals for suppressing synovial inflammation and ameliorating joint destruction in inflammatory arthritis. (A) Preparation of the reprogrammed siRNA/neutrophil cytopharmaceuticals with down-regulated TNF α . siTNF α was first condensed with protamine to form a complex (prot/siTNF α ), and then encapsulated by tertiary amine-derived cationic liposomes to gain a lipoplex (siTNF α /TP), followed by the uptake by neutrophils to obtain the reprogrammed siRNA/neutrophil cytopharmaceuticals (siTNF α /TP/NEs). (B) The in vivo mechanisms of siTNF α /TP/NEs against inflamed joints. (i) After intravenous injection into collagen-induced arthritis (CIA) mice, siTNF α /TP/NEs can migrate along a gradient of chemoattractants to the inflamed joints leveraging the chemotaxis of neutrophils. (ii) When arrive at the inflammatory joints, siTNF α /TP/NEs can release the encapsulated siTNF α /TP through the formation of NETs. Both the released siTNF α /TP and the apoptotic siTNF α /TP/NEs can be phagocytosed by macrophages, which effectively deliver the siTNF α to macrophages in inflamed joints, thus reducing the TNF α expression. (iii) The downregulation of TNF α level can modulate the inflammatory network and result in the relief of local inflammation and protection of cartilages.

Article Snippet: Scrambled siRNA (siNC) (sense strand: 5′- UucuccgaacgugucacgudTdT -3′, anti-sense strand: 3′- AcgugacacguucggagaadTdT -5′), siTNF α (anti-sense strand: 5′- CgtcgtagcaaaccaccaadTdT -3′), fluorecein-labeled scrambled siRNA (FAM-siRNA) and Cy5-labeled scrambled siRNA (Cy5-siRNA) were purchased from RiboBio (Guangzhou, China).

Techniques: Derivative Assay, Liposomes, In Vivo, Injection, Chemotaxis Assay, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Reprogrammed siTNF α /neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy

doi: 10.1016/j.apsb.2022.08.012

Figure Lengend Snippet: Preparation and characterization of siRNA/neutrophil cytopharmaceuticals (siTNF α /TP/NEs). (A) Schematic of the preparation of siTNF α /TP and the associate TEM images of prot/siTNF α , blank TA7 liposomes and siTNF α /TP, respectively. Scale bar: 100 nm. (B) Flow cytometry analysis of the purity of isolated neutrophils. Isolated neutrophils were double stained with PE anti-mouse CD11b and violen t- 421 anti-mouse Ly6G antibodies. (C) Morphological images of isolated neutrophils stained with Giemsa–Wright stain. Scale bar: 5 μm. (D) Schematic of the preparation of siRNA/TP/NEs and siRNA/TA7/NEs. (E) CLSM images of siRNA/TP/NEs after culturing at 37 °C for 8 h. The nuclei of neutrophils were stained with Hoechst 33342 (Blue), TA7 liposomes were stained with rhodamine B (Red), and siRNA was labeled with FAM (Green). The merged image is the overlay of the three individual images. Scale bar: 5 μm. (F) The fluorescent intensity of Cy5-siRNA in the siRNA/TP/NEs at different time, n = 3. (G) Percentage of migrated neutrophils in the lower chamber. Fresh neutrophils (NEs) were used as control. Data were presented as mean ± SEM and analyzed by Student's t- test, n = 3. ns, not significant. (H) The release profile of Cy5-siRNA from siRNA/TP/NEs in RPMI 1640 medium with 100 nmol/L PMA ( n = 3 per group).

Article Snippet: Scrambled siRNA (siNC) (sense strand: 5′- UucuccgaacgugucacgudTdT -3′, anti-sense strand: 3′- AcgugacacguucggagaadTdT -5′), siTNF α (anti-sense strand: 5′- CgtcgtagcaaaccaccaadTdT -3′), fluorecein-labeled scrambled siRNA (FAM-siRNA) and Cy5-labeled scrambled siRNA (Cy5-siRNA) were purchased from RiboBio (Guangzhou, China).

Techniques: Liposomes, Flow Cytometry, Isolation, Staining, Wright Stain, Labeling, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Reprogrammed siTNF α /neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy

doi: 10.1016/j.apsb.2022.08.012

Figure Lengend Snippet: Reprogrammed neutrophils (siTNF α /TP/NEs) with anti-inflammatory ability and cartilage protection. (A) Time-dependent endo/lysosomal escape of siTNF α /TP in neutrophils. The representative gray values of endosomes and siRNA following the white line were analyzed by image J. Endo/lysosomes were stained with Lyso-Tracker Red, siRNA was labeled with FAM, and the nuclei were stained with Hoechst 33342. Scale bar: 2 μm. (B) q-PCR assay of Tnfα mRNA in siTNF α /TP/NEs and siNC/TP/NEs with LPS stimulation. Data are presented as mean ± SEM and analyzed by two-way ANOVA test with Sidak's correction, n = 3. siNC/TP/NEs set as control. ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (C–D) q-PCR assays of (C) Tgf-β and (D) Mmp9 mRNA in neutrophils (NEs in the figure), siNC/TP/NEs and siTNF α /TP/NEs stimulated by LPS, respectively. Data are presented as mean ± SEM and analyzed by one-way ANOVA test with Turkey's correction, n = 3. NEs group was used as a control. ns, not significant, ∗∗∗ P < 0.001. The significance of pairwise group is shown on the top of the corresponding line. ### P < 0.001. (E) Schematic illustration of the in vitro model to evaluate the effect of siTNF α /TP/NEs on chondrocytes stimulated by IL1 β to mimic the RA environment. (F–G) q-PCR assays of (F) Adamts5 and (G) Mmp1 3 mRNA in chondrocytes (Chon) stimulated by IL-1 β . Data are presented as mean ± SEM and analyzed by one-way ANOVA test with Turkey's correction, n = 3. Chon group was used as a control. ns, not significant, ∗∗ P < 0.01; ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. The significance of pairwise group is shown on the top of the corresponding line. ## P < 0.01, ### P < 0.001, #### P < 0.0001.

Article Snippet: Scrambled siRNA (siNC) (sense strand: 5′- UucuccgaacgugucacgudTdT -3′, anti-sense strand: 3′- AcgugacacguucggagaadTdT -5′), siTNF α (anti-sense strand: 5′- CgtcgtagcaaaccaccaadTdT -3′), fluorecein-labeled scrambled siRNA (FAM-siRNA) and Cy5-labeled scrambled siRNA (Cy5-siRNA) were purchased from RiboBio (Guangzhou, China).

Techniques: Staining, Labeling, Control, In Vitro

Journal: Acta Pharmaceutica Sinica. B

Article Title: Reprogrammed siTNF α /neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy

doi: 10.1016/j.apsb.2022.08.012

Figure Lengend Snippet: The transfer of siTNF α to macrophages including BMDM and RAW264.7 cells under inflammatory conditions and the following TNF α gene silencing. (A) CLSM images of siTNF α /TP released from siTNF α /TP/NEs through NETs formation in mimic RA environment with IL-1 β (10 ng/mL). siTNF α /TP/NEs were stained with Hoechst 33342 (Blue) and citrullinated Histone H3 (Violet), which were the typical markers of NETs. TA7 liposomes were labeled with rhodamine B (RHO-TA7, Red), and siRNA was labeled with FAM (FAM-siRNA, Green). Scale bars: 5 μm. (B) CLSM images of BMDM and RAW264.7 cells after incubation with siTNF α /TP/NEs for 8 h siRNA was labeled with Cy5 (Cy5-siRNA, Red), neutrophils were stained by fluorescein isothiocyanate (FITC)-conjugated Ly6G (NEs, green). Arrowheads indicate the process of macrophages engulfing neutrophils. Scale bars: 5 μm. (C–F) Measurement of TNF α expression in (C, D) BMDM and (E, F) RAW264.7 cells after LPS (200 ng/mL) stimulation and quantification by q-PCR and ELISA, respectively. Data were presented as mean ± SEM and analyzed by one-way ANOVA test with Turkey's correction, n = 3. BMDM or RAW group was used as a control, respectively. ns, not significant, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. The significance of pairwise group is shown on the top of the corresponding line. # P < 0.05, ### P < 0.001, #### P < 0.0001.

Article Snippet: Scrambled siRNA (siNC) (sense strand: 5′- UucuccgaacgugucacgudTdT -3′, anti-sense strand: 3′- AcgugacacguucggagaadTdT -5′), siTNF α (anti-sense strand: 5′- CgtcgtagcaaaccaccaadTdT -3′), fluorecein-labeled scrambled siRNA (FAM-siRNA) and Cy5-labeled scrambled siRNA (Cy5-siRNA) were purchased from RiboBio (Guangzhou, China).

Techniques: Staining, Liposomes, Labeling, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Reprogrammed siTNF α /neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy

doi: 10.1016/j.apsb.2022.08.012

Figure Lengend Snippet: The biodistribution of Cy5-siRNA/TP/NEs in vivo . (A) In vivo images of arthritic joints over time after intravenous injection of free Cy5-siRNA, Cy5-siRNA/TP or Cy5-siRNA/TP/NEs into CIA mice, at a dose of 0.3 mg/kg, respectively. (B) Accumulation of Cy5-siRNA in the joints and different organs detected using the ex vivo imaging at 24 h post-injection of different formulations. (C) Frozen sections of synovium to observe the cellular distribution of Cy5-siRNA. Macrophages were marked by F4/80-FITC (Green) and nucleus was stained by Hochest 33342 (Blue). Scale bar: 5 μm.

Article Snippet: Scrambled siRNA (siNC) (sense strand: 5′- UucuccgaacgugucacgudTdT -3′, anti-sense strand: 3′- AcgugacacguucggagaadTdT -5′), siTNF α (anti-sense strand: 5′- CgtcgtagcaaaccaccaadTdT -3′), fluorecein-labeled scrambled siRNA (FAM-siRNA) and Cy5-labeled scrambled siRNA (Cy5-siRNA) were purchased from RiboBio (Guangzhou, China).

Techniques: In Vivo, Injection, Ex Vivo, Imaging, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Reprogrammed siTNF α /neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy

doi: 10.1016/j.apsb.2022.08.012

Figure Lengend Snippet: Evaluation of the in vivo anti-rheumatoid arthritis effect mediated by siTNF α /TP/NEs. (A) Schematic illustration of the administration regimen. On Day 0, mice were subcutaneously injected (s.c.) with complete Freunds adjuvant in the skin of the tail at the base of the tail. After 21 days, mice were subcutaneously injected with incomplete Freunds adjuvant in the skin of the tail at the base of the tail as the second immunization. Next, the CIA mice were intravenously injected (i.v.) with saline, siNC/TP/NEs, siTNF α /TP, free MTX, and siTNF α /TP/NEs every 3 days for eight times. (B) Arthritic score measurement in CIA mice after different treatment over time. (C) Thickness of paws of CIA mice after different treatment over time. (D) Concentration of TNF α protein in the serum of CIA mice after different treatment over time. Relative mRNA levels of (E) Tnfα , (F) Adamts5 , and (G) Mmp13 in the joints of CIA mice after different treatment. The data at different groups ( n = 3) were shown as mean ± SEM and analyzed by one-way ANOVA with Turkey's correction. Compared to Saline group: ns, not significant, ∗ P < 0.05, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. The significance of pairwise group is shown on the top of the corresponding line. ns, not significant, # P < 0.05, ## P < 0.01, #### P < 0.0001.

Article Snippet: Scrambled siRNA (siNC) (sense strand: 5′- UucuccgaacgugucacgudTdT -3′, anti-sense strand: 3′- AcgugacacguucggagaadTdT -5′), siTNF α (anti-sense strand: 5′- CgtcgtagcaaaccaccaadTdT -3′), fluorecein-labeled scrambled siRNA (FAM-siRNA) and Cy5-labeled scrambled siRNA (Cy5-siRNA) were purchased from RiboBio (Guangzhou, China).

Techniques: In Vivo, Injection, Adjuvant, Saline, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Reprogrammed siTNF α /neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy

doi: 10.1016/j.apsb.2022.08.012

Figure Lengend Snippet: The MRI, H&E-stained images, micro-CT, and safranin O-fast green stained images of joint tissues of normal mice and saline, siNC/TP/NEs, siTNF α /TP, MTX and siTNF α /TP/NEs treated-CIA mice. (A) T2-weighted fa t- suppressed MRI images of arthritic joints. White arrows indicate the inflammation site of synovitis. (B) Histological analysis by H&E staining of joint tissue sections. Scale bar: 100 μm. (C) Representative micro-CT images of arthritic joints. The red circles indicate periarticular osteophytes. (D) Proteoglycans assay by Safranin O-fast green staining of joint tissue sections. Scale bar: 50 μm.

Article Snippet: Scrambled siRNA (siNC) (sense strand: 5′- UucuccgaacgugucacgudTdT -3′, anti-sense strand: 3′- AcgugacacguucggagaadTdT -5′), siTNF α (anti-sense strand: 5′- CgtcgtagcaaaccaccaadTdT -3′), fluorecein-labeled scrambled siRNA (FAM-siRNA) and Cy5-labeled scrambled siRNA (Cy5-siRNA) were purchased from RiboBio (Guangzhou, China).

Techniques: Staining, Micro-CT, Saline

Journal: International Journal of Gynecological Cancer

Article Title: Long Noncoding RNA GAS5, Which Acts as a Tumor Suppressor via microRNA 21, Regulates Cisplatin Resistance Expression in Cervical Cancer

doi: 10.1097/IGC.0000000000001028

Figure Lengend Snippet: Expression of GAS5 and miR-21 in CC tissues and cell lines. Forty pairs of the tissue samples are gathered from 40 patients, including the cancer tissue and pericarcinomatous tissue. Quantitative real-time PCR was used to measure the expression of GAS5 and miR-21. The relative expression is presented as the fold change on the figure. A and B, Samples were classified by the clinical stage of CC (FIGO, 2009). C and D, Average relative expression of GAS5 and miR-21 in CC tissue compared with the normal pericarcinomatous tissue. E and F, Average relative expression of GAS5 and miR-21 in HeLa, SiHa, CaSki, and SiHa/cDDP CC cell lines. Data are means (SD) of 3 independent experiments. ** P < 0.01.

Article Snippet: The sequences were as follows: GAS5 siRNA1 (sense strand, 5’ CUUGCCUGGACCAGCUUAA dTdT 3’; antisense strand, 3’ dTdT GAACGGACCUGGUCGAAUU 5’), GAS5 siRNA2 (sense strand, 5’ GCUCUGGAUAGCACCUUAU dTdT 3’; antisense strand, 3’ dTdT CGAGACCUAUCGUGGAAUA 5’), and GAS5 siRNA3 (sense strand, 5’ GCAGUGGCCUUUGAAGCUU dTdT 3’; antisense strand, 3’ dTdT CGUCACCGGAAACUUCGAA 5’). microRNA 21 mimic and inhibitor (Ribobio, Guangzhou, China) were used for overexpressing and silencing the expression of miR-21.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: International Journal of Gynecological Cancer

Article Title: Long Noncoding RNA GAS5, Which Acts as a Tumor Suppressor via microRNA 21, Regulates Cisplatin Resistance Expression in Cervical Cancer

doi: 10.1097/IGC.0000000000001028

Figure Lengend Snippet: Identification of GAS5 as a target of miR-21 and effect of regulation to miR-21 target proteins PTEN and PDCD4 by GAS5. A, Expression of GAS5 after GAS5 lentivirus transfection. Four cervical cell lines were transfected with either GAS5 or blank lentivirus (Ctrl). The cells were harvested after screening the stable GAS5 transfected cell lines, and total RNA was isolated, followed by detecting the expression of GAS5 using qRT-PCR. B, Detection of miR-21 in the stable GAS5 transfected cell lines using qRT-PCR; experimental procedure was the same as in A. C and D, Three CC cell lines HeLa, SiHa, and CaSki were transfected with con-siRNA or GAS5 siRNA1, GAS5 siRNA2, or GAS5 siRNA3 for 24 hours; the expression of GAS5 or miR-21 was determined by qRT-PCR. E and F, Identification of the GAS5 expression in the 3 CC cell lines by transfecting miR-21 mimic and miR-21 inhibitor by qRT-PCR. G and H, Dual-luciferase reporter assay of GAS5 and miR-21 and the sequence of mutation site. WT, luciferase vector of GAS5; Mut, GAS5 mutant vector; WT+ miR-21, luciferase expression with vector of GAS5 after transfecting miR-21 mimic; Mut + miR-21, luciferase expression with mutant vector of GAS5. I, Identification of the basic level of PTEN and PDCD4 in 4 CC cells HeLa, SiHa, CaSki, and SiHa/cDDP. Cellular protein was isolated from the cells and used for Western blot. J and K, Relative protein levels (mean [SD], n = 3) of PDCD4 and PTEN were determined. L-Q, Effects of GAS5 and miR-21 on expression of the tumor suppressors PDCD4 and PTEN. Error bars represent SD, n = 3. * P < 0.05 or ** P < 0.01.

Article Snippet: The sequences were as follows: GAS5 siRNA1 (sense strand, 5’ CUUGCCUGGACCAGCUUAA dTdT 3’; antisense strand, 3’ dTdT GAACGGACCUGGUCGAAUU 5’), GAS5 siRNA2 (sense strand, 5’ GCUCUGGAUAGCACCUUAU dTdT 3’; antisense strand, 3’ dTdT CGAGACCUAUCGUGGAAUA 5’), and GAS5 siRNA3 (sense strand, 5’ GCAGUGGCCUUUGAAGCUU dTdT 3’; antisense strand, 3’ dTdT CGUCACCGGAAACUUCGAA 5’). microRNA 21 mimic and inhibitor (Ribobio, Guangzhou, China) were used for overexpressing and silencing the expression of miR-21.

Techniques: Expressing, Transfection, Isolation, Quantitative RT-PCR, Luciferase, Reporter Assay, Sequencing, Mutagenesis, Plasmid Preparation, Western Blot

Journal: International Journal of Gynecological Cancer

Article Title: Long Noncoding RNA GAS5, Which Acts as a Tumor Suppressor via microRNA 21, Regulates Cisplatin Resistance Expression in Cervical Cancer

doi: 10.1097/IGC.0000000000001028

Figure Lengend Snippet: Effect of GAS5 on proliferation inhibition, migration, and invasion in CC cells HeLa, SiHa, and CaSki. A-C, Cell proliferation CCK-8 assay. Three cell lines were firstly grown and transiently transfected with GAS5 lentivirus and GAS5 siRNA and then subjected to CCK-8 assay. * P < 0.05 versus control groups. Then, 3 cell lines were grown and transiently transfected with GAS5 lentivirus and GAS5 siRNA and then subjected to (D) migration and (F) invasion assay. E and G, Quantitative data of D and F, respectively. Error bars represent SD, n = 3. * P < 0.05; ** P < 0.01.

Article Snippet: The sequences were as follows: GAS5 siRNA1 (sense strand, 5’ CUUGCCUGGACCAGCUUAA dTdT 3’; antisense strand, 3’ dTdT GAACGGACCUGGUCGAAUU 5’), GAS5 siRNA2 (sense strand, 5’ GCUCUGGAUAGCACCUUAU dTdT 3’; antisense strand, 3’ dTdT CGAGACCUAUCGUGGAAUA 5’), and GAS5 siRNA3 (sense strand, 5’ GCAGUGGCCUUUGAAGCUU dTdT 3’; antisense strand, 3’ dTdT CGUCACCGGAAACUUCGAA 5’). microRNA 21 mimic and inhibitor (Ribobio, Guangzhou, China) were used for overexpressing and silencing the expression of miR-21.

Techniques: Inhibition, Migration, CCK-8 Assay, Transfection, Control, Invasion Assay

Journal: International Journal of Gynecological Cancer

Article Title: Long Noncoding RNA GAS5, Which Acts as a Tumor Suppressor via microRNA 21, Regulates Cisplatin Resistance Expression in Cervical Cancer

doi: 10.1097/IGC.0000000000001028

Figure Lengend Snippet: Suppression of tumor growth by GAS5 in the xenograft mouse model. SiHa cells were transfected with GAS5 lentivirus and NC-lentivirus and then injected into the left subaxillary, as described in the text. A, Tumor growth was measured every 3 days of injection. B and C, Tumors were then harvested on day 36 and weighed. D, Immunohistochemical evaluation of PTEN and PDCD4 in the tumor tissue derived from the GAS5 group and the NC group of the xenograft mice, respectively.

Article Snippet: The sequences were as follows: GAS5 siRNA1 (sense strand, 5’ CUUGCCUGGACCAGCUUAA dTdT 3’; antisense strand, 3’ dTdT GAACGGACCUGGUCGAAUU 5’), GAS5 siRNA2 (sense strand, 5’ GCUCUGGAUAGCACCUUAU dTdT 3’; antisense strand, 3’ dTdT CGAGACCUAUCGUGGAAUA 5’), and GAS5 siRNA3 (sense strand, 5’ GCAGUGGCCUUUGAAGCUU dTdT 3’; antisense strand, 3’ dTdT CGUCACCGGAAACUUCGAA 5’). microRNA 21 mimic and inhibitor (Ribobio, Guangzhou, China) were used for overexpressing and silencing the expression of miR-21.

Techniques: Transfection, Injection, Immunohistochemical staining, Derivative Assay

Journal: International Journal of Gynecological Cancer

Article Title: Long Noncoding RNA GAS5, Which Acts as a Tumor Suppressor via microRNA 21, Regulates Cisplatin Resistance Expression in Cervical Cancer

doi: 10.1097/IGC.0000000000001028

Figure Lengend Snippet: The effects of GAS5 and miR-21 on cisplatin-induced cytotoxicity in SiHa and SiHa/cDDP cells. Cells with stable GAS5 expression and cells (SiHa and SiHa/cDDP cells) were cultured for 24 hours after transfecting GAS5 siRNA, miR-21 mimic, and miR-21 inhibitor. Cells were treated with different concentrations of cisplatin (1, 2, 3, 4, 5, 6, 10, 15, and 30 μg/mL). A, C, E, and G, Effects of GAS5 and miR-21 on cytotoxicity of cisplatin in normal SiHa cells. B, D, F, and H, Effects of GAS5 and miR-21 on cytotoxicity of cisplatin in SiHa/cDDP cells. The cytotoxicity was then determined 24 hours after cisplatin treatment using the CCK-8 assay. Each data point represents the survival rate of the cells.

Article Snippet: The sequences were as follows: GAS5 siRNA1 (sense strand, 5’ CUUGCCUGGACCAGCUUAA dTdT 3’; antisense strand, 3’ dTdT GAACGGACCUGGUCGAAUU 5’), GAS5 siRNA2 (sense strand, 5’ GCUCUGGAUAGCACCUUAU dTdT 3’; antisense strand, 3’ dTdT CGAGACCUAUCGUGGAAUA 5’), and GAS5 siRNA3 (sense strand, 5’ GCAGUGGCCUUUGAAGCUU dTdT 3’; antisense strand, 3’ dTdT CGUCACCGGAAACUUCGAA 5’). microRNA 21 mimic and inhibitor (Ribobio, Guangzhou, China) were used for overexpressing and silencing the expression of miR-21.

Techniques: Expressing, Cell Culture, CCK-8 Assay

Journal: International Journal of Gynecological Cancer

Article Title: Long Noncoding RNA GAS5, Which Acts as a Tumor Suppressor via microRNA 21, Regulates Cisplatin Resistance Expression in Cervical Cancer

doi: 10.1097/IGC.0000000000001028

Figure Lengend Snippet: The effects of GAS5 and miR-21 on regulating the phosphorylation of Akt in SiHa/cDDP cells. SiHa/cDDP cells with stable GAS5 expression and SiHa/cDDP cells were cultured for 24 hours after transfecting GAS5 siRNA, miR-21 mimic, and miR-21 inhibitor. Cellular protein was isolated from the transfected cells and used for Western blot. A, D, F, and I, Relative protein levels (mean [SD], n = 3) of pAkt and Akt were determined. B and C, Quantitative data of A. E, Quantitative data of D. G and H, Quantitative data of F. J, Quantitative data of I. Error bars represent SD, n = 3. * P < 0.05 or ** P < 0.01.

Article Snippet: The sequences were as follows: GAS5 siRNA1 (sense strand, 5’ CUUGCCUGGACCAGCUUAA dTdT 3’; antisense strand, 3’ dTdT GAACGGACCUGGUCGAAUU 5’), GAS5 siRNA2 (sense strand, 5’ GCUCUGGAUAGCACCUUAU dTdT 3’; antisense strand, 3’ dTdT CGAGACCUAUCGUGGAAUA 5’), and GAS5 siRNA3 (sense strand, 5’ GCAGUGGCCUUUGAAGCUU dTdT 3’; antisense strand, 3’ dTdT CGUCACCGGAAACUUCGAA 5’). microRNA 21 mimic and inhibitor (Ribobio, Guangzhou, China) were used for overexpressing and silencing the expression of miR-21.

Techniques: Phospho-proteomics, Expressing, Cell Culture, Isolation, Transfection, Western Blot