Journal: Annals of Medicine and Surgery

Article Title: Deciphering the molecular mechanism of long non-coding RNA HIF1A-AS1 regulating pancreatic cancer cells

doi: 10.1097/MS9.0000000000002097

Figure Lengend Snippet: Overexpression (OE) of HIF1A-AS1 affects the proliferation, apoptosis of the pancreatic cancer (PC) cells (PANC-1). (A) RT-qPCR measure the mRNA level of HIF1A-AS1 in pancreatic cells (PANC-1), Real-time PCR showed that the levels of HIF1A-AS1 was significantly increased in the cells from OE group compared with the empty vector-controlled normal control (NC) cells. (B, C) OE of HIF1A-AS1 reduces significantly the viability of PANC-1 cells at 24, 48, and 72 h. (D) OE of HIF1A-AS1 promotes significantly apoptosis of PANC-1 cells assessment of cellular apoptosis using Annexin V‑fluorescein isothiocyanate staining coupled with flow cytometry. (E) Total percentage of apoptotic PANC-1 cells in each group are summarized with data presented as the mean ± SD of three independent experiments. (F) Western blotting reveal the full length caspase-3, Bax, P53, and PARP1 protein expression in PANC-1 cells. *** P <0.001. We used IMAGE J to analyze the expression of the target protein in western blot results. The specific data are as follows: (a) The protein expression increased about 22% for OE-Bax VS NC-Bax. (b) The protein expression increased about 20% for OE-caspase-3 VS NC-caspase-3. (c) The protein expression increased about 10% for OE-P53 VS NC-P53. (d) The protein expression increased about 34% for OE- PARP1 VS NC- PARP1.

Article Snippet: The human pancreatic cancer cell lines (PANC-1, PATU8988 and SW1990) and one normal cell line (HPDE6-C7) were provided by Procell (Wuhan, China).

Techniques: Over Expression, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Staining, Flow Cytometry, Western Blot, Expressing

Journal: Annals of Medicine and Surgery

Article Title: Deciphering the molecular mechanism of long non-coding RNA HIF1A-AS1 regulating pancreatic cancer cells

doi: 10.1097/MS9.0000000000002097

Figure Lengend Snippet: Overexpression (OE) of HIF1A-AS1 regulates migration of the pancreatic cancer (PC) cells (PANC-1). (A, B)OE HIF1A-AS1 inhibits migration of PANC-1 cells by transwell migration assay, and the properties of migration of cells were evaluated compared the NC group. (C, D) OE of HIF1A-AS1 inhibits migration of PANC-1 cells by clone formation assays with calculating the number of colonies compared the NC group. (E, F) OE of HIF1A-AS1 inhibits migration of PANC-1 cells by wound-healing assay at 0, 24, and 48 h post-injury time points after the wound was made with calculating the percentage of wound width compared the wound width at 0 h. ( n = 3 cultures, paired Student’s t -test, ± SD) ** P <0.01, *** P <0.001.

Article Snippet: The human pancreatic cancer cell lines (PANC-1, PATU8988 and SW1990) and one normal cell line (HPDE6-C7) were provided by Procell (Wuhan, China).

Techniques: Over Expression, Migration, Transwell Migration Assay, Wound Healing Assay

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA CTBP1-AS2 upregulates USP22 to promote pancreatic carcinoma progression by sponging miR-141-3p

doi: 10.3892/mmr.2022.12602

Figure Lengend Snippet: CTBP1-AS2 expression is significantly upregulated in pancreatic carcinoma. (A) The Gene Expression Profiling Interactive Analysis database was used to analyze the expression of CTBP1-AS2 in tumor tissues of patients with pancreatic carcinoma and normal pancreatic tissues. (B) RT-qPCR was used to detect CTBP1-AS2 expression in tumor tissues and adjacent normal tissues of patients with pancreatic carcinoma. (C) RT-qPCR was used to detect CTBP1-AS2 expression in normal human pancreatic duct epithelial cell line (HPDE6-C7) and pancreatic carcinoma cell lines. *P<0.05 vs. Normal, **P<0.01 vs. HPDE6-C7 and ***P<0.001 vs. Normal or HPDE6-C7. CTBP1-AS2, CTBP1 antisense RNA 2; RT-qPCR, reverse transcription-quantitative PCR; TPM, transcripts per kilobase of exon model per million mapped reads.

Article Snippet: Normal human pancreatic duct epithelial cell line (HPDE6-C7), human pancreatic carcinoma cell lines (Hs766T, SW1990, CAPAN-1) and 293T were obtained from the American Type Culture Collection; the JF305 pancreatic carcinoma cell line was purchased from Yuchi (Shanghai) Biological Technology Co., Ltd. (cat. no. SCO423).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA CTBP1-AS2 upregulates USP22 to promote pancreatic carcinoma progression by sponging miR-141-3p

doi: 10.3892/mmr.2022.12602

Figure Lengend Snippet: Relationship between CTBP1-AS2 expression and clinicopathological characteristics of patients with pancreatic carcinoma.

Article Snippet: Normal human pancreatic duct epithelial cell line (HPDE6-C7), human pancreatic carcinoma cell lines (Hs766T, SW1990, CAPAN-1) and 293T were obtained from the American Type Culture Collection; the JF305 pancreatic carcinoma cell line was purchased from Yuchi (Shanghai) Biological Technology Co., Ltd. (cat. no. SCO423).

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA CTBP1-AS2 upregulates USP22 to promote pancreatic carcinoma progression by sponging miR-141-3p

doi: 10.3892/mmr.2022.12602

Figure Lengend Snippet: CTBP1-AS2 knockdown suppresses pancreatic carcinoma cell proliferation, migration and invasion and induces cell apoptosis. (A) CTBP1-AS2 siRNAs were transfected into Hs766T and JF305 cell lines, and the transfection efficiency was verified by reverse transcription-quantitative PCR. CCK-8 assay was used for examining the proliferation of (B) Hs766T and (C) JF305 cell lines transfected with CTBP1-AS2 siRNAs. (D) EdU assay was used to examine the proliferation of Hs766T and JF305 cell lines following the transfection with CTBP1-AS2 siRNA. Transwell and Matrigel assays were used to detect the (E) migratory and (F) invasive abilities, respectively, of Hs766T and JF305 cell lines following si-CTBP1-AS2 transfection. Flow cytometry was used to detect the apoptosis rate of (G) Hs766T and (H) JF305 cell lines following the transfection with CTBP1-AS2 siRNA. *P<0.05, **P<0.01 and ***P<0.001 vs. si-NC. CTBP1-AS2, CTBP1 antisense RNA 2; EdU, 5-ethynyl-2-deoxyuridine; NC, negative control; si, short interfering RNA.

Article Snippet: Normal human pancreatic duct epithelial cell line (HPDE6-C7), human pancreatic carcinoma cell lines (Hs766T, SW1990, CAPAN-1) and 293T were obtained from the American Type Culture Collection; the JF305 pancreatic carcinoma cell line was purchased from Yuchi (Shanghai) Biological Technology Co., Ltd. (cat. no. SCO423).

Techniques: Knockdown, Migration, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, CCK-8 Assay, EdU Assay, Flow Cytometry, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA CTBP1-AS2 upregulates USP22 to promote pancreatic carcinoma progression by sponging miR-141-3p

doi: 10.3892/mmr.2022.12602

Figure Lengend Snippet: CTBP1-AS2 targets miR-141-3p to promote pancreatic carcinoma progression. (A) StarBase database showed that CTBP1-AS2 sequence contained the binding site complementary to miR-141-3p. RT-qPCR was performed to detect the expression level of miR-141-3p in pancreatic carcinoma (B) tissues and (C) cell lines. (D) Pearson's correlation analysis of CTBP1-AS2 and miR-141-3p expression levels in pancreatic carcinoma tissues. (E) RT-qPCR was used to examine miR-141-3p expression levels in Hs766T and JF305 cell lines following the transfection of siCTBP1-AS2-1. (F) RT-qPCR was used to examine miR-141-3p expression in Hs766T and JF305 cell lines following the transfection of miR-141-3p mimics or miR-141-3p inhibitor. (G) Dual-luciferase reporter assay was used to verify the inaction between miR-141-3p and CTBP1-AS2. CCK-8 assay was conducted to examine proliferation of (H) Hs766T and (I) JF305 cell lines following the co-transfection of si-CTBP1-AS2-1 and miR-141-3p inhibitor. (J) EdU assay was also used to examine proliferation in Hs766T and JF305 cell line following the co-transfection of si-CTBP1-AS2-1 and miR-141-3p inhibitor. Transwell and Matrigel assays were performed to detect (K) migratory and (L) invasive abilities, respectively, of Hs766T and JF305 cell lines following the co-transfection of si-CTBP1-AS2-1 and miR-141-3p inhibitor. (M) Flow cytometry was performed to detect the apoptosis of Hs766T and JF305 cells following the co-transfection of si-CTBP1-AS2-1 and miR-141-3p inhibitor. *P<0.05 vs. si-CTBP1-AS2-1, **P<0.01 vs. HPDE6-C7, inhibitor NC, mimics NC, si-NC or si-CTBP1-AS2-1. ***P<0.001 vs. Normal, HPDE6-C7, si-NC or mimics NC. CTBP1-AS2-1, CTBP1 antisense RNA 2 siRNA-1; EdU, 5-ethynyl-2-deoxyuridine; miR, microRNA; MUT, mutant; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; si, short interfering RNA; WT, wild-type.

Article Snippet: Normal human pancreatic duct epithelial cell line (HPDE6-C7), human pancreatic carcinoma cell lines (Hs766T, SW1990, CAPAN-1) and 293T were obtained from the American Type Culture Collection; the JF305 pancreatic carcinoma cell line was purchased from Yuchi (Shanghai) Biological Technology Co., Ltd. (cat. no. SCO423).

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Luciferase, Reporter Assay, CCK-8 Assay, Cotransfection, EdU Assay, Flow Cytometry, Mutagenesis, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA CTBP1-AS2 upregulates USP22 to promote pancreatic carcinoma progression by sponging miR-141-3p

doi: 10.3892/mmr.2022.12602

Figure Lengend Snippet: miR-141-3p directly targets USP22. (A) StarBase database search indicated that the 3′UTR of USP22 mRNA contained a target site for miR-141-3p, which was confirmed by luciferase reporter assay. (B) Western blotting (top) and RT-qPCR (bottom) were conducted to detect the expression levels of USP22 protein and mRNA, respectively, in pancreatic carcinoma cells transfected with miR-141-3p inhibitor. (C) Western blotting (top) and RT-qPCR (bottom) were used to detect USP22 protein and mRNA expression, respectively, in pancreatic carcinoma cell lines. (D) RT-qPCR was used to detect USP22 expression in pancreatic tumor tissues. Pearson's correlation analysis of (E) USP22 mRNA and miR-141-3p expression levels and (F) USP22 mRNA and CTBP1-AS2 expression levels in pancreatic carcinoma tissues. **P<0.01 vs. mimics NC and ***P<0.001 vs. mimics NC, HPDE6-C7 or Normal. CTBP1-AS2, CTBP1 antisense RNA 2; miR, microRNA; MUT, mutant; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; USP22, ubiquitin-specific protease 22; WT, wild-type.

Article Snippet: Normal human pancreatic duct epithelial cell line (HPDE6-C7), human pancreatic carcinoma cell lines (Hs766T, SW1990, CAPAN-1) and 293T were obtained from the American Type Culture Collection; the JF305 pancreatic carcinoma cell line was purchased from Yuchi (Shanghai) Biological Technology Co., Ltd. (cat. no. SCO423).

Techniques: Luciferase, Reporter Assay, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Mutagenesis, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Ubiquitin Proteomics

Journal: Journal for immunotherapy of cancer

Article Title: Oncolytic peptide LTX-315 induces anti-pancreatic cancer immunity by targeting the ATP11B-PD-L1 axis.

doi: 10.1136/jitc-2021-004129

Figure Lengend Snippet: Figure 3 LTX-315 downregulates PD-L1 expression in pancreatic cancer. (A) Representative images and further quantification of PD-L1 IHC staining in the immunocompetent mice treated with LTX-315. (B–F) Representative images and further quantification of flow cytometry analysis of PD-L1 expression in tumor cells (B), dendritic cells (C), granulocytic-MDSCs (D), monocytic-MDSCs (E), and macrophages (F) in tumors collected from immunocompetent mice. (G–I) LTX-315 inhibition of PD-L1 expression in a dose-dependent manner. PD-L1 expression in BxPC-3 (G), SW1990 (H), and KPC (I) cell treated with LTX-315 at increasing concentrations, individually analyzed by western blotting. (J–L) Inhibition of PD-L1 expression by LTX-315 in a time-dependent manner. PD-L1 expression in BxPC-3 (J), SW1990 (K), and KPC (L) cell treated with LTX-315 at increasing time points, individually analyzed by western blotting. (M–O) Inhibition of PD-L1 expression by LTX-315 regardless of the effect of cell death. PD-L1 expression in BxPC-3 (M), SW1990 (N), and KPC (O) cells treated with LTX-315 and CDDP, individually analyzed by western blotting. Results are presented as mean±SD of one representative experiment. *p<0.05, **p<0.01, ***p<0.001 by a two-tailed t-test; DC, dendritic cell; IHC, immunohistochemical; MDSCs, myeloid-derived suppressor cells; NS, not significant; PD-L1, programmed cell death ligand 1.

Article Snippet: D ow nloaded from 5Tang T, et al. J Immunother Cancer 2022;10:e004129. doi:10.1136/jitc-2021-004129 KPC, BXPC- 3, and SW1990 cells were stably transfected or co- transfected with human ATP11B Double Nickase Plasmid (sc- 411692- NIC, Santa Cruz), and ATP11B Double Nickase Plasmid (sc- 429296- NIC, Santa Cruz) and KPC and SW1990 were stably transfected with CMTM6 Double Nickase Plasmid (sc- 412381- NIC, Santa Cruz) and CMTM6 Double Nickase Plasmid (sc- 426447- NIC, Santa Cruz) according to the manufacturer’s instructions.

Techniques: Expressing, Immunohistochemistry, Flow Cytometry, Inhibition, Western Blot, Two Tailed Test, Immunohistochemical staining, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Oncolytic peptide LTX-315 induces anti-pancreatic cancer immunity by targeting the ATP11B-PD-L1 axis.

doi: 10.1136/jitc-2021-004129

Figure Lengend Snippet: Figure 6 ATP11B interacts with PD-L1 in a CMTM6-dependent manner. (A–B) ATP11B interaction with PD-L1 in vivo. Cell lysates from KPC and SW1990 cells were separately subjected to immunoprecipitation and western blotting using the indicated antibodies. (C) Lack of binding of ATP11B to PD-L1 in vitro. FLAG-tagged ATP11B and MYC-tagged PD-L1 purified from 293 T cell lysates and subjected to immunoprecipitation after co-incubation. (D) Positive correlation of ATP11B with CMTM6. Correlation of ATP11B and CMTM6 analyzed according to the pancreatic data sets of The Cancer Genome Atlas. (E–G) Representative images (E–F) and statistical results (G) of CMTM6 and ATP11B immunohistochemical staining in pancreatic cancer tissue microarray. (H) Western blotting of ATP11B, CMTM6, and PD-L1 expression in paired clinical tissue samples. (I–M) Interaction of ATP11B with PD-L1 in pancreatic cancer in a CMTM6-dependent manner. (I−J) cell lysates from KPC and SW1990 cells were separately subjected to immunoprecipitation and western blotting using the indicated antibodies. (K) Western blotting of ATP11B expression in pancreatic cancer cell lines after CMTM6 knockdown. (L–M) Cell lysates from CMTM6-KD KPC (L) and CMTM6-KD SW1990 (M) separately subjected to immunoprecipitation and western blotting using the indicated antibodies. CMTM6, CKLF-like MARVEL transmembrane domain containing 6; PD-L1 programmed cell death ligand 1.

Article Snippet: D ow nloaded from 5Tang T, et al. J Immunother Cancer 2022;10:e004129. doi:10.1136/jitc-2021-004129 KPC, BXPC- 3, and SW1990 cells were stably transfected or co- transfected with human ATP11B Double Nickase Plasmid (sc- 411692- NIC, Santa Cruz), and ATP11B Double Nickase Plasmid (sc- 429296- NIC, Santa Cruz) and KPC and SW1990 were stably transfected with CMTM6 Double Nickase Plasmid (sc- 412381- NIC, Santa Cruz) and CMTM6 Double Nickase Plasmid (sc- 426447- NIC, Santa Cruz) according to the manufacturer’s instructions.

Techniques: In Vivo, Immunoprecipitation, Western Blot, Binding Assay, In Vitro, Purification, Incubation, Immunohistochemical staining, Staining, Microarray, Expressing, Knockdown