Journal: Molecular Cancer

Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

doi: 10.1186/s12943-018-0843-8

Figure Lengend Snippet: Expression analysis of HULC, miR15a, PTEN in human liver cancer tissue. a (left&middle ) The representative analytic results of in situ hybridization for HULC and ( right ) immunohistochemistry staining for PTEN in human liver cancer tissue and their paired adjacent noncancerous tissues from the same patient (DAB stainning, original magnification× 100). b The representative analytic results of RT-PCR with HULC primers in liver cancer tissue ( c ) and its paracancerous liver tissues ( p ) respectively. β-actin as internal control. c The Real-time RT-PCR results of mature miR15a in liver cancer tissue ( c ) and its paracancerous liver tissues ( p ), respectively. U6 as internal control. d The representative analytic results of western blotting with anti-PTEN in liver cancer tissue ( c ) and its paracancerous liver tissues ( p ), respectively. β-actin as internal control

Article Snippet: Real-time RT-PCR-based detection of mature miR-15a and U6 snRNA was achieved with the miRNA Detection kit (including a universe primer, U6 primers, Qiagen) and miR15a specific upsteam primers (Origene, USA).

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: Molecular Cancer

Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

doi: 10.1186/s12943-018-0843-8

Figure Lengend Snippet: HULC inhibitsthe expression and mature miR15a. a ( left) RNA Immunoprecipitation (RIP) with anti-METTL3 followed by RT-PCR with pri-miR15a primers in Hep3B cell line. IgG RIP as negative control. RT-PCR for pri-miR15a as INPUT. ( right) quantitive RIP analysis. b ( left) RIP with anti-m6A followed by RT-PCR with pri-miR15a primers in liver cancer cells. IgG RIP as negative control. RT-PCR for pri-miR15a as INPUT. ( right) quantitative RIP analysis. c ( left) RIP with anti-DGCR8 followed by RT-PCR with pri-miR15a primers in Hep3B cell line. IgG RIP as negative control. RT-PCR for pri-miR15a as INPUT. ( right) quantitative RIP analysis. d ( left) RIP with anti-Droha followed by RT-PCR with pri-miR15a primers in liver cancer cells. IgG RIP as negative control. RT-PCR for pri-miR15a as INPUT. ( right) quantitive RIP analysis. e Super-EMSA (gel-shift) with biotin-pre-miR15a probe and anti-Exportin5 antibody. The intensity of the band was examined by Western blotting with anti-Bioton. HistoneH3 as internal control. f Biotin-pre-miR15a pulldown followed by Western blotting with anti-Dicer, anti-ago2 Biotin as INPUT and β-actin as internal control. g Northern blotting analysis of miR15a in liver cancer cell Hep3B cell lines. h The real-time PCR detection of mature miR15a in liver cancer cells. U6 as internal control. i The real-time PCR detection of mature miR15a in Hep3B cell lines infected with rLV and rLV-miR15a respectively. Each value was presented as mean ± standard error of the mean (SEM).**, P < 0.01. j Westernbloting with anti-P62, anti-Jun, anti-Notch, anti-PTEN, anti-mTOR. β-actin as internal control

Article Snippet: Real-time RT-PCR-based detection of mature miR-15a and U6 snRNA was achieved with the miRNA Detection kit (including a universe primer, U6 primers, Qiagen) and miR15a specific upsteam primers (Origene, USA).

Techniques: Expressing, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Electrophoretic Mobility Shift Assay, Western Blot, Northern Blot, Real-time Polymerase Chain Reaction, Infection

Journal: Molecular Cancer

Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

doi: 10.1186/s12943-018-0843-8

Figure Lengend Snippet: HULC promotes autophagy in liver cancer cells. a (left) Chromatin Immunoprecipitation (CHIP) with anti-RNApolII followed by PCR with LC3 promoter primers in Hep3B cell line. IgG CHIP as negative control. LG3 promoter as INPUT. ( right) quantitative CHIP analysis. b (left) RT-PCR with LC3 primer in Hep3B cell line. ( right) Real-time RT-PCR analysis. c (left) Western blotting with anti-LC3 in Hep3B cell line. β-actin as internal control. ( right) density analysis of band. d Western blotting with anti-sirt1 in Hep3B cell line. e Co-Immunoprecipitation (IP) with anti-Ac Ab followed by Western blotting with anti-LC3 in Hep3B cell line. IgG IP as negative control. INPUT refers to Western blotting with. Anti-LC3. f (left) Western blotting with anti-LC3 and anti-Sirt1 in Hep3B cell lines transfected with pcDNA3,pcDNA3-Sirt1,and pcDNA3-Sirt1 mutant respectively. ( right) density analysis of band. g (left) Western blotting with anti-LC3 and anti-Sirt1,and RT-PCR with HULC primers in Hep3B cell line. ( right) density analysis of band. h Thereal-time PCR detection of mature miR15a in liver cancer cells. i (left) Western blotting with anti-LC3 and RT-PCR with HULC primers in Hep3B cell line. ( right) density analysis of band

Article Snippet: Real-time RT-PCR-based detection of mature miR-15a and U6 snRNA was achieved with the miRNA Detection kit (including a universe primer, U6 primers, Qiagen) and miR15a specific upsteam primers (Origene, USA).

Techniques: Chromatin Immunoprecipitation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Transfection, Mutagenesis

Journal: Molecular Cancer

Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

doi: 10.1186/s12943-018-0843-8

Figure Lengend Snippet: HULC inhibits PTEN through ubiquitin–proteasome system mediated by autophagy-P62. a Western blotting with anti-PTEN and RT-PCR with PTEN primer in Hep3B cell line. β-actin as internal control. b Western blotting with anti-PTEN and RT-PCR with PTEN primer in Hep3B cell lines transfected with pCMV6-A-GFP plus pCMV6-XL5-PTEN (FL), pCMV6-A-GFP-HULC plus pCMV6-XL5-PTEN (FL),respectively. c (left) Co-Immunoprecipitation (IP) with anti-62 followed by Western blotting with anti-PTEN in Hep3B cell lines transfected with pCMV6 -A-GFP plus pCMV6 -XL5-PTEN (FL), pCMV6-A-GFP-HULC plus pCMV6 -XL5-PTEN (FL), pCMV6-A-GFP-HULC plus pGFP-V-RS--Sirt1 plus pCMV6-XL5-PTEN (FL), and pCMV6-A-GFP-HULC plus pLV-miR15a plus pCMV6-XL5-PTEN (FL), respectively. ( right) density analysis of band. d (left) Co-Immunoprecipitation (IP) with anti-HA followed by Western blotting with anti-PTEN in the Hep3B cell line. INPUT refers to Western blotting with. Anti-PTEN. ( right) density analysis of band. e (left) Co-Immunoprecipitation (IP) with anti-HA followed by Western blotting with anti-PTEN in the Hep3B cell line. ( right) density analysis of band. f (left) Cells were incubated with 50 μM MG132 (Sigma) for 6 h at proper after transfection. Western blotting with anti-PTEN in Hep3B cell lines transfected with pCMV6-A-GFP plus pCMV6-XL5-PTEN (FL). g . The assay of PTEN 3'-UTR luciferase activity in Hep3B cells infected with pCMV6-A-GFP or pCMV6-A-GFP-HULC

Article Snippet: Real-time RT-PCR-based detection of mature miR-15a and U6 snRNA was achieved with the miRNA Detection kit (including a universe primer, U6 primers, Qiagen) and miR15a specific upsteam primers (Origene, USA).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunoprecipitation, Incubation, Luciferase, Activity Assay, Infection

Journal: Molecular Cancer

Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

doi: 10.1186/s12943-018-0843-8

Figure Lengend Snippet: The schematic illustrates a model of Long noncoding RNA HULC promotes liver cancer cell growth by inhibition of PTEN via autophagy cooperation to miR15a. HULC decreases mature miR15a and inhibits P62 expression. Furthermore, HULC overexpression increased the expression of LC3I and LC3II (a autophagy marker) dependent on sirt1 (a deacetylase) Notably, HULC enhanced the interplay between LC3 and ATG3.Furthermore, HULC also increased the expression of becline-1(autophagy related gene). Therefore, HULC increased the cellular autophagy by activating sirt1, specifically, under starvation. Furthermore, HULC reduced the expression of PTEN, β-catenin and increased the expression of SAPK/JUNK,PKM2,CDK2, NOTCH1,C-Jun in Hep3B cells. Strikingly, HULC inhibited PTEN through ubiquitin–proteasome system mediated by autophagy-P62. Therefore, HULC activates AKT-PI3K-mTOR pathway via PTEN reduction in liver cancer cells

Article Snippet: Real-time RT-PCR-based detection of mature miR-15a and U6 snRNA was achieved with the miRNA Detection kit (including a universe primer, U6 primers, Qiagen) and miR15a specific upsteam primers (Origene, USA).

Techniques: Inhibition, Expressing, Over Expression, Marker, Histone Deacetylase Assay