Journal: Hepatology Communications

Article Title: NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

doi: 10.1002/hep4.2019

Figure Lengend Snippet: Efficient down‐regulation of NPRL2 significantly increased the activity of the rag GTPases via the mammalian target of rapamycin (mTOR) pathway and inhibited autophagy in hepatocellular carcinoma (HCC) cells. (A) The protein expression levels of NPRL2, NPRL3, and DEPDC5 were relatively high and similar in HepG2, Hep3B, and Huh7 cell lines, as determined by western blotting ( n = 3). (B) NPRL2 down‐regulation was successfully performed and significantly decreased the NPRL2 protein expression in HepG2, Hep3B, and Hul7 cells using the small interfering RNA (siRNA) system. The NPRL2 protein expression significantly reduced in HepG2 cells compared with that in Hep3B and Huh7 cells. (C) We performed knocked down NPRL2, NPRL3, and DEPDC5 in the HepG2 cells using the siRNA system. The siRNA sequences successfully and significantly reduced the expression of NPRL2, NPRL3, and DEPDC5 in the HepG2 cells with NPRL2, NPRL3, and DEPDC5 down‐regulation compared to those with NPRL3 and DEPDC5 down‐regulation. The protein expression of NPRL2 and NPRL3 in the HepG2 was significantly decreased in the HepG2 cells with NPRL2 down‐regulation compared to those with NPRL3 and DEPDC5 down‐regulation. (D) The protein expression of NPRL2, NPRL3, and DEPDC5 was reduced but similar to that following the NPRL2, NPRL3, and DEPDC5 down‐regulation in the Hep3B cells. (E) We knocked down NPRL2 in HepG2 cells using the short hairpin RNA (shRNA) systems. NPRL2, NPRL3, and DEPDC5 protein expression was significantly reduced in the HepG2 cells (lines 1 and 2). (F) Activation of rag a, rag C, mTOR, and 4E‐binding protein (4E‐BP1) but not p‐S6K in the HepG2 cells with NPRL2 knockdown ( n = 3). (G) LC3‐II protein expression significantly decreased and p62 protein expression significantly increased in the HepG2 cells with NPRL2 knockdown ( n = 3). GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; KD, knockdown.

Article Snippet: The membranes were blocked with BlockPro 1 min protein‐free blocking buffer and incubated with the following primary antibodies at 4°C overnight: anti‐NPRL2 (sc‐376986; Sigma‐Aldrich), anti‐NPRL3 (ab121346; Abcam, Cambridge, MA), anti‐DEPDC5 (GTX133570; GeneTex), anti‐Rag A (D8B5; Cell Signaling Technology), anti‐Rag C (D8H5; Cell Signaling Technology), anti‐phospho‐mTOR (Ser2448; Cell Signaling Technology), anti‐phospho–4E‐binding protein (4E‐BP1) (Thr37/46; 2855; Cell Signaling Technology), anti‐phospho‐S6K (Cell Signaling Technology), anti‐LC3 (NB‐100‐2220; Novus Biologicals), anti‐p62 (H0008878‐M01; Abnova), and anti‐GAPDH (NB‐300‐221; Novus Biologicals) antibody.

Techniques: Activity Assay, Expressing, Western Blot, Small Interfering RNA, shRNA, Activation Assay, Binding Assay

Journal: Hepatology Communications

Article Title: NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

doi: 10.1002/hep4.2019

Figure Lengend Snippet: Knockdown of NPRL2 significantly promoted the proliferation, migration, and colony formation in HCC cells. (A) NPRL2 down‐regulation of HepG2 cells by the shRNA system significantly increased the cell proliferation rate compared with that in the vehicle group on days 3, 6, and 9. Cell proliferation increased up to 6‐fold on day 9. The growth curves and their derivatives were determined by MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) assay ( n = 3). (B) The migration of HepG2 cells significantly increased after NPRL2 down‐regulation by shRNA for 72 h (scale bar: 200 μm; n = 3). (C,D) HepG2 cells with NPRL2 down‐regulation by the shRNA system exhibited 3‐fold to 4‐fold increased colony formation ability compared with that in the vehicle group ( n = 3). (E–H) NPRL2, NPRL3, and DEPDC5 down‐regulation in the hep G2 cells by the siRNA system significantly increased the cell proliferation, migration, and colony formation ability compared with that in the vehicle group ( n = 3). The cell proliferation, migration, and colony formation were significantly increased by siRNA‐mediated NPRL2 down‐regulation compared with those following the NPRL3 and DEPDC5 down‐regulation in the HepG2 cells. (I–L) NPRL2, NPRL3, and DEPDC5 down‐regulation in the Hep3B cells by the siRNA system significantly promoted the cell proliferation, migration, and colony formation ability compared with that in the vehicle group ( n = 3). The cell proliferation, migration, and colony formation were significantly increased by the siRNA‐mediated NPRL2 down‐regulation compared with those following the NPRL3 and DEPDC5 down‐regulation in the Hep3B cells. Statistical analysis was performed by one‐way analysis of variance (ANOVA). * p < 0.05; ** p < 0.01 compared with the counterpart group.

Article Snippet: The membranes were blocked with BlockPro 1 min protein‐free blocking buffer and incubated with the following primary antibodies at 4°C overnight: anti‐NPRL2 (sc‐376986; Sigma‐Aldrich), anti‐NPRL3 (ab121346; Abcam, Cambridge, MA), anti‐DEPDC5 (GTX133570; GeneTex), anti‐Rag A (D8B5; Cell Signaling Technology), anti‐Rag C (D8H5; Cell Signaling Technology), anti‐phospho‐mTOR (Ser2448; Cell Signaling Technology), anti‐phospho–4E‐binding protein (4E‐BP1) (Thr37/46; 2855; Cell Signaling Technology), anti‐phospho‐S6K (Cell Signaling Technology), anti‐LC3 (NB‐100‐2220; Novus Biologicals), anti‐p62 (H0008878‐M01; Abnova), and anti‐GAPDH (NB‐300‐221; Novus Biologicals) antibody.

Techniques: Migration, shRNA

Journal: Hepatology Communications

Article Title: NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

doi: 10.1002/hep4.2019

Figure Lengend Snippet: Knockdown of NPRL2 in human HepG2 cells promoted tumor growth in vivo . (A) HepG2 cells with or without NPRL2 KD were subcutaneously inoculated into BALB/c nude mice. The images show mice with transplanted tumors from the indicated groups at the end of the study. (B) In the NPRL2 down‐regulation group compared with the counterpart group, the tumor growth curve was significantly increased on days 21–49, and the tumor volume was increased by up to 8‐fold on Day 30. There were 4 and 8 mice in the vehicle group and NPRL2 KD group, respectively. (C) Transplanted tumors from the indicated groups. (D) The tumor weight in the NPRL2 down‐regulation group was significantly increased by up to 3‐fold of that in the vehicle group at the end of the study. (E) Hematoxylin and eosin staining of the transplanted tumors in both groups indicated malignant tumors. (F–H) in the orthotopic xenograft mouse model, HepG2 cells with or without NPRL2 KD were implanted into the liver of BALB/c nude mice ( n = 4). The images show transplanted tumors from the indicated groups. The tumor diameter (0.8 ± 0.2 cm vs. 2.3 ± 0.4 cm, p < 0.001) and intrahepatic metastasis number (0.25 ± 0.5 vs. 13 ± 3.5, p < 0.001) were significantly increased in the NPRL2 down‐regulation group compared with those in the vehicle group at 3 weeks. (I) The images show NPRL2, NPRL3, DEPDC5, rag a, rag C, mTOR, 4EBP1, LC3, and p62 in the immunohistochemical (IHC) staining of transplanted tumors from the indicated groups (upper and low panel, ×20). (J) Quantification of the IHC staining results revealed that the protein expression levels of NPRL2, NPRL3, DEPDC5, and LC3 were significantly lower and those of rag a, rag C, mTOR, 4E‐BP1, and p62 were significantly higher in the NPRL2 KD group than in the counterpart group. Data are shown as number (%). All data are presented as the mean ± SEM (* p < 0.05; ** p < 0.01).

Article Snippet: The membranes were blocked with BlockPro 1 min protein‐free blocking buffer and incubated with the following primary antibodies at 4°C overnight: anti‐NPRL2 (sc‐376986; Sigma‐Aldrich), anti‐NPRL3 (ab121346; Abcam, Cambridge, MA), anti‐DEPDC5 (GTX133570; GeneTex), anti‐Rag A (D8B5; Cell Signaling Technology), anti‐Rag C (D8H5; Cell Signaling Technology), anti‐phospho‐mTOR (Ser2448; Cell Signaling Technology), anti‐phospho–4E‐binding protein (4E‐BP1) (Thr37/46; 2855; Cell Signaling Technology), anti‐phospho‐S6K (Cell Signaling Technology), anti‐LC3 (NB‐100‐2220; Novus Biologicals), anti‐p62 (H0008878‐M01; Abnova), and anti‐GAPDH (NB‐300‐221; Novus Biologicals) antibody.

Techniques: In Vivo, Staining, Immunohistochemical staining, Immunohistochemistry, Expressing

Journal: Hepatology Communications

Article Title: NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

doi: 10.1002/hep4.2019

Figure Lengend Snippet: NPRL2, NPRL3, DEPDC5, LC3, p62, and mTOR were predictive factors for disease‐free survival (DFS) and overall survival (OS) in patients with HCC after surgical resection. (A) Among 300 patients with HCC, 79 exhibited recurrence and 62 experienced mortality. Low NPRL2, NPRL3, and DEPDC5 protein expression in the tumors was significantly associated with higher recurrence and mortality ( p < 0.05). Data are shown as numbers (%). (B) In patients, low NPRL2, NPRL3, and DEPDC5 protein expression was significantly associated with worse DFS as revealed by Kaplan–Meier analysis ( p < 0.05). (C) Low NPRL2, NPRL3, and DEPDC5 protein expression in patients was significantly associated with worse OS, as determined by Kaplan–Meier analysis ( p < 0.05). (D) Low LC3, high p62, and high mTOR protein expression in patients was significantly associated with worse DFS according to Kaplan–Meier analysis ( p < 0.05). (E) In patients, low LC3, high p62, and high mTOR protein expression was significantly associated with worse OS as determined by Kaplan–Meier analysis ( p < 0.05).

Article Snippet: The membranes were blocked with BlockPro 1 min protein‐free blocking buffer and incubated with the following primary antibodies at 4°C overnight: anti‐NPRL2 (sc‐376986; Sigma‐Aldrich), anti‐NPRL3 (ab121346; Abcam, Cambridge, MA), anti‐DEPDC5 (GTX133570; GeneTex), anti‐Rag A (D8B5; Cell Signaling Technology), anti‐Rag C (D8H5; Cell Signaling Technology), anti‐phospho‐mTOR (Ser2448; Cell Signaling Technology), anti‐phospho–4E‐binding protein (4E‐BP1) (Thr37/46; 2855; Cell Signaling Technology), anti‐phospho‐S6K (Cell Signaling Technology), anti‐LC3 (NB‐100‐2220; Novus Biologicals), anti‐p62 (H0008878‐M01; Abnova), and anti‐GAPDH (NB‐300‐221; Novus Biologicals) antibody.

Techniques: Expressing

Journal: Hepatology Communications

Article Title: NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

doi: 10.1002/hep4.2019

Figure Lengend Snippet: Univariate analyses of factors associated with recurrence and mortality

Article Snippet: The membranes were blocked with BlockPro 1 min protein‐free blocking buffer and incubated with the following primary antibodies at 4°C overnight: anti‐NPRL2 (sc‐376986; Sigma‐Aldrich), anti‐NPRL3 (ab121346; Abcam, Cambridge, MA), anti‐DEPDC5 (GTX133570; GeneTex), anti‐Rag A (D8B5; Cell Signaling Technology), anti‐Rag C (D8H5; Cell Signaling Technology), anti‐phospho‐mTOR (Ser2448; Cell Signaling Technology), anti‐phospho–4E‐binding protein (4E‐BP1) (Thr37/46; 2855; Cell Signaling Technology), anti‐phospho‐S6K (Cell Signaling Technology), anti‐LC3 (NB‐100‐2220; Novus Biologicals), anti‐p62 (H0008878‐M01; Abnova), and anti‐GAPDH (NB‐300‐221; Novus Biologicals) antibody.

Techniques:

Journal: Hepatology Communications

Article Title: NPRL2 down‐regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression

doi: 10.1002/hep4.2019

Figure Lengend Snippet: Multivariate analyses of factors associated with recurrence and mortality

Article Snippet: The membranes were blocked with BlockPro 1 min protein‐free blocking buffer and incubated with the following primary antibodies at 4°C overnight: anti‐NPRL2 (sc‐376986; Sigma‐Aldrich), anti‐NPRL3 (ab121346; Abcam, Cambridge, MA), anti‐DEPDC5 (GTX133570; GeneTex), anti‐Rag A (D8B5; Cell Signaling Technology), anti‐Rag C (D8H5; Cell Signaling Technology), anti‐phospho‐mTOR (Ser2448; Cell Signaling Technology), anti‐phospho–4E‐binding protein (4E‐BP1) (Thr37/46; 2855; Cell Signaling Technology), anti‐phospho‐S6K (Cell Signaling Technology), anti‐LC3 (NB‐100‐2220; Novus Biologicals), anti‐p62 (H0008878‐M01; Abnova), and anti‐GAPDH (NB‐300‐221; Novus Biologicals) antibody.

Techniques: