Mmp10

The hBMECs transfected with pcDNA3.1-lncRSPH9-4, si-lncRSPH9-4, or miR-17-5p mimics, or miR-17-5p inhibitors were lysed in RIPA buffer with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), sonicated and centrifuged at 10,000 g for 10 min at 4 °C. The insoluble debris was removed and protein concentration in the supernatant was measured using the BCA protein assay kit (NCM Biotech, Suzhou, China). The protein samples were then separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked in 5% BSA in Tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature and then incubated overnight with primary antibodies against ZO-1, Occludin, Claudin-5, MMP3 (Abcam, Cambridge, MA, USA), MMP1, β-actin (Proteintech, Chicago, IL, USA), or MMP10 (ABclonal, Wuhan, China). The blots were subsequently washed and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Biodragon, Beijing, China) at 37 °C for 1 h. The blots were visualized with the Super electrochemiluminescence (ECL) Prime kit (US Everbright, Suzhou, China) and were densitometrically analyzed using the Image Lab software (Bio-Rad, Hercules, CA, USA).

HCC samples and the corresponding adjacent nontumour liver tissues were collected from adult patients with HCC who underwent curative resection at the Tongji Hospital of Tongji Medical College (Wuhan, China). A preoperative clinical diagnosis of HCC was based on the diagnostic criteria of the American Association for the Study of Liver Diseases and haematoxylin & eosin staining of the samples was also performed. All of the samples were selected with distinctive pathologic diagnosis and none of the patients received any preoperative chemotherapy or radiotherapy. These tissues were stained for E‐cadherin (Cell Signaling Technology, 3195), MMP10 (Abclonal, A3033), P4HA2 (Abcam, ab233197), ITGA5 (Abcam, ab150361), MMP9 (Cell Signaling Technology, 13667), MT1X (Proteintech, 17172‐1‐AP) and SPP1 (Abclonal, A1499) expression. Furthermore, negative control for IHC staining was shown in Figure S8. IHC staining was performed using the Dako Envision Plus System (Dako) according to the manufacturer's instructions. The IHC staining intensity was scored as 0 (negative); 1 (weak); 2 (medium); 3 (strong). The percentage of positive cells was scored from 0 to 4 (0%, 1%‐25%, 26%‐50%, 51%‐75%, 76%‐100%). Overall score ranging from 0 to 12 was calculated by multiplying the above two scores, resulting in a negative (0‐3) staining or a positive (4‐12) staining for each example.