Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3p and underlying mechanism.
OBJECTIVE
To investigate the mechanism by which miR-204-3p inhibits the growth of hepatocellular carcinoma (HCC) tumor endothelial cells (TECs).


METHODS
Flow cytometry was used to identify HCCTECs and analyze their purity. Differentially expressed miRNAs in HCC TECs as compared to normal hepatic sinusoidal endothelial cells (HSECs) were examined using the HmiOA v4 Human miRNA OneArray microarray. miR-204-3p showed the most significant decrease in expression and was further studied. Over-expression of miR-204-3p was achieved using lentiviral transduction into TECs of HCC. The biological changes in HCC TECs before and after transduction were detected using MTT and apoptosis assays. The association between miR-204-3p and fibronectin 1 (FN1) was determined using the dual luciferase activity assay. Changes in FN1 protein expression before and after transduction were detected using Western blot analysis.


RESULTS
Microarray results showed that compared to normal HSECs, 15 miRNAs were differentially expressed in HCC TECs, including 6 miRNAs with increased expression and 9 miRNAs with decreased expression. Among them, miR-204-3p showed the most significant decrease in expression (log2 = -1.233477, P = 0.000307). Over-expression of miR-204-3p in HCC TECs via lentiviral transduction significantly inhibited the proliferation of HCC TECs and promoted apoptosis. Results from the dual luciferase activity experiment showed that the luciferase intensity in the wild type FN1 group was significantly inhibited (P < 0.05), while that in the mutant FN1 group was not obviously affected. This observation indicated that FN1 was one of the potential targets of miR-204-3p. After over-expression of miR-204-3p in HCC TECs, Western blot analysis showed that the expression of FN1 protein was significantly inhibited.


CONCLUSIONS
MiR-204-3p acts on its potential target gene, FN1, and inhibits its expression, thus blocking the adhesion function of FN1 in promoting the growth of TECs.
Zhong-Hui Cui, Shi-Qiang Shen, Zu-Bing Chen, Chao Hu, Department of General Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China.
Zhong-Hui
ZH
Cui
Zhong-Hui Cui, Shi-Qiang Shen, Zu-Bing Chen, Chao Hu, Department of General Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China.
Shi-Qiang
SQ
Shen
Zhong-Hui Cui, Shi-Qiang Shen, Zu-Bing Chen, Chao Hu, Department of General Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China.
Zu-Bing
ZB
Chen
Zhong-Hui Cui, Shi-Qiang Shen, Zu-Bing Chen, Chao Hu, Department of General Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China.
Chao
C
Hu
D015703:Antigens, CD / Q000378:metabolism; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000098:blood supply* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D048430:Cell Shape; D016000:Cluster Analysis; D000071063:Endoglin; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000098:blood supply* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection
D015703:Antigens, CD; D014408:Biomarkers, Tumor; C064206:ENG protein, human; D000071063:Endoglin; C067625:FN1 protein, human; D005353:Fibronectins; C549404:MIRN204 microRNA, human; D035683:MicroRNAs; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011956:Receptors, Cell Surface
Fibronectin 1; Hepatic sinusoidal endothelial cells; MiRNA microarray; Mir-204-3p; Tumor vascular endothelial cells of hepatocellular carcinoma