Screening And Identification Of Metastasis-associated MicroRNAs In Human Hepatocellular Carcinoma

Background and aim:miRNA is a kind of short non-code small RNA, which plays inhibition after transcription through binding mRNA 3'UTR region, and then regulate cellular apoptosis, differentiation, metabolism, motility and participate in each step of tumorigenesis. In recent years, some studies confirm that the differential expression of microRNA is related with metastasis, Responsiveness of drug treatment and prognosis in liver cancer. This study focus on investigating the differential expressed miRNAs in invasive-metastasis cascade of HCC and exploring the biological function of miR-612 in human liver cancer metastasis, in order to help us to further understand the molecular mechanism of liver cancer, and offer clues for personalized treatment of liver cancer.Methods:miRNAs microarray was used to detect microRNA expression between Primary liver cancer foci and lung metastasis. According to the differences multiples of SAM, screen the special differential miRNAs spectrum in liver cancer intrusion and metastasis chain, and then utilize bioinformatics analysis to construct biological events regulatory network of differential expressional miRNAs; Using Poly (A) real-time PCR to detect the expressional level of miRNAs; Using real-time PCR and Western blot to detect the level of mRNA and protein of gene expression; Using Trans-well and BD Matrigel to observe cellular mobility and invasion ability; Using bioinformatics analysis to forecast the target genes of miR612 and conduct function enrichment analysis of signal pathway; construction Dual-Luciferase reporter vector to directly verify the target gene of miR612. miRIDINA mimic, hairpin inhibitor were employed and transient transfected cell to overexpress and seal up the miR612. AKT2 selective inhibitor VIII was used to seal up miR612 so as to block the activation of downstream signal pathway. Use nude mouse model by tail vein injection to observe the lung metastasis ability of tumor.Result:1) There are 32 differential expressional miRNAs in liver cancer invasive-metastasis cascade,15 miRNAs up-regulated and 17down-regulated; Through cluster analysis, the 32 differential expressional miRNAs could be classified into two unique expression profile:primary lesion and metastatic lesion; the 32 differential expressional miRNAs mainly take part in 18 biological events via acting on their target genes.2) The expression levels of miR612 among HCCLM3, MHCC97H, MHCC97L, HepG2 and SMMC-7721 presents an gradually heighten trend, which in SMMC-7721 is 15times as much as in HCCLM3; the level of miR612 shows relation of inverse proportion.3) The levels of miR612 among HCCLM3, MHCC97H and MHCC97L display relation of inverse proportion with migration and intrusion ability; among different subclones of HCCLM3, the levels of miR612 show significant difference, what's more is that this difference negatively correlate with migration and intrusion ability of different subclones.4) miRIDINA mimics transient transfection can markedly inhibit migration and intrusion ability of HCCLM3 and HepG2 cells, while hairpin inhibitor by sealing up the expression of miR612 in the above-mentioned cells after 72 hours can significantly increase their migration and intrusion ability.5) Enrichment bioinformatics Analysis of miR612 target genes and signal pathways by using miRanda, TargetScan and mirarget2 reveals AKT2 may be the important target gene for miR612 to produce biological effect.6) Western blot results show reverse relation between miR612 and AKT2 level in HCCLM3, MHCC97H, MHCC97L, HepG2 and SMMC-7721. Overexpressing miR612 can strikingly reduce the AKT2 protein level in HCCLM3 and HepG2 cells, whereas, sealing up miR612 could generate opposite result; which indirectly validates that AKT2 is the functional target gene of miR612.7) using AKT2 selective depressant can obviously reverse the increase of cellular migration and intrusion ability of HCCLM3 and HepG2 caused by miR612 inhibitor, which suggest "AKT2 disabled" may play a similar function as endogenous miR612, and further check the prediction that AKT2 is the functional target gene of miR612.8) After transfecting Dual-Luciferase reporter into HCCLM3 and SMMC-7721, Firefly luciferase activity of HCCLM3 was significantly higher than SMMC-7721. Co-transfection of Dual-Luciferase reporter and miRIDINA mimic was able to obviously lower the luciferase activity, on the contrary, Co-transfecting Dual-Luciferase reporter and miR612 inhibitor was capable of increasing luciferase activity in SMMC-7721, which further verified AKT2 is the target gene of miR612.9) The result of nude mouse model through tail veil injection exactly match experiments in vitro, Overexpressing miR612 notably suppress the formation of lung metastases in nude mouse, the result was just the opposite if sealing up miR612,which indicate miR612 can restrain the experimental metastasis in vivo of human liver cancer.10) Down-regulating the miR612 level led to spindle-like change of HCCLM3, HepG2 and SMMC-7721, however, over-expressing the miR612 level resulted in cobblestone-like change.11) The results of qRT-PCR, Cellular immune fluorescence and WB revealed that morphologic changes of hepatoma cells caused by miR612 down-regulation was relative to E-cad down-regulation and Vimentin up-regulation, vice versa. It indicated miR612 down-regulation induced EMT of hepatoma cells.12) After AKT selective depressant VIII block the activation of AKT2, The results of qRT-PCR, Cellular immune fluorescence and WB revealed that VIII could obviously reverse E-cad down-regulation and Vimentin up-regulation, which prompted miR612 regulated the EMT generation of hepatoma cells via AKT2.13) After differential-expressing miR612 (up-regulation/down-regulation), the results of qRT-PCR suggested SIP1 (ZEB2) standed out from the crowd among Twist, Slug, Snail and ZEB1/2 which were transcription factor participated in modifying EMT regulation. WB further confirmed that sealing up miR612 could significantly up-regulate the level of SIP1, while over-expressing miR612 as well as VIII could down-regulate the protein level of SIP1. It indicated AKT2-SIP1 signal pathway took part in regulation of liver cancer by miR612.Conclusion:In present study, we demonstrate that multiple miRNAs are differential expressed in invasive-metastasis cascade of HCC. There miRNAs affect the biological function of HCC cells and involve in HCC metastasis through negative regulating protein coding genes (PCGs). This study firstly clarified the function of miR612 which participated in the regulation of tumor metastasis, suggesting miR612 would be a key molecule and would be proven as a clinically effective target for intervening in tumor metastasis.