The Effect Of UC001kfo On Invasion And Metastasis Of Human Hepatoma Cell Line HepG2

BackgroundHepatocellular carcinoma(HCC) is one of the high incidence of malignancy in the world, the mortality of liver cancer ranks third in the world, in our country, the situation is even grimmer, ranks the second in the deaths, more than half of the world, about11million people die from liver cancer per year, there have been more clinical methods for the treatment of liver cancer, mainly include surgical resection, interventional radiologist, radiotherapy and chemotherapy, radiofrequency ablation, liver transplantation, in which the efficacy of surgical are worldwide recognized, but most of the patients suffered from distant metastasis before diagnosed, losing the chance of operation, even radical resection, in five years there are still more than60%of patients occur recurrence and metastasis, the rate of recurrence and metastasis is higher. Yet to have a breakthrough in the field of treatment of liver cancer. Therefore, to targete invasion and metastasis of liver cancer is extremly significant to improve the survival time and the life quality of liver cancer patients.With the large-scale gene sequencing, it was found that less than2%of the human genome is transcribed into protein-coding RNA, more than98%of gene transcription is non-protein-coding RNA (noncoding RNA, ncRNA), based on the nucleotides that non-coding RNA contains, the length of more than200nt of ncRNA called long non-coding RNA (lncRNA), lncRNA has been considered to be a byproduct of the gene transcription with no biological function. However, recent studies indicate that lncRNA also takes a critical role in gene expression, and most of the known protein-coding genes have one or more corresponding lncRNAs. It provides a new perspective for us to understand the disease process, lncRNA has been a indispensability link.In the previous study, a highly expressed genes in HCC tissue named UC001kfo was found. UCOO1kfo is a new gene, isn’t included in pubmed database, only is interpreted as non-coding RNA in UCSC and Ensembl database. Target gene prediction results show that the target gene of UCOO1kfo is ACTA2which is the coding gene of a-SMA, and UC001kfo plays a role by regulatinga-SMA.a-SMA is highly conserved in the biological evolutionary, in different species the a-actin sequence is very close, the function is involved in cell motility, maintenance of cell morphology, constitute the cytoskeleton and is one of the main constituent of the cell pseudopodia. studies have shown that a-SMA is related with prognosis of patients and has been to considered as a mark of invasion or EMT. a-SMA is the direct Implementers of cell motility and metastasis.Recent research reveal that tumor occurrence and metastasis is a multi-step, multi-part process. At the molecular level it shows that Tumor signaling pathway is network-shaped distribution, therefor to prevent a single link can’t prevent the transmitting of tumor signaling pathway, the other nodes well compensates and replace this part of the function and eventually lead to the failure of the original treatment. a-SMA-targeting treatment can avoid the middle of the tumor intricate signaling processes and avoid the poor prognosis from the source.ObjectiveTo explore the impact on invasion and metastasis capacity of human hepatoma cell line HepG2and its effect on the regulation of a-SMA.Methods1. Design of siRNAsiRNA is designed by siRNA-designed tools according to sequences of UCOOlkfo, analyzed by BLAST to get rid of other genes which have homology sequence with uc001kfo, then performed RNA structural analysis software. At last gained three UC001kfo-siRNA, meanwhile, to design both negative control and Fam mark control.2. Transfect human hepatoma cell line HepG2Using liposomal transfection technology to Transfect UC001kfo-siRNA into HepG2, then undergo related experiments and testing after24to48hours after Transfected.3. Detection of target Respectively detect the expression of UCOOlkfo and a-SMA after RNA interference and perform correlation analysis; to detect the migration by Cell healing test, detect the invasion by the transwell test, to detect cell population dependency and the proliferation by Tablet cloning experiments and cell proliferation by MTT.4. Statistical analysisusing SPSS v18.0statistical software analysis, measurement data expressed as mean±standard deviation (x±s), between two groups was compared using t-test, Rate were compared using the chi-square test and correlation analysis between factors using Spearman correlation analysis.Results1. siRNA interference detection efficiencythree siRNAs were respectively transfected into HepG2, then dectect the expression of UC001kfo and select interference efficiency strongest siRNA, the interference efficiency is about65%.2. cell invasion and metastasisCell healing test show that the negative control and the siRNA group were respectively (17.23±0.28) μm and (10.45±0.53)μm after transfected48hours, the difference was statistically significant (P<0.01); transwell test results show that the siRNA group wearing the membrane cells (56±10) was significantly less than the negative control group (141±14)(P<0.01).3. detection of proliferative abilityClonal colony formation test results show that the clone formation rate of RNA interference group and negative control group respectively is (0.36+0.12) and (0.64±0.07), compared with the negative control, the RNA interference group is significantly lower(P<0.05). MTT assay shows the cell proliferation inhibition rate of RNA interference group is significantly higher than the negative control group, HepG2proliferation was significantly lower after interfered12h.4.RT-qPCR and related analysisRT-qRCR results show that the expression of siRNA group UCOOlkfo is0.35±0.22and a-SMA is0.44±0.19relative to the negative control group, both are statistically significant (P<0.05) compared with the negative control group, the correlation analysis showed that the expression between a-SMA and UC001kfo exists a positive correlation (r=0.868, p=0.001)Conclusions1. Using RNA interference technology to transfect the specific siRNA fragments into HepG2cells can successfully mediate UCOOlkfo silence, so as to achieve the effect of UC001kfo knockout. 2. HepG2migrate and invasion, colony formation and proliferation ability is significantly reduced after the UCOOlkfo is down-regulated.3. There are a good correlation between UCOOlkfo and a-SMA, and UCOOlkfo play the role of gene regulation through a-SMA.