| BACKGROUND&OBJECTIVEHepatocellular carcinoma (HCC) is one of the common cancers in the world. Although with the constant improvement of the level of diagnosis and treatment, the curative effect of hepatocellular carcinoma patients increasingly significant, but the survival rate was not significantly improved, its main reason is that many HCC patients have been diagnosed with metastasis at beginning. Therefore, it is an urgent task to work out the metastasis-associated factors and find out the preventive and therapeutie methods.Tiaml (T lymphoma invasion and metastasis1) gene is one of nucleic acid Dbl family guanine conversion factor (guanine nucleotide exchange factor, GEFs), which regulate cytoskeletal reorganization through participation in multiple signals, including cell cycle progression, gene transcription, cell migration and cell adhesion and other life activities, and play an important role in tumor growth, invasion and metastasis. In our previous study, we first investigated Tiaml protein expression in213cases of hepatocellular carcinoma which have complete clinical follow-up data. We found that Tiaml was up-regulated in hepatoecllular carcinoma tissues, and had no expression in normal hepatic tissue, indicating Tiaml maybe regarded as the important marker for clinical detection of HCC; combined evaluation of Tiam protein high expression and clinieal follow-up data analysis showed that Tiaml had close relationship with prognosis and metastasis of hepatocellular carcinoma patients. On the basis of preliminary studies, we have carried out further experiments Tiaml functions in HCC. We found that Tiaml gene overexpression can promote the proliferation, migration and invasion of MHCC97L cell lines with low metastatic potential; we also observed that down-regulation of Tiaml can inhibit the proliferation, migration and invasion of HCCLM6cell lines with high metastatic potential in vitro, and reduced the proportion of S phase. The study results suggest that Tiaml is an important HCC metastasis-associated gene. However, regulatory mechanism of Tiaml expression in HCC metastasis is unclear.MicroRNAs (miRNAs) are a class of about22nt long, non-coding and single-stranded RNA molecules. Recent research findings have showed that they can combined with3’UTR of the target gene mRNA, resulting in target gene mRNA degradation or translational repression in the post-transcriptional level. Studies have indicated that miRNA involved in cell proliferation, apoptosis, differentiation, invasion, migration and angiogenesis generation of tumors by regulating the expression of oncogenes and/or tumor suppressor genes, and closely correlated with the occurrence, development and prognosis of cancers.In this study, we will explore the miRNAs which involved in the regulation of Tiaml gene in HCC. It will provide new clues to the molecular mechanism of Tiaml in HCC invasion and metastasis, and establish Tiaml and miRNAs as new target for early metastatic diagnostic markers and novel therapeutic strategies.METHOD1. Identification the miRNAs interacting Tiaml(1) Three classic databases including TargetScan, DIANA-microT and miRanda were used to forecast the miRNAs with search terms of Tiaml, miRNAs with high predictive values (at least predicted by three databases) were selected as candidate miRNAs;(2) In six HCC cell lines, RT-PCR was used to detect the expression of candidate miRNAs or Tiaml respectively;(3) Luciferase reporter system was used to validate the direct binding of Tiaml and the screened miRNAs;(4) RT-PCR or in situ hybridization method was used to detect the expression of miRNA in30fresh HCC tissues and212cases of hepatocellular carcinoma samples. Spearman correlation and clinical pathological analysis were then carried out.2. The function of target miRNAs in HCC invasion and metastasis(1) The target miRNA inhibitor was transfected transiently into MHCC97L cell lines; Western blot was performed out to detect the expression of Tiaml;(2) The targeted miRNA lentiviral vector was designed and established. After viral packaging, it was infected into HCCLM3cell lines. The stable miRNA-expressing HCC cell lines were obtained by limited dilution method, and then were used to detect the expression of Tiaml by Western blot;(3) CCK-8assay, would healing assay and in vitro invasion assay were carried out to detect cell proliferation and invasion abilities in vitro after transfection of targeted miRNA inhibitor.(4) CCK-8assay, would healing assay and in vitro invasion assay were carried out to detect cell proliferation and invasion abilities in vitro after transfection of targeted miRNA-overexpression vector;(5) In in vivo experiment, the effects of targeted miRNAs were observed on the growth of cutaneous tunlors and metastatic abilities in vivo.RESULT1. Identification the miRNAs interacting Tiaml(1) Results of three common databases showed that miR-141and miR-106b had the high predictive value;(2) RT-PCR results showed that in30HCC tissues, the expressions of miR-141and Tiaml had the opposite trend, but there was no significant correlation between miR-106b and Tiaml expression. Thus, miR-141may be the interacting miRNA of Tiaml;(3) Luciferase reporter system showed that the luciferase activity was decreased significantly in miR-141group compared to blank group(P=0.005) or NC group (P=0.014) after transfection of wild-type Tiaml psi-CHECK2plasmid. But the luciferase activity had no significant difference between miR-141inhibitor group and NC group or blank group (P=0.267, P=0.496). There was no significant difference of luciferase activity between miR-141group and blank group (P=0.446) after transfection of Mut Tiaml3’UTR plasmid. The above results indicate that miR-141inhibits the activity of luciferase through the binding sites of Tiaml3’UTR region;(4)In30fresh pair of HCC tissues, RT-PCR showed that the expression of miR-141in HCC tissues was significantly lower than normal liver tissues (P<0.01). In situ hybridization showed that miR-141is expressed mainly in the cell nucleus. According score, the total high expressive rate of miR-141in212cases of hepatocellular carcinoma was42.9%(91/212), low expression rate was58.2%(121/212). Spearman correlation analysis showed a significant negative correlation between Tiaml and miR-141in the212cases of HCC (r=-0.426, P<0.01); miR-141expression was significantly associated with metastasis (P=0.027), However, there was no correlation between miR-141and gender, age, tumor size, degree of differentiation, cirrhosis, recurrence, serum HBsAg, serum AFP (P>0.05). By follow-up analysis, hepatocellular carcinoma patients with low expression of miR-141had32.0months of median survival time; however, patients with high expression of miR-141had48.0months of median survival time, the survival time had signifieant difference between the two groups (P=0.002). Univariate analysis showed that low expression of miR-141, tumor size, metastasis, tissue differentiation and serum AFP value is an important factor affecting the survival time of patients with hepatocellular carcinoma (P=0.002; P=0.000; P=0.000; P=0.000; P=0.000; P=0.000) Multivariate analysis results showed that low expression of miR-141, tumor size, tumor grade, metastasis and serum AFP might play a role in predicting the overall survival in HCC patients (P=0.035; P=0.000; P=0.016; P=0.016; P=0.000).2. The effect of miR-141inhibitor on MHCC97L cell behaviors in vitro(1) RT-PCR results showed that the miR-141expression was significantly difference among the three groups (F=11.823, P=0.026); Western Blot results showed that the expression of Tiaml was significantly up-regulated after transfecting miR-141inhibitor into MHCC97L cells compared to NC group and blank group;(2)CCK-8assay showed that compared to blank or NC group, the proliferative abilities of MHCC97L cells were significantly enhanced in miR-141inhibitor group (P<0.01);(3) Would healing assay and cell invasion in vitro assay showed that compared to blank or NC group, cell migration abilitis of MHCC97L cells were significantly enhanced in miR-141inhibitor group, and it is time-dependent(F=102.07, p=0.000); Cell invasion capacity of MHCC97L cells transfected with miR-141inhibitor was also increased (F=174, P<0.01). The data indicated that inhibition of miR-141in MHCC97L cells was associated with enhanced migratory and invasive ability;3. The effect of miR-141overexpession on HCCLM3cell behaviors in vitro and in vivo(1) We successfully constructed miR-141overexpression lentiviral vectors with a green fluorescent protein reporter gene, and infected it into HCCLM3cell lines with high metastatic potential, which named M3/miR-141+, while the control cell lines named M3/mock, without any treatment of the cells named M3. Real-time RT-PCR results showed that compared with M3/mock and M3group, the expression of miR-141was significantly difference (F=6.379, P=0.037); Western Blot results showed that compared with M3/mock and M3group, Tiaml protein expression in M3/miR-141+group was significantly decreased;(2) CCK-8assay showed that compared with M3/mock and M3group, cell proliferation in M3/miR-141+group was significantly decreased (P<0.01);(3) Would healing assay and cell invasion in vitro assay showed that compared to blank or NC group, cell migration abilitis of MHCC97L cells were significantly enhanced in miR-141inhibitor group, and it is time-dependent (F=112.04, p=0.000); Compared with M3/mock and M3group, the invasive ability of M3/miR-141+cells was significantly decreased, the difference was statistically significant (F=11.10, p=0.002);(4) We observed the ability of proliferation and metastasis of liver cancer in vivo after stable expression of miR-141. The M3/mock cells and M3/miR-141+cells were inoculated subcutaneously in nude mice with bilateral. Through30consecutive days of observation, we found that the growth of subcutaneous tumors in nude mice in M3/miR-141+group were markedly reduced compared to M3/mock group (F=34.667, P=0.000), the decrease in M3/miR-141+cell growth starting from day15, up to1.6-fold decrease in mice of tumor volume at day30after cell injection.To unambiguously clucidate the declined effect of miR-141on HCC metastasis, we performed the metastasis assay in vivo through the lateral tail-vein injection. Mice were sacrificed2months later beeause the mice of M3/miR-141+group were moribund. In M3/miR-141+group, only8%(1/12) of animals had a small lung metastasis; but in M3/mock group,58.3%(7/12) of animals had lung metastasis. Metastases were not found in other organs and tissues of the animals.These data indicated that miR-141overexpression in liver M3cell was associated with decreasing migratory and invasive ability.Conclusion1. MiR-141is the novel target gene of Tiaml;2. MiR-141can inhibit the ability of proliferation, migration, invasion, metastasis of HCC cells by targeting Tiaml. |
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