| Objective This study aimed to explore the expression, function and mechanism of miR-128-3p in HCC, and determine the correlation of miR-128-3p expression with clinicopathological factors.Methods In the present study, qRT-PCR was used to detect the expression of miR-128-3p in HCC tissues and cells, and then we further analyzed the correlation between miR-128-3p expression and clinicopathological factors and prognosis. In addition, miR-128-3p mimics and negative control (NC) were transfected into human HCC cell lines. Proliferation, colony formation, migration and cell cycle of the transfected cells were measured by CCK-8 assays, colony-forming assays, Transwell assays and flow cytometer, respectively. Mechanically, the potential target gene of miR-128-3p was predicted by bioinformatics, and the impact of miR-128-3p overexpression on its potential target gene and related signaling pathway in HCC cell were validated by qRT-PCR and Western blot. Furthermore, to further reveal the molecular mechanism of miR-128-3p in HCC, the potential target gene was detected in HCC tissues by qRT-PCR, and the correlation between the expression of target gene and the expression of miR-128-3p in HCC tissues was investigated.Results (1)The results of qRT-PCR in HCC tissues and cell lines showed that the expression of miR-128-3p was downregulated in 65.30%(47/72 aired) HCC samples compared to that in their matched controls (P<0.05), and was decreased in all HCC cell lines (QGY-7703, SK-hep1, QGY-7404, SMMC-7721, Huh7, HepG2) compared to HL-7702 (P<0.05).(2)The relationship between miR-128-3p expression and clinicopathological factors and prognosis was analyzed by statistical approach. We found that low expression of miR-128-3p strongly correlated with tumor size and TNM (P< 0.05), whilst not correlated with other factors such as age, HBV, portal vein tumor thrombus. Additionally, survival analysis demonstrated that Lower miR-128-3p expression in HCC tissues significantly correlated with shorter survival of HCC patients (P<0.05).(3)The effect of miR-128-3p on HCC cell biological function was observed. We found that the proliferation and colony formation of HCC cells transfected with miR-128-3p mimes was significantly inhibited compared with that of NC transfected cells. Cell cycle analysis of the transfected cells by FACS indicated that miR-128-3p overexepression can induce cell cycle arrest in GO/Gl phase (P <0.01). Moreover, Transwell assay displayed the number of migration of HCC cells with overexpression miR-128-3p was significantly fewer than that of NC. These results showed that miR-128-3p plays a tumor-suppressor role in HCC.(4)To elucidate the underlying molecular mechanism of miR-128-3p in HCC, the target gene of miR-128-3p was predicted by bioinformatics, and further validated by qRT-PCR and Western blot. The results prove that PIK3R1 is the most likely target gene. Furthermore, Phosphorylation of the essential molecules in the PI3K/AKT pathway were analyzed by Western blot. Our results showed that the protein level of p85a, the phosphorylation of AKT, mTOR, p70S6K were inhibited by miR-128-3p overexpression in HCC cell. Finally, to confirm the relevance of our findings in vitro, PIK3R1 mRNA expression was identified in the same 72 HCC samples too. The results of qRT-PCR revealed that PIK3R1 mRNA expression was upregulated in 68.06% (49 of 72) HCC, and Pearson’s correlation coefficient analysis showed an inverse correlation between PIK3R1 and miR-128-3p expression in HCC samples. This suggested that reduced miR-128-3p may weaken its effect on the inhibition of PIK3R1 expression in HCC patients.Conclusion miR-128-3p is commonly downregulated in HCC and is closely associated with prognosis of HCC patients. MiR-128-3p could also act as a tumor suppressor by silencing PIK3R1 to regulate PI3K/AKT signalling pathways, so that affecting HCC proliferation and metastasis. These results suggested that miR-128-3p could be a potential therapeutic target for HCC treatment. |
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