| Hepatopoietin Cn(HPPCn) was isolated and identified by our laboratory as a novel cytokine specifically promoting the proliferation of hepatocytes. HPPCn upregulates the DNA synthesis and cell division of hepatocytes in vitro, and promotes liver regeneration in mouse model with 70% partial hepatectomy. It has been confirmed in our previous research that HPPCn protects liver fibrosis induced by CCl4 in rat model. The current study investigates the protective effects of HPPCn on ethanol-induced chronic liver injury as well as the regulatory relationship between HPPCn and micro RNA(mi RNA, mi R) in the metastasis of hepatocellular carcinoma(HCC), elucidating the role of HPPCn-mi RNA interaction in HCC metastasis. 1. HPPCn protects liver from chronic injury via sphingosine kinase(Sph K)pathway activation. China suffers from various types of liver diseases widely with increasing incidences of liver fibrosis and cirrhosis caused by excessive alcohol consumption. Long term alcohol exposure also induces epigenetic alteration, resulting in the transition of liver cells into cells with properties of stem cells or progenitors, which is associated with the development of HCC. Therefore, it is of great social significance to investigate the intervention targets and therapeutic strategies for alcoholic liver disease(ALD). Our previous studies revealed the protective effects of recombinant human HPPCn protein(rh HPPCn) on ethanol-induced acute liver injury in mouse model. We found that HPPCn protected liver from oxidative stress and lipid peroxidation caused by ethanol. In addition, it has been reported recently that sphingosine kinase(Sph K)/sphingosine 1-phosphate(S1P)/S1 P receptors(S1PRs) signalling axis plays a role in liver injury and fibrosis. Our researches showed that HPPCn could activate Sph K signalling pathway in hepatocytes. There are two isoforms of Sph K, Sph K1 and Sph K2. In our experiments, the therapeutic effects of Sph K1 gene delivery was comparable to that of rh HPPCn administration in a mouse short term ethanol feeding model. These studies indicate that Sph K1/S1P/S1 PRs may mediate the protective effects of HPPCn against ethanol-induced liver oxidative injury. On the basis of previous works, the first objective of the present study is to investigate the protective effects of HPPCn on chronic liver injury in a transgenic mice(HPPCnliver+/+) model with liver-specific HPPCn overexpression and verify the contribution of Sph K1/S1P/S1 PRs signalling axis in the protective effects of HPPCn. The long term ethanol feeding model was established using male FVB HPPCnliver+/+ mice, with wide-type mice as control. The protective effects of HPPCn on ethanol induced liver injury were examined by the detection of alanine transaminase(ALT) release in serum and the histopathological observation of liver tissue. The content of malondialdehyde(MDA) in liver was analysed to illustrate the hepatic lipid peroxidation. Hepatic collagen deposition, total hydroxyproline(Hyp) as well as the expression of relevant genes were studied to evaluate the extent of liver fibrosis. Our results showed that HPPCn was overexpressed specifically in the liver of HPPCn transgenic mouse. Compared with WT control, HPPCn overexpression in liver prevented ALT release, pathological alteration and MDA production, indicating the protective effects of HPPCn on chronic liver injury induced by ethanol. Moreover, HPPCn transgenic mice exhibited alleviated liver fibrosis after long term ethanol feeding, with less collagen deposition, Hyp content and expression of smooth muscle α-actin(α-SMA) and pro-collagen α1(I) [Colα1(I)]. Subsequently, we analysed the expression of Sph K1/S1P/S1 PRs signalling axis. Our results showed that ethanol feeding elevated the activity of hepatic Sph K and S1 P expression in HPPCn transgenic mice but not in WT mice. Compared with Con-fed counterpart and WT mice, the expression of Sph K1 in HPPCn transgenic mice was promoted on both early and advanced stage of ethanol feeding, while the expression of Sph K2 remained unchanged. The variation of Sph K1 in WT mice after ethanol feeding presented little statistical significance. These results indicated that the increased Sph K activity and S1 P level was associated with HPPCn overexpression. Additionally, S1 PRs expression detection showed that after 3-day ethanol feeding, the relative m RNA expression of S1PR3 was promoted in HPPCn transgenic mice. After 8-week ethanol feeding, the relative m RNA expression of S1PR2 in WT mice was downregulated while that in HPPCn transgenic mice was upregulated significantly. These results suggested that HPPCn activated different S1 PRs in acute and chronic liver injury. In addition, our results demonstrated that HPPCn promoted the phosphorylation of Erk1/2 pathway in hepatocytes via Sph K activation, depending on S1PR3. 2. Mi R-17-92 is involved in the HCC growth and metastasis induced by HPPCn. In our previous research on HCC therapy, we found that when challenged with DEN to induce HCC, the liver-specific overexpression of HPPCn in HPPCn transgenic mice upregulated the expression of oncogenic c-myc, which might be responsible for the increased lung metastasis incidence of HCC. It has been reported that mi RNAs which is a type of non-coding small molecule single-strain RNA plays an important role in the oncogenesis and development of HCC. And a number of mi RNA clusters also exhibit expression abnormality in liver cancer cells and thus contribute to oncogenesis and tumour progression by regulating important signalling pathways such as P53, PTEN, c-myc, making them potential targets for cancer therapy. The abnormal expression of the highly conserved mi R-17-92 has been reported recently to be associated with the development and metastasis of various types of malignant cancers, but the regulatory effect of this gene cluster on HCC biology and the underlying mechanisms remain elucidative. We hypothesize that there is interactivity among HPPCn, c-myc and mi R-17-92. Therefore, the second objective of this study focuses on the regulatory effect of HPPCn/c-myc/mi R-17-92 in the context of HCC growth and metastasis, analysing the rationality of using this pathway as a target for HCC therapy. In this regard, we detected the differential expression profile of mi RNA in HCC tissues of HPPCn transgenic mice. The results showed that compared with WT control, the expression of mi R-17-92 was significantly upregulated in HPPCn transgenic mice. We then examined the expression of HPPCn/c-myc/mi R-17-92 in HCC cell lines with different metastasis potential as well as in HCC tissues from patients to investigate the regulatory relationship among them. Their relationship were further verified in HCC cells treated with rh HPPCn or RNAi targeting HPPCn and c-myc respectively. Lentivirus vectors were deployed to establish a HCC cell line with stable mi R-17-92 overexpression, which was used in subcutaneous tumour baring model and splenic injection metastasis model to investigate the effects of mi R-17-92 on the proliferation and metastasis of HCC. Next, we established a mi R-17-92 cluster knockout mouse model(mi R-17-92Δ/Δ) to study its role in the induced HCC oncogenesis. Our results demonstrated that the expression of mi R-17-92 was positively related to the metastasis potential of HCC cells as well as the expression of HPPCn and c-myc. We found that HPPCn knockdown resulted in the downregulation of mi R-19 a, mi R-20 and mi R-92 in HCC cells; while rh HPPCn treatment upregulated c-myc and mi R-17-92 cluster. And c-myc knockdown diminished the promoting effect of HPPCn on mi R-17-92 expression. Clinical samples from patients with HCC also presented the significant differential expression of mi R-17-92 between cancerous and paracarcinoma tissue, which was associated with the expression of HPPCn and c-myc. In clinical tumour tissues, we observed significant differential expression of mi R-17-92 between cancerous and paracarcinoma lesions, which was correlated to the expression of HPPCn and c-myc. These results suggest that HPPCn inhibits the expression of targets genes of mi R-17-92 via elevating the expression of this cluster. Although our research did not demonstrate clearly the impact of mi R-17-92 overexpression mediated by lentivirus vectors on the proliferation of HCC cells, we verified that mi R-17-92 overexpression promoted the migration and metastasis of HCC cells in vitro and in vivo. In our splenic injection metastasis model, we observed obvious more incidences of liver, lung and abdomen abdominal cavity metastasis of HCC in mi R-17-92 overexpression group. In the induced HCC oncogenesis model, we found that mi R-17-92 knockout downregulated the expression of oncogenic c-mys and HCC marker alpha-fetoprotein(AFP). Our research demonstrate that HPPCn regulates mi R-17-92 and its downstream target genes via c-myc, and HPPCn/c-myc/mi R-17-92 signalling system is vital to the metastasis of HCC, making this system a potential target in HCC therapy. |
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