| Hepatocellular carcinoma is one of the most common fatal diseases in the world,and its five-year survival rate is only 7%.The onset of HCC is insidious,and early diagnosis is difficult.About 90% of patients are diagnosed as liver cancer in the middle and late stage,and they lose the opportunity of surgery,and the prognosis is very poor.Despite the development of specific treatment strategies,the prognosis of HCC is still poor.Conventional chemotherapy drug treatment not only has great toxic and side effects,but also can not significantly alleviate the disease progression or prolong the life of patients.At present,it is urgent to study the pathogenesis and key regulatory factors of HCC in order to improve the early diagnosis of HCC and develop new therapeutic drugs for liver cancer,improve the therapeutic efficacy of liver cancer,delay tumor recurrence and metastasis,and improve the prognosis of patients.Numerous studies have suggested that dysregulated long noncoding RNAs(lnc RNAs)contributed to development and progression of many cancers.Lnc RNA OIP5 antisense RNA 1(lnc RNA OIP5-AS1)has been reported to be increased in several cancers.However,the roles of lnc RNA OIP5-AS1 in LIHC remain to be investigated.In this study,we demonstrated that lnc RNA OIP5-AS1 was up-regulated in LIHC tissue specimens and its overexpression was associated with the poor survival of patients with LIHC.Furthermore,loss-of function experiments indicated that lnc RNA OIP5-AS1 promoted LIHC cell proliferation and inhibited cell apoptosis both in vitro and in vivo.Moreover,binding sites between lnc RNA OIP5-AS1 and hsa-mi R-26a-3p as well as between hsa-mi R-26a-3p and EPHA2 were confirmed by luciferase assays.Finally,rescue assay was performed to prove the effect of lnc RNA OIP5-AS1-hsa-mi R-26a-3p-EPHA2 axis on LIHC cell biological behaviors.Based on all above findings,our results suggested lnc RNA OIP5-AS1 promoted LIHC cell proliferation and invasion via regulating of hsa-mi R-26a-3p/EPHA2 axial.There is increasing evidence that liver cancer stem cells(LCSCs)contribute tohepatocellular carcinoma(HCC)initiation and progression.Micro RNA(mi RNA)plays a significant functional role by directly regulating respective targets in LCSCs-triggered HCC,however,little is known about the function of the mi RNA-302 family in LCSCs.In this part,mi RNAs microarray was used to detect the mi RNAs involved in LCSCs maintenance and differentiation.Biological roles and the molecular mechanism of mi R-302a/d and its target gene E2F7 were detected in HCC in vitro.The expression and correlation of mi R-302a/d and E2F7 in HCC patients was evaluated by quantitative PCR and Kaplan–Meier survival analysis.We found that the mi RNA-302 family was downregulated during the spheroid formation of HCC cells and patients with lower mi R-302a/d expression had shorter overall survival(OS)and progression-free survival(PFS).Moreover,E2F7 was confirmed to be directly targeted and inhibited by mi R-302a/d.Furthermore,concomitant low expression of mi R-302a/d and high expression of E2F7 correlated with a shorter median OS and PFS in HCC patients.Cellular functional analysis demonstrated that mi R-302a/d negatively regulates self-renewal capability and cell cycle entry of liver cancer stem cells via suppression of its target gene E2F7 and its downstream AKT/?-catenin/CCND1 signaling pathway.Our data provide the first evidence that E2F7 is a direct target of mi R-302a/d and mi R-302a/d inhibits the stemness of LCSCs and proliferation of HCC cells by targeting the E2F7/AKT/?-catenin/CCND1 signaling pathway.In the third part of this paper,we hybridized conditionally overexpressed c Myc mice with Alb-Cre tool mice to form spontaneous liver cancer in the liver,which was used for the establishment of liver cancer model and the follow-up mechanism research,and the preliminary study on the therapeutic effect of lornafanib combined with degrasy on Alb-c Myc spontaneous tumorigenic liver cancer mice.Our results showed that the number of tumor nodules on the liver surface of the mice treated with lornafanib combined with degrasy was significantly reduced,and the median survivaltime was prolonged.It is suggested that the combination of lornafanib and degrasy has no obvious toxic and side effects,and has certain therapeutic effect on spontaneous tumorigenesis mice of c Myc.In terms of mechanism,we found 2655 dif m RNAs,96 dif mi RNAs and 158 dif lncrnas in the Alb-c Myc group compared with the control group.Enrichment analysis of differentially expressed genes related to m RNA confirmed that genes involved in chromosome stability and protein degradation may play an important role in the pathogenesis of hepatocellular carcinoma.Further experiments confirmed that compared with the control group,lnc RNA OIP5-AS1 and E2F7 were significantly up-regulated in Alb-c Myc spontaneous tumor bearing mice,but significantly decreased after lornafanib combined with degrasyn treatment;mi R-26a-3p,mi R-302a/d and EPHA2 were significantly down regulated in the liver cancer tissues of Alb-c Myc spontaneous tumor bearing mice,and significantly increased after lornafanib combined with degrasyn treatment.These results suggest that lonafanil combined with Degrasyn has the therapeutic effect of Alb-c Myc spontaneous tumor bearing mice,and its mechanism involves the regulatory effect on the expression of lnc RNA OIP5-AS1,mi R-26a-3p,EPHA2,mi R-302 a /d and E2F7.In conclusion,this study revealed the molecular mechanism of lnc RNA OIP5-AS1 in regulating the malignant biological behavior of hepatoma cells by regulating hsa-mi R-26a-3p/EPHA2 axis and mi R-302a/d targeting E2F7/Akt/?-Catenin/CCND1 signaling pathway,and the therapeutic effect and mechanism of lornafanib combined with degrasyn in Alb-c Myc spontaneous tumor bearing mice.The therapeutic effect and mechanism of Lonafani combined with Degrasyn on Alb-c Myc spontaneous tumor bearing mice were evaluated and explained,which can provide theoretical and practical basis for the diagnosis and treatment of liver cancer. |
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