| Globally,primary liver cancer is a malignant tumor with the sixth incidence and the third fatality rate,and its incidence continues to rise.Hepatitis B,Hepatitis C infection,cirrhosis,excessive alcohol consumption and aflatoxin intake are recognized risk factors for primary liver cancer.China,as a major hepatitis B epidemic,is a region with high incidence of liver cancer.According to epidemiological research statistics,in 2015 alone,the number of new liver cancer cases in China reached 460,000.At present,liver cancer resection is still the first choice for liver cancer treatment.However,due to the unclear symptoms of early liver cancer and hidden growth characteristics,most patients are already in the middle and late stages of the disease when they are diagnosed.The available treatment options are limited and have little effect.Studies have shown that the occurrence and development of liver cancer is a multi-step,multi-factor complex process,driven by the accumulation of various genetic and epigenetic changes.However,the specific molecular mechanisms underlying the development of liver cancer are largely unknown.Therefore,exploring the pathogenesis of liver cancer and clarifying the key molecular events behind the occurrence of liver cancer are important for the early diagnosis and effective treatment of liver cancer.Pathology and molecular biology studies have shown that embryonic development and tumorigenesis are similar,sharing many key signaling pathways and regulatory factors.Oncofetal molecules are refered to be highly expressed in the early stages of embryo development and tumorigenesis,but lowly or even not expressed in adulthood.The special expression pattern of oncofetal molecules that are abnormally activated in tumors indicates that oncofetal molecules may play an important role in the initiaction and progression of tumors and are potential targets for diagnosis and treatement of tumors.In HCC research,alpha-fetoprotein(AFP)is the first reported oncofetal molecule and has been widely used in clinical diagnosis as a serum marker.Recently,many noval nocofetal molecules,such as phosphatidylinositol proteoglycan-3(GPC3),spalt like transcription factor(SALL4)and liver leukemia factor(HLF),have been discovered,providing new clues for cancer diagnosis and treatment.In our previous research,through comparing the expression profiles of mouse fetal liver tissues,adult liver tissuses and liver caner tissues,we found that not only coding genes but also non-coding transcripts showed oncofetal characteristics.The expression of lnc RNA-PVT1 and lnc Ptn-dt are gradually decressed during liver development and reactivated in liver cancer.They play important roles in the progression of liver cancer.However,samples selected in our previous research was mouse liver tissues,the large differences of non-coding RNAs between different species restricted us to discover more clinically valuble nocofetal molecules.In addition,we only focused on long non-coding RNA and whether other kinds of non-coding RNA also have oncofetal characteristics remains to be further studied.In order to enrich our understanding of oncofetal molecules,we have performed the following studies.In the first study,through microarray technology and bioinformatics analysis,we screened long non-coding RNA that is highly expressed in both human fetal liver and cancer liver tissues.Through the research,we found that the expression of lnc RNA MIR4435-2HG was high in fetal liver,significantly decreased in adult liver,and abnormally activated in liver cancer tissues.Further detection of MIR4435-2HG expression in liver cancer tissues showed that the expression level of MIR4435-2HG in liver cancer tissues was significantly higher than that of adjacent tissues,and the high expression of MIR4435-2HG in liver cancer tissues indicateds a poor prognosis of patients and a longer survival time after surgery.The expression of lnc RNA is regulated by many factors.M6 A modification is the most abundant epigenetic regulatory element in RNA and has been proved to be involved in the regulation of embryonic development and liver cancer progression.Therefore,we proposed that the m6 A modification may participate in the activation of MIR4435-2HG in liver cancer.The results of bioinformatic prediction and RNA immunoprecipitaion showed that m6 A modification exists in MIR4435-2HG.Further interference experiments and dual fluorescence reporter experiments confirmed that MIR4435-2HG was activated by METTL3 in a m6 A dependent way in liver cancer.Through functional experiments,we found that overexpression of MIR4435-2HG signifianctly promoted the proliferatioin of HCC cells both in vitro and in vivo.We confirm that MIR4435-2HG is a nocofetal molecule.Then,we further discussed the specific mechanism of promoting cancer effect of MIR4435-2HG.Directly binding to proteins is an important way for lnc RNA to participate in the regulation of life activities.Through RNA pulldown experiment,we found that MIR4435-2HG directly binds to NOP58,a member of C/D box RNA protein complex.Protein synthesis inhibition and Western blot experiments showed that overexpression of MIR4435-2HG upregulated the expression level of NOP58.Meanwhile,the results of ubiquitination level detection showed that MIR4435-2HG inhibited the degration of NOP58 in liver cancer cells by reducing the ubiquitination level of NOP58.Further more,the rescue experiments showed that the pro-cancer effect of MIR4435-2HG depends on its regulation of NOP58 expression.During the progression of liver cancer,up-regulation of MIR4435-2HG inhibits the degration of NOP58,increases the methylation modification of r RNA and ribosome biosynthesis,thereby promoting the proliferation of liver cancer cells.In the second part,we explored the relationship between circ PVT1,derived from the exon of the nocofetal gene lnc RNA-PVT1,and liver cancer.We detected the relative expression of circ PVT1 in fetal liver,adult liver and 70 pairs of tumor and adjacent tissues.Real-time PCR results showed that similar to lnc RNA-PVT1,the expression of circ PVT1 in fetal liver and liver cancer was significantly higher than that in adult liver tissues.Meanwhile,patients with higher expression of circ PVT1 showed worse prognosis and had shorter survival time.Through functional experiments,we found that overexpression of circ PVT1 significantly promoted the proliferation and metastasis of liver cancer cells.Thus,we could conclude that circ PVT is a oncofetal gene.In further mechanism research,bioinformatic analysis and dual flurescence reporter experiments showed that circ PVT1 specifically adsorbs mi R-203 through base complementary pairing.At the same time,real-time PCR and western blot analysis showed that circ PVT1 repressed mi R-203 expression and decreased the downstream target of mi R-203,HOXD3.Overexpression of mi R-203 mimics in liver cancer cells confirmed that circ PVT1 promoted the proliferation and metastasis of liver cancer cells through regulation of mi R/HOXD3 pathway.In summary,we focused on “oncofetal gene” and conducted in-depth research on nocofetal non-coding RNA in HCC.Lnc RNA MIR4435-2HG and circ RNA PVT1 are identified as oncofetal molecule.They provide new clues for explaining the initiation of liver cancer and are of great significance for HCC diagnosis and treatment. |
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