| Background:China is a large country of liver cancer,the number of new incidence and death cases accounts for more than half of the world every year.Therefore,improving the diagnosis and treatment and reducing the incidence rate and mortality of liver cancer are important issues of the Chinese health and Health Committee and liver specialists.In China,Primary liver cancer is currently the fourth most common malignant tumor and the second leading cause of cancer death.In our country,75% ～ 85% of primary liver cancer cases are hepatocellular carcinoma(HCC),64% of HCC patients are at stages of CNLC-Ⅱand Ⅲ(Barcelona liver cancer clinical stage(BCLC)-stage B and C)at the initial diagnosis,and the median survival time is about 11 months.The majority of middle and late stage patients lost the chance of surgical resection,the resection rate is only around 30%,and it is easy to relapse and metastasis after surgery.The 5-year survival rate was less than 20%.In recent years,significant progress has been made in the non-surgical systemic treatment of HCC.In particularly,antiangiogenic drugs combined with immunotherapy for advanced or unresectable HCC can achieve an objective remission rate of about30%,and the median survival time of patients can be increased to about 20 months.On the other hand,local treatment methods such as transcatheter arterial chemoembolization(TACE),hepatic arterial infusion chemotherapy(HAIC)and radiotherapy have not only achieved better results in reducing tumor and controlling tumor thrombus,but also improved patient survival through the improvement of technology and drugs and the combination with other treatment methods.However,these treatment methods have great individual differences and limitations,the disease control rate is still not very ideal,and only part patients can benefit from it.To further explore the mechanism of the occurrence and development of liver cancer and find the key molecular signal transduction pathways regulating the growth of cancer cells and liver immune microenvironment will provide new ideas and potential targets for the diagnosis of liver cancer and the development of new drugs.Long non-coding RNAs(lnc RNA)is an important part of gene regulation.It can regulate the occurrence and development of tumor cells through epigenetic,transcriptional regulation and post transcriptional regulation.In recent years,many studies have found that lnc RNAs are involved in the occurrence and development of a variety of cancers.The expression of long-chain noncoding RNA(lnc RNA)DIO3OS decreases in a variety of cancers,it can participate in the regulation of tumor cell activity and proliferation through a variety of signal pathways such as m RNA and hedgehog factor(Hh),but its correlation with HCC is seldom reported.Aims:In this study,we aim to explore the functional role and molecular mechanism of lnc RNA DIO3 OS in regulating the development of HCC by detecting the expression of lnc RNA DIO3 OS in human HCC tissues and hepatoma cell lines,and the effect of DIO3 OS overexpression on the growth and activity of hepatoma cells,so as to explore the new mechanism of hepatocarcinogenesis and development and provide potential new targets for the treatment of liver cancer.Method:First,we obtained differentially expressed lnc RNAs,m RNAs and micro RNAs(mi RNAs)through databases GSE101728 and GES57555.Confirmed that the expression of differential gene lnc RNA DIO3 OS was lower in HCC tissues.Subsequently,a series of in vitro experiments were carried out with hepatoma cell lines Hep G2 and bel-7405.DIO3 OS was enhanced in hepatoma cells to observe its behavior changes,and then the location of DIO3 OS was determined through Lncatlas(http://lncatlas.crg.eu/)and FISH test.Immunoblotting to detect the expression of genes related to Hedgehog(Hh)signal pathway.The effect of overexpression of DIO3 OS on tumor growth was also verified.Further,a series of in vitro experiments were carried out with hepatoma cell lines Hep G2 and BEL-7405 to construct overexpression or silencing plasmids.The expression of related RNA was detected by RT-q PCR,the survival of cells was detected by CCK-8 and edu staining,and the apoptosis of cells was detected by flow cytometry and Hoechst 33258 staining.The migration and invasion of hepatoma cells were detected by transwell cell culture.Fluorescence in situ hybridization was used to determine the localization of DIO3 OS in cells.Luciferase reporter gene was used to detect the binding relationship between mi R-328 and DIO3 OS and HHIP.The expression of Hh signal pathway related proteins in HCC cells was detected by Western Blot.In addition,the effect of DIO3 OS expression level on tumor growth was verified in vivo by subcutaneous liver cancer tumor model in nude mice,and the effect of overexpression of DIO3 OS on tumor cell proliferation was detected by Ki67 immunohistochemistry.The specimens of human HCC tissues and adjacent tissues were obtained from 31 patients with HCC who were surgically removed and pathologically confirmed in the hospital from 2018 to 2019.Result:The expressions of lnc RNA DIO3 OS were lower in either human HCC tissues and hepatoma cell lines Hep G2 and BEL-7405.DIO3 OS overexpression inhibited the proliferation,invasion and migration of Hep G2 and BEL-7405 cells,and promoted the apoptosis of Hep G2 and BEL-7405 cells.DIO3 OS knockout promoted the proliferation of LO2 cells,significantly increased the ability of migration and invasion of LO2 cells,and significantly inhibited the occurrence of apoptosis.In vivo,DIO3 OS gene overexpression significantly inhibited the growth of subcutaneous hepatoma tumor in nude mice.We have also demonstrated that DIO3 OS was located in the cytoplasm,and negatively correlated with the expression of mi R-328.The expression of HHIP in human tumor tissues decreased significantly.Overexpression of DIO3 OS significantly increased the expression of HHIP.While HHIP was positively correlated with DIO3 OS and negatively correlated with mi R-328.DIO3 OS can destroy the Hh signal pathway.After overexpression of DIO3 OS,the expression of Gli1,Gli2 and Gli3 decreased significantly.It is suggested that DIO3OS/mi R-328/HHIP may be a regulatory network regulating the growth,migration and invasion of hepatoma cells.At the same time,DIO3 OS and mi R-328 form a regulatory loop.Overexpression of DIO3 OS gene leads to the decrease of mi R-328 expression,enhances the expression of HHIP and inhibits the Hh pathway.Conclusion:Our results showed that DIO3 OS expression was lower in both human HCC tissues and hepatoma cell line.Overexpression of the DIO3 OS gene significantly inhibited the proliferation,invasion,and migration of human HCC cells,and increased their apoptotic rate,and inhibited the growth of tumor in vivo.Lnc RNA DIO3 OS interacts with the HHIP dependent Hh signaling pathway.DIO3 OS inhibits the proliferation,migration and invasion of hepatoma cells by negatively regulating mi R-328 and inhibiting Hh signaling pathway,which further suggests that Lnc RNA DIO3 OS may be an effective potential target for the treatment of HCC.DIO3OS/mi R-328/HHIP may be a regulatory network for the growth,migration and invasion of hepatoma cells. |
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