| Background and Objectives:Hepatocellular carcinoma(HCC)is a common malignant tumor that seriously endangers human health.It ranks fifth in the global incidence of malignant tumors and third in the mortality rate.Surgical resection and radiofrequency ablation are the first choice for the treatment of HCC.However,the pathogenesis of HCC is complex,leading to recurrence of HCC patients after operation or radiofrequency ablation,and resistance to radiotherapy and chemotherapy.The treatment effect is still not ideal and the prognosis is poor.With the development of cancer research,it has been found that cells in cancer tissue are in different stages of differentiation.Few of them can self-renew and differentiate into multiple cells.They are called Cancer Stem Cells(CSCs),or Cancer Initiating Cells(CICs).According to the theory of cancer stem cells,a small number of tumorigenic cell subsets in cancer tissue have the ability of infinite proliferation,self-renewal and multi-differentiation,and are susceptible to chemotherapeutic drugs,which may be the main cause of tumor recurrence,metastasis and drug resistance.Liver Cancer Stem Cells(LCSCs),a group of cells with self-renewal and differentiate-on ability,which can significantly affect the occurrence,development,recurrence,metastasis and drug resistance of hepatocellular carcinoma,were also found in hepatocellular carcinoma tissues.Therefore,to analyze the mechanism of self-renewal of hepatocellular carcinoma stem cells,and to develop targeted anti-hepatocellular carcinoma stem cell therapies is the focus of current research on hepatocellular carcinoma.Long non-coding RNA(lnc RNA)is widely distributed in eukaryotic cells and is an important part of epigenetic regulation mechanism.Many kinds of lnc RNA have been found in hepatocellular carcinoma and play an important role in the occurrence and metastasis of HCC.However,the understanding of the types of lnc RNA and the signaling pathways in CSCs regulated by different lnc RNA is still insufficient,and more in-depth and extensive exploration is needed.SAMMSON is a long-chain non-coding RNA located on chromosome3p13-3p14.Studies have confirmed that the abnormal expression of lnc RNA SAMMSON in melanoma is related to the occurrence and development of cancer cells.The survival of melanoma cells requires SAMMSON.Knocking down the expression of SAMMSON can reduce the survival ability of melanoma cells.However,the role of SAMMSON in hepatocellular carcinoma and hepatocellular carcinoma stem cells remains unclear.In previous experiments,we found that the expression of SAMMSON in hepatocellular carcinoma tissues was higher than that in adjacent tissues,and the expression of SAMMSON in advanced hepatocellular carcinoma was higher than that in early hepatocellular carcinoma.This phenomenon is highly consistent with the characteristics of cancer stem cells,suggesting that the high expression of SAMMSON may be intrinsically related to hepatocellular carcinoma stem cells.Based on the literature review and previous studies,we hypothesize that SAMMSON may be involved in the maintenance of self-renewal of hepatocellular carcinoma stem cells.The main objectives of this study are: 1.To detect the expression of SAMMSON in hepatocellular carcinoma tissues and hepatocellular carcinoma stem cells;2.To clarify the role of SAMMSON in self-renewal of hepatocellular carcinoma stem cells;3.To explore the specific mechanism of SAMMSON in self-renewal and maintenance of hepatocellular carcinoma stem cells.Methods:A:The expression of SAMMSON in hepatocellular carcinoma tissues and hepatocellular carcinoma stem cells1.Collect 19 specimens of hepatocellular carcinoma tissues and corresponding para-cancerous tissues from patients undergoing hepatectomy.The expression of SAMMSON in different tissues was detected by real-time quantitative PCR and immunohistochemistry.The relationship between SAMMSON expression and clinicopathological characteristics and prognosis was analyzed.2.Hepatocellular carcinoma cell spheres were obtained by serum-free culture.CDl33+ hepatocellular carcinoma stem cells and CD133-non-stem cells were separated by flow cytometry.The expression of SAMMSON in the two groups of cells was detected by real-time quantitative PCR.3.Suspension balling test was used to collect cancerous and non-cancerous spheres,and real-time quantitative PCR and fluorescence in situ hybridization were used to detect the difference of SAMMSON expression between the two groups.The localization of SAMMSON in cells was detected by nuclear-cytoplasmic separation assay.B:The role of SAMMSON in self-renewal of hepatocellular carcinoma stem cells1.Silent SAMMSON ASO cells were constructed using antisense oligonucle-otides.The effects of knocking down SAMMSON on stem cell-like characteristics of hepatocellular carcinoma(HCC)were investigated by balling test and cell invasion test.Different concentrations of SAMMSON silencing cells were injected into BALB/c nude mice to complete the tumorigenesis experiment in vivo.The tumorigenesis rate and weight of the tumors were counted to verify the results in vitro.2.Over-expression of SAMMSOM cells(oe SAMMSON)was constructed by lentivirus vector.The effects of over-expression of SAMMSON on stem cell-like characteristics of hepatocellular carcinoma(HCC)were detected by balling and cell invasion experiments.Different concentrations of SAMMSON overexpressed cells were injected into BALB/c nude mice to complete the in vivo tumorigenesis experiment of hepatocellular carcinoma cells,and the tumorigenesis rate and weight changes were counted to verify the results in vitro.C:Mechanisms of SAMMSON in regulating self-renewal of hepatocellular carcinoma stem cells1.The effect of SAMMSON silencing and overexpression on self-renewalrelated signaling pathways of hepatocellular carcinoma stem cells was detected by PCR array.2.The related signaling pathways affecting SAMMSON were identified by bioinformatics,and further validated by spheroid formation,Western blot and double luciferase reporter gene experiments.Results:1.SAMMSON is highly expressed in hepatocellular carcinoma and hepatocellular carcinoma stem cells,and mainly located in the nucleus.The results of q PCR showed that the expression of SAMMSON gene was gradually increased in adjacent tissues,early and advanced hepatocellular carcinomas,and was the highest in advanced hepatocellular carcinomas.Immunohistochemical results showed that the expression of SAMMSON in hepatocellular carcinomas was significantly higher than that in adjacent tissues.The expression of SAMMSON in advanced hepatocellular carcinomas was higher than that in early hepatocellular carcinomas,which was consistent with the results of Hepatocellular carcinoma stem cells were sorted by cell surface marker CD133,and then SAMMSON expression was detected by real-time quantitative PCR.The results showed that the expression of SAMMSON in CD133(+)hepatocellular carcinoma stem cells was significantly higher than that in CD133(-)non-hepatocellular carcinoma stem cells.The results of balling test and in situ hybridization showed that the expression of SAMMSON in carcinomatous spheres was significantly higher than that in non-carcinomatous spheres.Nuclear plasmid isolation assay detected the intracellular localization of SAMMSON,confirming that SAMMSON was localized in the nucleus,not in the mitochondria.2.SAMMSON promotes self-renewal of hepatocellular carcinoma stem cells After silencing SAMMSON,the globular formation ability of hepatocellular carcinoma stem cells was impaired and the invasive ability was weakened.The tumorigenic experiment in nude mice showed that the ability of hepatocellular carcinoma formation was impaired in vivo,which inhibited the formation and growth of hepatocellular carcinoma in animals.Overexpression of SAMMSON enhanced the ability of spherogenesis and invasion of hepatocellular carcinoma stem cells.Tumor formation experiments in nude mice showed that the ability of hepatocellular carcinoma formation in vivo was enhanced and the proliferation of hepatocellular carcinoma was promoted.The high expression of SAMMSON significantly promoted the self-renewal ability of hepatocellular carcinoma stem cells.3.SAMMSON stimulates the self-renewal of hepatocellular carcinoma stem cells by activating Wnt/beta-catenin signaling pathway.The expression of Wnt/beta-catenin target gene decreased after SAMMSON silencing and increased after SAMMSON overexpression,suggesting that SAMMSON is associated with the activation of Wnt/beta-catenin signaling pathway.The results of double Luciferase Report experiment,spheroid formation experiment and Western blot test support this conclusion.Conclusion:1.Compared with the adjacent tissues of hepatocellular carcinoma,the expression level of long-chain non-coding RNA SAMMSON in HCC tissues was significantly increased,and the expression level of SAMMSON in advanced hepatocellular carcinoma was higher than that in early hepatocellular carcinoma,which was related to the poor prognosis of malignant tumors.2.SAMMSON is also highly expressed in hepatocellular carcinoma stem cells,and it was found that SAMMSON is mainly located in the nucleus,not in the cytoplasm.3.Silencing SAMMSON could significantly inhibit the stem,invasion,proliferation and tumorigenesis of HCC cell lines,and the self-renewal ability of HCC stem cells was impaired after silencing.4.Overexpression of SAMMSON can significantly enhance the ability of stem,invasion,proliferation and tumorigenesis of HCC cell lines.Overexpression of SAMMSON can enhance the self-renewal ability of hepatocellular carcinoma stem cells and promote the occurrence of hepatocellular carcinoma.5.SAMMSON promotes self-renewal of hepatocellular carcinoma stem cells by regulating the expression of c-MYC and CND2 proteins and activating the Wnt/beta-catenin signaling pathway. |
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