| Research background and purpose:Hepatocelluar carcinoma(HCC)is the most common liver primary malignant tumor,its incidence rate ranks fifth place in the cancer of the world,at the same time,it is also one of the most common cancer-related cause of death.HCC has high degree of malignancy.Since HCC is not obvious clinical symptoms in the early stage,many HCC patients are already in their terminal stage once diagnosed.Currently,great progress has been made for the treatment of HCC,and there are a lot of therapeutic methods that can be selected,surgery still remains the preferred one.However,due to the low resection rate and high recurrence rate of surgery,the therapeutic effect of HCC is not ideal.Thus,there is an urgent need to elucidate the mechanisms of HCC development,progression,invasion and metastasis process to further develop more effective early diagnosis and therapeutic methods.In recent years,along with the deepening research on cancer stem cells(CSCs),increasing evidence show that the heterogeneity of tumor is closely related to CSCs differentiation.In tumor tissues,although CSCs only account for a small proportion,the existing studies suggest that not only it has the ability of self-renewal,unlimited proliferation and multi-directional differentiation,but also it plays a key role in the process of tumor multi-step metastasis and drug resistance relapse.In liver cancer,tumor cells with stem cell-like properties are called as liver cancer stem cells(LCSCs).It is imperative to uncover the regulatory mechanisms of LCSCs and identify potential therapeutic targets to provide new ideas for the treatment of HCC.Kruppel like factor 8(KLF8)that participated in a variety of important gene transcription plays an important role in HCC occurrence,proliferation,invasion and other malignant biological behaviors.In previous studies,we first discovered that Wnt/β-catenin pathway is one of KLF8 protein’s potential downstream signaling pathways in HCC cell,and the activity disorders of Wnt/β-catenin in HCC cells may be directly related to the high expression of KLF8 protein.Therefore,abnormal protein expression level of KLF8 may be directly associated with HCC cancerization,invasion and metastasis.But so far,KLF8 research in stem cells is still limited,and its regulation on LCSCs has not been reported.The purpose of this study is to explore the possibility for KLF8 to become the new type of LCSCs marker,and to clarify its value in clinical drug indications and combination chemotherapy,so as to provide new ideas for HCC diagnosis and treatment.Research methods:1.Collection of clinical data and samples:it collected 80 cases of HCC patients who had surgical treatment in March 2008 to November 2011 in our hospital,including their clinical data and tumor tissue samples.Then,it analyzed the relationship between KLF8 expression and patient’s prognosis by detecting the KLF8 expression levels in patients’tumor tissues,and combined with recorded follow-up data.2.The molecular mechanism of KLF8 on HCC cell line stem cell-like properties:using a low-adhesion balling culturing experiment to enrich LCSCs,we detected the KLF8 expression levels in LCSCs by qRT-PCR;using lent virus to interfere KLF8 expression in HCC cell lines,we performed qRT-PCR,flow cytometry assay and balling ability assay to explore the effects on HCC cell line stem cell-like properties after KLF8 knockdown,and then further explore the interrelation between KLF8 and Wnt/p-catenin signaling pathway in HCC cell line stem cell-like properties regulation.3.The effects of KLF8 in drug-resistance hepatoma cells:using flow cytometry assay,CCK8 assay and WB to detect the sensitivity of low KLF8 expression HCC cell lines to clinical commonly used drugs,namely Sorafenib and Cisplatin.Experiment results:1.Using qRT-PCR to detect the mRNA expression level of KLF8 in 80 cases of HCC tissues and the corresponding para-carcinoma tissues.The result showed that the expression of KLF8 is lower in para-carcinoma tissues than that in 76.3%(61/80)of HCC tissue;Using WB to detect 22 cases of HCC tissues and the corresponding para-carcinoma tissues.The result showed that in 81.8%(18/22)of samples,the protein expression level of KLF8 in tumor tissues is significantly higher than that in para-carcinoma tissues;Using qRT-PCR to detect the mRNA expression level of KLF8 in tumor tissues,para-carcinoma tissues and portal vein tumor thrombus(PVTT)in 40 cases of HCC patients.The result showed that the expression of KLF8 in PVTT is significantly higher than that in HCC and para-carcinoma tissues;using detection and statistical analysis on the tissue samples and follow-up records in 80 patients with HCC.The result showed that the prognosis of KLF8(+)patients is worse.2.Using low adhesion balling culture and qRT-PCR detection,we found that the mRNA expression level of KLF8 in balling cells is significantly higher than that in adherent cells in three different HCC cell lines(SMMC-7721,MHCC-LM3,Hep G2);by isolating corresponding balling cells from primary HCC cells,we found that the mRNA expression level of KLF8 in primary balling cells is also higher than that in primary adherent cells in HCC patients;using qRT-PCR testing,we found that the mRNA expression level of KLF8 in HCC cells of CD24(+),CD90(+)and EpCAM(+)is significantly higher than that in HCC cells of CD24(-),CD90(-)and EpCAM(-).3.Using qRT-PCR testing,we found that the mRNA expression level of EpCAM,CD24,CD90 and CD133 in both HCC cell lines(SMMC-7721 and MHCC-LM3)is significantly reduced after KLF8 knockdown;and then use flow cytometry to analyze and sort out the corresponding HCC cells,we found that it can reduce the EpCAM(+)cells proportion in LM3 cell lines after KLF8 knockdown;using 6 pore plate low adhesion balling experiment and vitro experiments and extracorporal low adhesion limiting dilution experiment,we found that the balling ability of KLF8(-)HCC cell lines is significantly inhibited,whose balling number is significantly fewer than the control cell lines;using qRT-PCR test,we confirmed that the genes of stem cell-like properties of a series of HCC cells,such as Lin28,Bamil,Sox-2,OCT4 and Nanog are also significantly down-regulated after KLF8 knockdown.4.Using qRT-PCR detection,we found that the mRNA expression of KLF8 in Sorafenib and Cisplatin HCC cells is significantly higher than that in normal HCC cells;using flow cytometer detection,we found that the apoptotic rate of KLF8(-)HCC cell lines at a fixed concentration of Sorafenib(5μM),and Cisplatin(2μg/ml)is significantly increased compares with the controls;using CCK8 assay,we found that the apoptotic rate of KLF8(-)HCC cells is significantly increased comparing with the controls in a time-depend manner;PARP is a common mark of apoptosis.Using WB,we found that the PARP protein expression of KLF8(-)HCC is significantly increased under the action of certain concentration of Sorafenib(10μM)or Cisplatin(2μg/ml).5.Using WB,we found that the expression levels ofβ-catenin in KLF8(-)HCC cell lines is significantly reduced;using qRT-PCR detection,we found that the mRNA level of P-catenin downstream molecules,such as C-myc,Cyclin D1 and Survivin in HCC cells is significantly reduced after KLF8 knockdown;FH535 is a common Wnt/β-catenin signal transduction inhibitor,flow cytometry assay is conducted after adding FH535(40nM),we found that the proportion of EpCAM(+)cells in KLF8(-)HCC cell lines and EpCAM(+)cells in KLF8(+)HCC cell lines treated with FH535 were little changed;low adhesion balling experiment further confirms that FH535 offsets the difference in the balling ability between KLF8(-)HCC cell lines and control cell lines.Conclusion:1.The expression of KLF8 is high in HCC and distant metastasis tissues.HCC patients with high expression of KLF8 have worse prognosis.2.KLF8 is highly expressed in LCSCs,and promotes the stem cell-like properties of HCC cell lines by Wnt/β-catenin signaling pathway.3.KLF8 promotes the drug resistance of HCC cells to Sorafenib and Cisplatin. |
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