Expression Of Krüppel-like Factor 8 Promotes Tumor Growth, Invasion And Indicates Early Recurrence And Poor Prognosis In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is the third most common cause of cancer-related death.Although survival of patients with HCC has been improved by advances in surgical techniques and perioperative management, long-term survival following surgical resection remains unsatisfactory due to the high rate of recurrence and metastasis.Advances in treatment of this disease are likely to stem from a better understanding of its biology and behavior.As biological and clinical behaviors of cancer are affected by multiple molecular pathways that are under the control of transcription factors,improved understanding of how these transcription factors affect cancer biology may lead to an improved ability to predict clinical outcomes and the discovery of novel therapeutic strategies. Kr(u|¨)ppel-like factors(KLFs) constitute a family of nuclear proteins that modulate gene transcription.Emerging evidence suggests that KLFs may be critical factors in tumor development,growth,and metastasis.One of these family members,KLF8,is a CACCC-box binding protein that associates with C-terminal binding protein(CtBP) to repress transcription.Recent studies in breast cancer indicated that KLF8 participates in oncogenic transformation and induces the epithelial-to-mesenchymal transition(EMT),which is an important mechanism during tumor invasion transformation.We found that KLF8 is highly expressed in HCC tissues compared with peritumoral tissues based on transcript profiles,and this result suggested that KLF8 may play an important role in hepatocarcinogenesis.Until now,no studies have reported a role of KLF8 in HCC or the clinicopathological significance of this factor.Here,we examined KLF8 expression in stepwise metastatic potential human HCC cell lines and tumor tissues by quantitative real-time polymerase chain reaction(qRT-PCR),western blot,and immunochemistry analyses.To investigate the possible mechanism,KLF8 expression was knocked down in a HCC cell line with high metastatic potential(HCCLM3 cells) with small-interfering RNA(siRNA),and then the differential gene profiles were investigated by cDNA microarray analysis.The effects of KLF8 depletion were observed in vitro and in vivo.To explore the clinical effects of this factor,KLF8 expression was detected in 314 HCC patients using tissue microarrays(TMAs).Together,our data indicates that KLF8 promotes HCC cell proliferation,invasion,and migration.High expression of KLF8 is strongly associated with early recurrence and poor prognosis in HCC patients after curative resection.Part one Expression of KLF8 in different liver cell lines and in HCC tissuesObjective:This study investigated expression of KLF8 in differenr liver cell lines and in HCC tissues to evaluate the role of KLF8 in HCC development and progression.Methods:The KLF8 level in different liver cell lines was respectively determined by quantitative real-time PCR, Immunocytochemistry and Western blot.The KLF8 mRNA level in different HCC tissues was determined by RT-PCR.Using immunohistochemistry,we investigated KLF8 expression patterns in Hepatocellular carcinoma(HCC) tissue specimens,metastases,normal liver tissue specimens,and liver cirrhosis tissue specimens.Results:We found that KLF8 protein expressed in the nuclei of cancer cells,strong KLF8 expression was detected in tumor cells,whereas no or very weak KLF8 expression was detected in normal cells surrounding or within the tumors.Metastatic potential HCC cell lines have higher KLF8 mRNA and protein level than primary HCC cell lines.The same results appeared in KLF8 mRNA level of different HCC tissues.Metastasis specimens exhibited the highest level of KLF8 expression(83.3%strong positive;P＜0.001),and primary tumor tissue specimens had higher level of KLF8 expression than normal and liver cirrhosis tissue specimens(P＜0.001).Conclusion:The overexpression of KLF8 may be related to the carcinogenesis and development of HCC.Part two Clinical significance of overexpression of KLF8 in hepatocellular carcinoma Objective:To investigate the clinical significance of overexpression of KLF8 in hepatocellular carcinoma.Methods:The qRT-PCR and westernblot were used to detect the expression of ELF8 in HCC tissues,the TMA was used to detect the expression of KLF8 in 314 HCC patients,then the clinical significance of overexpression of KLF8 was analyzed by SPSS11.5.Results:In tissue samples,patients suffering HCC recurrence exhibited higher KLF8 mRNA expression levels than those without recurrence (p=0.001).According to western blot analysis,KLF8 was highly expressed in five patients with recurrence,KLF8 is also present in 3 of 5 without recurrence,but only one of these the level of expression is high.The expression of KLF8 was classified as strong positive in 162 cases(51.6%, 162/314) and weak positive or not stained in 152 cases(48.4%, 152/314).KLF8 was found to be prognostic for OS(p=0.040) and TTR (p=0.006).The 5-year OS rate and tumor recurrence probabily in the KLF8-negative group were 60.3%and 41.9%,compared with 48.7%and 59.8% for the KLF8-positive group(p=0.038 and p=0.005).Multivariate analysis indicated that KLF8 was an independent prognosticator for OS (p=0.037) and TTR(p=0.018).We further investigated the predictive value of KLF8 within clinical subgroups(AFP≤20 ng/ml vs.＞20 ng/ml,TNM stageⅠvs.Ⅱ-Ⅲ,BCLC stage O+A vs.B+C,EdmondsonⅠ-Ⅱvs.Ⅲ-Ⅳ,and early recurrence vs.later recurrence).The prognostic significance of KLF8 occurred in HCC patients with TNM stageⅠ,BCLC stage O+A,AFP≤20ng/ml and Edmondson stageⅠ-Ⅱ.The 5-year tumor recurrence probabily of KLF8-positive patients was significantly increased compared with KLF8-negative patients in TNM stageⅠ(54.6%vs.35.3%,p=0.008).The 5-year tumor recurrence probabily of KLF8-positive patients was significantly increased compared with KLF8-negative patients in BCLC stage O+A(57.0%vs.37.9%,p=0.009).In the AFP≤20 ng/ml group,the 5-year tumor recurrence probabily were 41.5%and 60.3%in the KLF8-neagtive and KLF8-positve samples,respectively(p=0.027).In the Edmondson stageⅠ-Ⅱgroup,the 5-year tumor recurrence probabily were 36.5%and 54.3%for the KLF8-neagtive and KLF8-positve samples, respectively(p=0.005).According to the recurrence time,the prognostic significance of KLF8 was useful in the early recurrence group (p=0.001) but not in the later recurrence group(p=0.623).KLF8 positve status was positively correlated with early recurrence(p=0.008).Conclusion:KLF8 may be a new target for prognostic prediction and adjuvant treatment of HCC patients HCC after operation.Part three Role of KLF8 in HCCLM3 cells proliferation and invasion in vitro,and its possible mechanism.Objective:To investigate the function of KLF8 in HCC development and progression and its potential mechanism.Methods:KLF8 level in HCCLM3 was down regulated by small interference RNA,then proliferation and invasiveness of HCCLM3 was measured by MTT reduction assay and Matrigel invasion assay;we performed a microarray assay to compare the differential gene expression profiles between KLF8 siRNA-treated and scrambled siRNA-treated HCCLM3 cells.To verify the microarray findings, several differentially expressed genes were further analyzed by qRT-PCR. Other related genes were observed by RT-PCR and Western blot.Results: In western blots,the expression of endogenous KLF8 was knocked down by 90%after KLF8 siRNA treatment for 72 h.Based on trans-well matrigel invasion assays,the number of invasive cells in the KLF8 siRNA-treated group decreased significantly compared with those in the control group (p＜0.001).The MTT assay revealed that cell proliferation was significantly suppressed from 3 day post-transfection (p＜0.001).According to microarray analysis,453 genes were differentially expressed between the KLF8 siRNA-treated HCCLM3 cells and the scrambled siRNA-treated HCCLM3 cells.Of these,229 genes were upregulated(ratio＞2.0),and 224 genes were downregulated(ratio＜0.5). Many of these genes were related to tumor metastasis and regulation of cell migration.The RT-PCR results correspond well to the microarray data. Conclusion:The KLF8 may promote proliferation and invasion of HCCLM3, and involved in many pathways which are important in tumor development and progression. Part four Effects of KLF8 depletion in vivoObjective:To investigate the effects of KLF8 depletion on HCCLM3 carciogenesis and lung metastasis in nude mice.Methods:pGCSIL-KLF8 small hairpin RNA(shRNA),a KLFS-RNAi lentiviral vector was constructed, GFP-lentiviral vector(pGCSIL-GFP) was used as a negative control.The lentiviral vectors and pHelper were co-transfected into 293T cells.The culture supernatants were collected,concentrated,and used as a virus stock.The lentivirus was transfected into the HCCLM3 cells.HCCLM3 cells (5×10~6) that had been transfected with KLF8-RNAi lentivirus were implanted subcutaneously into the flanks of nude mice.HCCLM3 cells transfected with GFP-lentivirus were used as a negative control.All the mice were sacrificed 6 weeks later,and the weight of tumors was calculated. Lungs were removed and embedded in paraffin.The paraffin blocks were consecutively sectioned and stained with haematoxylin and eosin.Based on the cell number in the maximal section of the metastatic lesion,the lung metastases were classified into four grades:GradeⅠ,HCCLM3 cells≤20;GradeⅡ,20-50;GradeⅢ,50-100;and GradeⅣ,＞100.Results: The tumors in the KLF8 siRNA-treated group had more necrotic foci than those in the control group.The tumor weights in the control group were significantly greater than that in the KLF8 siRNA group(3.67±0.75g vs.1.02±0.78g,p＜0.001).The incidences of lung metastasis in the KLF8 siRNA group and the control group were 16.7%and 100%,respectively (p=0.015).The metastatic foci of the KLF8 siRNA group were GradeⅠ,while most of metastatic loci in the controls were GradeⅢ-Ⅳ.Conclusion:KLF8 depletion could inhibit HCCLM3 carcinogenesis and lung metastasis in nude mice in vivo.