The Role Of Transcriptional Repressor Zinc Finger And Homodomain Box2in The Development And Therapy Of Hepatocelluar Carcinoma

Hepatocellular carcinoma (HCC) is one of the major malignant diseases, and is the fifth most prevalent carcinoma and the third most common cause of mortality in the world. At present, the patients with HCC in China take about55%of the whole cases in the world, and more and more new cases of the disease occure in young people. Different kinds of methods including liver transplantation (LT), microwave treatment and intervention treatment (DIT) have been invlolved in HCC therapy. However, the effects are limited. Surgical resection is still the most effective way for HCC therapy although the recurrence rate after surgical resection is about70%. Therefore, it is urgent to study the mechanisms of HCC and explore new ways for HCC therapy.Transcription factor ZHX2(zinc finger and homodomain box2) was first cloned in mice, and its human homologous was successful cloned in2003. ZHX2could not only form homodimers by himself, but also form heterodimers with the other member of ZHX family(ZHX1, ZHX3) or NF-YA (A submit of nuclear factor Y) and function as a transcription repressor. Consistently, ZHX2inhibits the promoter activity of cdc25C dependent on NF-YA in SL2cells. It has been demonstrated the ZHX2-mediated downregulation of expression of the biomarkers of HCC, such as AFP(a-Fetoprotein), GPC3(Glypican-3) and H19, indicating the involvement of ZHX2in HCC. However, the founction of ZHX2in HCC development is completely unknown. Here we aim to explore the roles of ZHX2in the development of HCC, and to study the potential application of ZHX2in clinical treatment of HCC. Part Ⅰ ZHX2inhibits HCC cell growth by repressing transcription of Cyclin A and Cyclin EIt has been demonstrated that ZHX2could inhibit the expression of several HCC markers, including AFP and GPC3in BalB/cJ mice. However, it is completely unknown whether ZHX2is involved in HCC development. In order to address that, we designed in vitro and in vivo studies as following:Ⅰ. ZHX2inhibites the proliferation of HCC cell lines both in vitro and in vivo.1. ZHX2inhibites the proliferation of HCC cell lines in vitro. pcZHX2was transfected in HCC cell lines (HepG2and HepG2.2.15) with low endogenous ZHX2and shRNAs (ps1674or ps2360) were transfected in HCC cell lines (SMMC7721and QSG7701) with high endogenous ZHX2. Cell growth was analyzed by CCK8at24hour intervals after transfection. ZHX2overexpression reduced proliferation over a four-day period in both HepG2and HepG2.2.15cells(p<0.001). Consistently, reducing ZHX2levels by transfecting shRNAs (pS1674and pS2360) in SMMC7721and QSG7701cells significantly enhanced cell proliferation(p<0.001).2. ZHX2decreases the colony formation ability of HCC cell lines. ZHX2overexpression and knockdown cell models were obtained as above. Then the transiently transfected cells were plated into6-well plates and cultured for8～12days. Colonies were stained with crystal violet and counted under a microscope. ZHX2overexpression led to a significant decrease in the number of colonies formed in HepG2and HepG2.2.15cell lines(p<0.001), while the shRNA-mediated knockdown of ZHX2significantly increased the number of colonies formed in QSG7701and SMMC7721cell lines(p<0.001).3. ZHX2inhibites the proliferation of HCC cell in vivo. Aliquots of HepG2.2.15cells (1×107) were transplanted subcutaneously into the nude mice. When tumors had reached a size of0.5cm in diameter, they were injected with pcDNA3.0or pcZHX2(20μg/100μl) every four days for4times. Tumor size was monitored every other day by measuring two diameters perpendicular to each other with calipers. Mice were scarified at20day after injection and the tumors were isolated for weight measurement. Cell proliferation in the tumor was assayed by immunoperoxidase staining of tumor sections with an anti-Ki-67antibody. RT-PCR analysis demonstrated that pcZHX2-injected tumors had increased ZHX2mRNA levels compared to pcDNA3.0-injected tumor s(p<0.05). Injection of pcZHX2significantly inhibited the tumor growth (p<0.05) and tumor weight(p<0.001). Immunohistochemical analysis demonstrated less Ki-67staining in pcZHX2-treated tumors(p<0.001).II. ZHX2leads to G0/G1arrest in HCC cell lines by down regulating the transcription of Cyclin A and Cyclin E.1. ZHX2overexpression leads to G0/G1arrest in HCC cell lines. pcZHX2was transfected in HepG2to overexpress ZHX2and shRNAs (ps1674or ps2360) were transfected in SMMC7721to knockdown ZHX2. Cells were collected48h after transfection for cell cycle analysis. Results showed that the cell number in G0/G1stage was increased in ZHX2overexpressed groups (67.2%±0.14%) compared with control group (58.2%±2.12%), but decreased in ZHX2shRNA treated groups (54.3%±0.28%, ps1674;56.4%±2.82%, ps2360)compared with control groups (66.4%±4.03%)2. ZHX2inhibits the expression of Cyclin A and Cyclin E. The total protein and RNA were collected from ZHX2overexpressed HCC cells or ZHX2-knockdown HCC cells. Results of western blot and RT-PCR showed that ZHX2downregulated the expression of Cyclin A and Cyclin E, but not Cyclin D1, p21and p27.Ⅲ. ZHX2inhibites the transcription of Cyclin A and Cyclin E by binding to its promoter regions.1. ZHX2inhibits the promoter activities of Cyclin A and Cyclin E. Total genomic DNA was extracted from human peripheral blood as template, and PCR were used to amplify the promoter region of Cyclin A(-505～+361) and Cyclin E(-402～+72) were with specific primers. Then PCR products were cloned into pGL3-basic to prepare the promoter reporter plasmids of cyclin A and cyclin E, named as pGL3-Ap and pGL3-Ep respectively. In order to detect the effects of ZHX2on cyclin A and cyclin E promoter activities, report plasmid pGL3-Ap (0.25μg) or pGL3-Ep (0.25μg) was co-transfected with pcZHX2(0.75μg) in HepG2respectively. Results of dual luciferase assay showed that ZHX2significantly decreased the promoter activities of Cyclin A and Cyclin E(p<0.01).2. ZHX2binds to the promoter regions of Cyclin A and Cyclin E confirmed by ChIP. HepG2cells were plated at6dish were transfected with pcZHX2(4μg) or pcDNA3.0(4μg) respectively.48h after transfection, cells were collected and sonicated until the DNA was sheared to an average size of200～1000bp. Supernatants obtained after centrifugation were used for immunoprecipitations using anti-HA antibody or control IgG. DNA purified from the immunoprecipitates was used for PCR amplification. Results showed that ZHX2could bind with the promoter region of Cyclin A and E but not Cylcin D since the specific promoter regions of Cyclin A and E could be detected by PCR with DNA purified from HA-immunoprecipitates.Ⅳ. Compared to paracancerous sections, ZHX2nuclear expression was significantly decreased in HCC tissues which relates well with HCC development.1. ZHX2nuclear expression level in liver carcinoma tissues is lower than that of adjacent noncancerous tissues, and relates to the tumor pathological grades.82liver carcinoma tissues and78matched adjacent noncancerous tissues were collected for immunohistoochemical staining with anti-ZHX2antibody. Cytoplasmic and nuclear staining is reported separately according to the German semiquantitative scoring system. Nuclear expression of ZHX2was reduced in human HCC samples compared to adjacent non-tumor tissues (53.7%vs70.5%, p=0.0282) but not the total level of ZHX2. Small tumors (diameter less than5cm) also had reduced nuclear levels of ZHX2than control tissue. Same results were obtained by western blot. ZHX2nuclear expression positively related to the tumor pathological grade.2. Nuclear ZHX2expression is negatively related to Cyclin A, Cyclin E and Ki67expession. Serial sections from liver tissues were stained with Cyclin A, Cyclin E and Ki67. The expression of these genes is negatively related to ZHX2.3. Nuclear ZHX2expression significantly correlates with HCC progressing. Tissue microarray was purchased and stained with anti-ZHX2and anti-CD31antibody. The staining is reported separately according to the German semiquantitative scoring system. Statistical analysis demonstrated that ZHX2nuclear expression is negatively related to MVD (microvascular density), and positively related to the survival of patients.V. Nuclear location of ZHX2determines its inhibitaroy effects on HCC cell proliferation.1. Recombinant fusion protein encoded by pZHX2(242-446) but not pZHX2(242-439) locolizes in the nuclear. The truncated ZHX2plasmids were constructed by inserting PCR products of ZHX2into pEGFP-N1respectively named pZHX2(242-439) and pZHX2(242-446). The plasmids (pZHX2, pEGFP-N1, pZHX2(242-439) and pZHX2(242-446)) were transfected into HEK293and CHO. The location of truncated ZHX2was confirmed by western blot and immunofluorescence. Results showed that truncated ZHX2encoded by pZHX2(242-446) located in the nuclear.2. pZHX2(242-446) but not pZHX2(242-439) inhibites the activity of pGL3-Ap and pGL3-Ep. pZHX2(242-439)(0.75μg) or pZHX2(242-446)(0.75μg) were respectively co-transfected with pGL3-Ap (0.25μg) and pGL3-Ep (0.25μg).48h later, the total protein were collected and then the luciferase activity was detected. pZHX2(242-446) inhibited the promoter activity of Cyclin A and Cyclin E.3. pZHX2(242-446) but not pZHX2(242-439) inhibites the growth of HCC cell lines in vivo and in vitro. The plasmids (pEGFP-N1, pZHX2(242-439) and pZHX2(242-446)) were transfected into HepG2for cell growth curve analysis. As expected, pZHX2(242-446) but not pZHX2(242-439) inhibited the proliferation of HepG2cells. For in vivo assay, aliquots of HepG2.2.15cells (1×10) were transplanted subcutaneously into the male BalB/c nude mice. When tumors reached a size of0.5cm in diameter, mice were injected with pEGFP-N1, pZHX2(242-439) or pZHX2(242-446)(20μg/100μl) every four days for3times. Tumor size was monitored every other day and the tumors were isolated after the last injection for weight measurement. Results showed that EGFP-ZHX2(242-446) inhibited the weight and the growth of the tumor in nude mice.In conclusion, ZHX2inhibited the growth of HCC by down-regulating the expression of Cyclin A and Cyclin E, which related to the location of ZHX2. Nuclear localization of ZHX2was reduced in human HCC samples, even in small tumors (diameter≤5cm), compared to adjacent non-tumor tissues. Moreover, reduced nuclear ZHX2significantly correlates with reduced overall survival times of patients, high levels of microvascularization and hepatocyte proliferation.Part II ZHX2enhances sensitivity of HCC cells to chemotherapyChemotherapy is one of the important methods in HCC therapy. However, HCC cells are well known as chemoresistant tumor cells. The drug resistance of HCC largely limits the use of chemotherapy. Therefore, it is urgent to explore new methods to enhance drug sensitivity of HCC. It has been report that ZHX2expression in multiple myeloma were positively relate to the effects of chemotherapy indicating the potential of ZHX2in chemotherapy. Here, in this study, some useful conclusions were drawn as follows:I. ZHX2significantly enhances the sensitivity of HCC cell lines to CDDP.1. ZHX2obviously enhances the CDDP-induced cell death. HepG2, HepG2.2.15and BEL7402were used to overexpress ZHX2by tranfection thenology. The transfected cells were treated with CDDP or not for24hours, then detected the OD value by CCK-8. The results showed that the viarability of combination treatment of ZHX2and CDDP is lower than the ZHX2(p<0.001) or CDDP treated alone group(p <0.01) and the control group(p<0.001).2. ZHX2significantly decreses the IC50values of CDDP in HCC cell lines. The ZHX2overexpress ion models of HepG2, HepG2.2.15and BEL7402were obtained as above. Then the transfected cells were treated with different concentration of CDDP for24hours. The IC50values of CDDP were calculated using GraphPad Prism5. The results deterimied that overexpression of ZHX2decreased the IC50value of CDDP both in ZHX2low expression cell lines (HepG2and HepG2.2.15) and the ZHX2high expression cell lines (BEL7402)(p<0.05).3. ZHX2increases the number of apoptosis cells induced by CDDP. HepG2cells were transfected with pcZHX2or pcDNA3.0, and then were treated with CDDP or not. The treated cells were stained by PI or Annexin V/PI, and then analyzed the apoptosis cells by flow cytometry. The dates showed that combination of ZHX2overexpression and CDDP significantly increased the proportion of apoptosis cells. Consistently, the same results were obatained by staining of DAPI or Hochest33258,(p<0.001)4. ZHX2overexpression plus CDDP cooperatively induces caspase-dependent apoptosis. HepG2cell were plated into6-well plates, and then treated as above. The whole cell extracts prepared were involved for detection of ZHX2, caspase-3, caspase-9, PARP, Bcl-2and β-actin. Cytoplasmic extracts were used to detect cytochrome c release in cytoplasma. CDDP treatment led to increased cytoplasm cytochrome c, more cleaved caspase-3, cleaved caspase-9and cleaved PARP. Moreover, combination treatment of pcZHX2and CDDP induced additive effects on these apoptotic proteinsII. ZHX2enhances the ability of CDDP-inhibited tumor growth in vivo.1. Combination treatment of ZHX2and CDDP inhibites tumor growth in vivo. Xenograft animal model was constructed as part one. Mice were divided randomly into four groups (six mice per group) and treated by intratumor injection of combination of pcZHX2(20μg) and CDDP (80μg) or intratumor injection of pcDNA3.0(20μg) and CDDP (80μg) at3days interval for four times. The tumor volume and weight were calculated. The combination of ZHX2overexpression plus CDDP cooperatively inhibited tumor growth more obviously than treatment with pcZHX2or CDDP alone. Accordance to the growth curve of tumors, the final weight of tumors were significantly reduced in mice with combination treatment of pcZHX2plus CDDP compared with mice treated with ZHX2overexpression or CDDP alone(p <0.05).2. Combination treatment of ZHX2and CDDP induces more apoptosis in xenograft tumor sections. Xenograft tumor sections were prepared for immunohistochemistry and transferase-mediated dUTP nicked labeling (TUNEL) analysis. The expression of ZHX2is higher in pcZHX2and pcZHX2/CDDP injection groups than pcDNA3.0and pcDNA3.0/CDDP groups detected by immunohistochemistry. The results with TUNEL assay revealed that the combination of ZHX2overexpression and CDDP resulted in significant increased apoptosis in tumor sections than that treated with pcZHX2or CDDP alone(p<0.001).Ⅲ. ZHX2enhances drug sensitivity in HCC cells by inhibiting the transcription ofMDR1. The overexpression of MDR1is an important reason of tumor cell resistance to chemical drugs. The expression of MDR1was regulated by NF-YA which could combine with ZHX2. ZHX2may take part in the regulation of MDR1expression. In order to define the mechanism of ZHX2enhance the sensitivity of tumor cells to chemical drugs, the following experiments were done.1. ZHX2decreases the exclusion ability of cells. ZHX2overexpression modle of HepG2was obatained as above, and the collected cells were treated with PI or ADM (Doxorubicin) for4h. Fluorescence intensity of cells was determined by flow cytometric analysis. The fluorescence intensity of pcZHX2transfected is higher than pcDNA3.0transfected(p<0.05).2. ZHX2inhibites the expression of MDR1in transcriptional level. The ZHX2overexpression cell models were constructed by liposome transfection technique. The total RNA was extracted. RT-PCR was performed to detect the expression of MDR1. The expression of MDR1in pcDNA3.0transfected group is higher than pcZHX2transfected group.3. ZHX2inhibites the activity of MDR1promoter dependent on NF-YA. The PCR products of MDR1promter core region inserted into pGL3-basic named pGL3-Mp. pcZHX2was co-transfected with pGL3-Mp and then the luciferase activity was detected. pcZHX2inhibited the promoter activity of MDR1and the inbition was encrease with concerntration of pcZHX2. pcZHX2, pGL3-Mp and siNF-YA were co-transfected in HepG2determined that ZHX2inhibited the activity of MDR1promoter dependent on NF-YA.4. ZHX2significantly enhances the sensitivity of HCC cell line to chemical drugs. ZHX2overexpression HepG2cells were respectively treated with CDDP,5-Fu and ADM at different concentrations. The OD value was detected every24h and the cell viability was calculated. The date showed that the ZHX2overexpression groups were more sensitive to chemical drugs than the control groups, especaily at24h and48h.In conclusion, ZHX2enhanced the sensitivity of HCC cells to chemodrugs such as CDDP. This enhancement is at least partially dependant on the negative regulation of MDR1transcription. ZHX2increased the CDDP-induced apoptosis both in vitro and in vivo by mitochondrial/caspase pathway.