| The First Section: The expression and significance of PPARγin hepatocellular carcinoma Objective To study the expression of peroxisome proliferators-activated receptor gamma (PPARγ) mRNA and protein in hepatocellular carcinoma (HCC) and its association with clinicopathologic features, angiogenesis, and prognosis of HCC. Methods PPARγmRNA and protein were detected with half quantitative reverse transcription PCR (RT-PCR) and Western blot in hepatocyte line L02 and hepatoma carcinoma cell lines HepG2, HepG2.2.15 and HCCLM3. Furthermore, PPARγmRNA and protein in 34 cases of HCC, including HCC tissues and corresponding peri-tumor tissues, were detected with RT-PCR and immunohistochemical method, anti-CD34 and multiplicity staining of anti-CD34 combined with periodic acid-schiff (PAS) were utilized to stain these specimens, and the significance of PPARγin HCC were analyzed. Results PPARγmRNA express in all cell lines, its relative gray scale score is 0.905±0.028, 1.547±0.071, 1.887±0.085 and 1.497±0.061 in L-02, HepG2, HepG2.2.15 and HCCLM3, respectively. It is statistically significant difference between L02 and hepatoma carcinoma cell lines (F=118.84, P<0.05), the expression of PPARγin HepG2.2.15 is highest, comparisons between the other HCC lines, it is statistically significant (F=25.33, P<0.05). PPARγprotein levels are coincidence with mRNA. 27 cases, PPARγmRNA in HCC tissues is higher than their paired non-tumor tissues; all of the HCC tumor cells were positive stained for PPARγprotein, expressed in cytoplasm and/or nucleus of hepatocytes. Hepatocytes of peri-tumor stained negative or weakly positive in cytoplasm, grade of positive staining in two groups is significant (x2=39.62, P<0.05). The clinical characteristic of HCC, such as tumor size, tumor capsule, tendency to recurrence, the background of HBV infection and cirrhosis, is correlated with PPARγmRNA levels and the grade of positive staining; but the patients'ages, gender, Okuda and pTNM stages, serum AFP levels, degrees of histological differentiation and metabasis by follow ups, is not associated with the expression of PPARγ. There is negative correlation between the expression of PPARγand the total life span in HCC patients, it is significant, P value tested by Log rank of Kaplan-Meier curve analysis in various mRNA levels and protein grading is 0.047 and 0.003, respectively. MVD (Microvessel density) in tumor tissues are higher than non-tumor tissues, it is statistically significance (t=18.925, P<0.05). High MVD is correlated with tumor size (P=0.010), tumor capsule breakdown (P=0.001), tendency to recurrence (P=0.004), metastases during follow up (P=0.008) and low differentiation type (P=0.001). The expression of VM (Vascularization mimicry) exists in 7/34 specimens of HCC, MVD in positive specimens of VM are higher (t=16.373, P<0.05). The expression of VM is correlated with clinical TNM stage (P=0.001), tendency to recurrence (P=0.029), and low differentiation type (P=0.001). There was a significant positive correlation between PPARγand high MVD expression (r=0.519, P<0.05), but there was no correlation between PPARγand MV expression (r=0.169, P<0.05). Kaplan-Meier analysis indicated that PPARγoverexpression, high MVD and VM expression were associated with patient's worse disease-free survival; P value tested by Log-rank is 0.013, 0.036 and 0.022, respectively. According to Cox multivariate survival analysis, PPARγand MVD were the independent prognostic factors of HCC (both Pmuti<0.05). Conclusions PPARγover-expressed in HCC, it may be associated with the carcinogenesis and development of HCC; it may be an index to judge prognosis of HCC. [Key Words] PPARγ; Hepatocellular carcinoma; Survival analysis; Biological characteristic; Angiogenesis; Vascularization mimicry The Second Section: Construction, identification and screening of shRNA targeted on PPARγgene Objective To construct shRNA targeted on PPARγgene with pBAsi-hU6-DNA plasmids, and to observe the inhibition efficacy of PPARγactivation after HCCLM3 cells were transfected by shRNA. Methods Duplicate bands DNA sequence were synthesized in accordance with human PPARγ1 gene cDNA sequence, to construct recombinant of PPARγ(pshPPARγ) with pBAsi-hU6-DNA plasmids, then free endotoxin extracted, and assessed by sequencing. pshPPARγrecombinants were transfected into HCCLM3 cells by DOTAP hangosome methods, RT-PCR and Western blot was used to detect the inhibition efficacy of PPARγactivation in 40h and 4w post-transfection. Results pshPPARγis coincidence with targeted sequence, after 40h pshPPARγwere transfected into HCCLM3 cells, the expression of PPARγmRNA and protein were inhibited comparing with normal cells and cells transfected by neo-DNA plasmids (F=29.47, P<0.01). In positive clone cells selcted with 600μg/ml G418 (in 4w), PPARγmRNA and protein also were inhibited significantly by pshPPARγ(F=75.22, P<0.01), their inhibition rate in RT-PCR test is 80.5% and 82.3%, respectively. Conclusions pshPPARγcan lastingly inhibit PPARγactivation in HCCLM3 cells; it may be a tool to study the mechanisms of PPARγof hepatocellular carcinoma in vitro or in vivo. [Key Words] RNA interference; short hairpin RNA; transfection; expression; recombinant The Third Section: Gene silencing targeted on PPARγaffect proliferation and apoptosis of hepatocellular carcinoma Objective To observe the effect on proliferation and apoptosis of HCCLM3 cells with short hair-pin RNA targeted on PPARγgene in vitro and in vivo, and to explore the potential mechanisms. Methods After HCCLM3 cells were transient transfected with pshPPARγplasmid, growth curves were detected by MTT methods, apoptosis rate were analyzed by flow cytometry, and morphocytology of apoptotic HCCLM3 cells were observed by TUNEL methods. Then, animal models were constructed with HCCLM3 cells stable transfected with pshPPARγ, PPARγmRNA and protein in tumor tissues were detected with RT-PCR and Western blot or immuocytochemistry (ICC) methods, growth curves and volumes of tumor were observed, and morphocytology of apoptotic cells were detected by TUNEL method. Moreover, the expression of PCNA and wt-P53 were stained by ICC methods, and MMP2, TIMP2 andβ1 integrin were detected by RT-PCR in vitro and in vivo. Results Cell growth was inhibited in pshPPARγgroup by 71.5%, apoptosis also increased significantly, FCM show the apoptosis ratio is 23.2±4.2%, it is statistically significance between pshPPARγgroup and control group (3.3±0.9% vs 3.1±0.7 %) (F=48.32, P<0.01), morphocytology test is the same as FCM. PPARγmRNA and protein decreased significantly in tumor tissues of pshPPARγgroup, inhibition rate reach to 82%. Stage of latency lengthen, and growth were inhibited, volumes of tumors in pshPPARγgroups are 1.86±0.65 cm3, but 4.86±1.15 cm3 in control groups, apoptosis in situ of tumors increased, it is statistically significance, P<0.05. In vitro and in vivo, PCNA protein decreased, and wt-P53 protein increased. In HCCLM3 cells of pshPPARγgroup, MMP2 and integrinβ1 mRNA decreased significantly, but TIMP2 increased. Conclusions Inhibition of PPARγactivation can decrease the proliferation in HCC. It maybe regulate the balance of MMP2 and TIMP2 via integrin signal way, then decrease the expression of PCNA, and promote wild-type P53 to induce the apoptosis of HCCLM3 cells. Key Words Proliferation; Apoptosis; Proliferating cell nuclear antigen; Wild-type P53; Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; Integrin The Fourth Section: Gene silencing targeted on PPARγinfluence metastasis and angiogenesis of hepatocellular carcinoma Objective To observe the effect on invasion, metastasis, and angiogenesis in HCC when PPARγactivation was inhibited by shRNA targeted at PPARγgene of HCCLM3 cells in vitro and in vivo, and to explore the potential mechanisms. Methods Boyden chambers were utilized to observe the effect on cell adhesion, migration, invasion and locomotion of HCCLM3 cells, meanwhile, the effect on lumens formed by ECV 304 cells was observed in models which HCCLM3 cells co-cultivated with ECV 304 cells post-transient transfected in vitro. Animal models of cancerometastasis were established by HCCLM3 cells stable expressed pshPPARγor not with nude mouse, pulmonary metastasis (PM) was assessed by HE staining and immuocytochemistry (ICC) staining with hepatocyte, graded and counted in serial sections of lung tissues. MVD and VM in transplantation tumor were stained by immuocytochemistry staining of CD34 and multiplicity staining with CD34 combined with periodic acid-schiff (PAS). Integrinβ1 expression in lung tissues, VEGF and TSP1 expression in tumors was detected by ICC staining and RT-PCR methods. Results Cells crawled over semipermeable membrane of boyden chambers were 68±7 in pshPPARγgroups, it is less than control groups (281±10 and 302±14), it is statistical significance (F=548.72, P<0.01). Cells permeated through ECM were 17±3; it is also less than control groups (F=258.19, P<0.01). Time when cells healed the wound were 6.40±1.14d, it is longer than control groups (F=17.58, P<0.01). Ability of cell adhesion decreased significantly, after 2h, cell counts are 49±9, 83±13 and 88±11 in pshPPARγgroup and two control groups, respectively, it is statistical significance (F=17.39, P<0.01). Lumens formed by ECV 304 cells were weakened in pshPPARγgroup. PM in pshPPARγgroup were smaller than in control group, and total amounts in lung tissues were also less (t=18.69, P<0.05). MVD in transplantation tumors are scarce and punctiform, while in control group, MVD are intensive and annuliform, it is statistically significance between two groups, P<0.05. VM exist in transplantation tumor, sparse and tiny in pshPPARγgroup, meanwhile, VEGF mRNA and protein decreased significantly, TSP1 increased. Integrinβ1 expression in lung tissues was not difference between two groups. Conclusions PPARγinhibition can decrease the ability of invasion, metastasis and angiogenesis in HCC; it may be via integrin signal way, to regulate the balance of MMP2 and TIMP2, and affect the secretion of VEGF/TSP1.
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