The Expression And Mechanism Of LDOC1 In Papillary Thyroid Carcinoma

Papillary thyroid carcinoma(PTC) is a differentiated malignant tumor originated from thyroid follicular epithelial cells, is also the most common pathological type of thyroid carcinoma. The incidence of thyroid cancer is rising in recent years, and PTC grows faster than others. The patients of PTC which seriously threatening people’s health is becoming younger, more female. PTC doesn’t show out typical clinical symptoms except that thyroid nodules are slow growing, therefore it is easy to be misdiagnosed, and thus missed the best treatment period. Although the overall prognosis of PTC is good, but there are still a few PTC very aggressive, appeared on the tumor invasion and lymph node metastasis in early stage, but also prone to repeated recurrence and metastasis after operation. Therefore, the urgent issue we faced in clinical and basic research of PTC is to discuss the related target molecules and molecular mechanisms in the occurrence and development of PTC, to explore new effective PTC molecular biology diagnosis, treatment and prognosis of markers.Leucine zipper down-regulated in cancer 1(LDOC1) is differentially expressed in RNA genes, discovered by Nagasaki using differential display technology in 1999.LDOC1 gene was mapped on Xq27 and encodes a protein with leucine zipper structure domain and SH3-binding mode ordered with homodimer or heterodimer to regu Late gene transcription formed by the leucine zipper domain with othertranscription factors, also through the SH3 binding combined with signal pathway proteins involved in intracellular signal transduction. It was reported that LDOC1 was differentially expressed in prostate cancer and hepatocellular carcinoma tissues.Another study shows that the expression of LDOC1 in cervical cancer and ovarian cancer is decreased due to DNA methylation. LDOC1 can lead to neoplastic transformation of oral mucosa cells, as it was reported. LDOC1 in some tumors and inflammatory diseases is differentially expressed, closely related to tumor formation and cell apoptosis, however, these results in different cell types, different disease status are not entirely consistent. Until now, the expression distribution and clinical value of LDOC1 in PTC is not clear. And the research is lacking in the effect of LDOC1 on PTC cell biology function, at home and abroad.Nuclear factor-κB(NF-κB) is the key transcription factor related to many cytokines expression, plays an important role in the immune response, cell proliferation, apoptosis, invasion, migration and angiogenesis formation aspects, and a number of research evidences indicate that NF-κB continued activation in a variety of tumor cells including PTC. At present, there are some reports about the effect of LDOC1 on NF- κB activity, but these results are not consistent. Nagasaki’s research shows that in the pancreatic cancer cell line Bx PC-3, LDOC1 inhibits NF-κB transcription activity which stimulated by TNFα and PMA in a dose dependent manner. Lee et al. confirmed that in OC3 and TW2.6 cells, LDOC1 effectively inhibited NF-κB transcriptional activity which stimulated by TNFα in a dose dependent manner through the NF-κB luciferase reporter assay, immunoblotting furtherly confirmed that LDOC1 down-regulated the expression of p65 protein in a dose dependent manner. Research on biliary atresia by song et al. indicate that LDOC1 increases NF-κB activity, activation of inflammatory cytokines, leading to biliary epithelial cell injury. For PTC, whether LDOC1 effects NF-κB activity has not been reported.Transforming growth factor-β plays an important role in many aspects of cell growth and differentiation, tissue repair and immune function etc.. TGF-β is a double-edged sword, which could exhibit bidirectional function of tumor promotion or inhibition in different environments. As for thyroid tumors, TGF-β mediatedgrowth inhibition was deprived by NF-κB activation. After giving NF-κB inhibitors,TGF-β recovers growth inhibitory and apoptosis inducing effect. We speculate that if LDOC1 affects the activity of NF-κB of PTC cells, will it resume inhibitory reaction of PTC cells to TGF-β signal? Therefore, we carried out the following research in this experiment.Part one The expression and clinical significance of LDOC1 in papillary thyroid carcinoma ObjectiveTo observe the expression of LDOC1 in PTC tissues, PTC cell lines and normal thyroid tissues, and to explore the potential clinical value of LDOC1 in PTC.Methods1. Collecting fresh 126 cases of PTC cancer tissue specimens, 47 cases of contralateral normal thyroid tissue, 56 cases of PTC tissues and 33 normal tissues of paraffin-embeded blocks.2. Using immunohistochemical and q RT-PCR method to detect the expression of LDOC1 protein and m RNA in PTC carcinoma tissue and normal thyroid tissue,and analyzing the relationship between the LDOC1 expression level and the clinicopathologic parameters of PTC patients.3. Detecting the expression of LDOC1 in PTC cells to provide a theoretical basis for gene therapy research.4. Detecting the expression of P65 in PTC samples with IHC. Analyzing the correlation between LDOC1 and P65 expression.Results1. The expression level of LDOC1 m RNA in PTC cancer was lower than that of normal tissue, the difference was statistically significant(P < 0.01). According to the tumor T status in TNM staging of AJCC standard, LDOC1 m RNA level decreased in T1, T2, T3 and T1 > T2 >T3, the difference was statistically significant(P < 0.05).2. The positive expression rate of LDOC1 in PTC was lower than that of normal thyroid tissue, the difference was statistically significant(P < 0.01). The expression of nuclear P65 in PTC cancer tissue was higher than that of normal thyroid tissue, the difference was statistically significant(P < 0.01). Spearman correlation analysis showed that LDOC1 was negatively correlated with the expression of nuclear P65 in the PTC tissue(χ2 = 7.458, P = 0.006).3. Compared with the normal thyroid tissue, the expression level of LDOC1 m RNA and protein decreased in PTC cells, the difference was statistically significant(P < 0.05), in which TPC-1 cells decreased more obviously.Conclusion1. The lower expression of LDOC1 in PTC tissue and its’ relating to primary tumor size, suggests that it may participate in the process of the occurrence and development of PTC.2. LDOC1 was negatively correlated with the expression of nuclear P65 in the PTC tissue.Part two The Effects of LDOC1 on biological behaviour of papillary thyroid cancer cells TPC-1.ObjectiveProviding a theoretical basis for further study on the molecular mechanisms involved in the pathogenesis of PTC through in vivo and in vitro experimental study about the contribution of LDOC1 on TPC-1 cells.Methods1. Infecting TPC-1 cells with exogenous h LDOC1 c DNA through recombinant lentiviral vector technology. Using the fluorescence microscope to observe the infection effect, assessing the infection efficiency by flow cytometry. Detecting the level of LDOC1 in Lv-LDOC1, Lv-NC and UNT groups with q RT-PCR and western blot.2. Inoculating cells 300 per pore in six-pore plate, observing colony forming ability 14 days later.3. Detecting the changes of apoptosis rate of UNT, LV-NC and LV-LDOC1 by TUNEL after the up-regulated expression of LDOC1.4. Through observing the healing velocity of the artificial scratch on monolayer cell to indirectly evaluate the cell migration ability.5. Observing the changes of invasion ability of TPC-1 cells after LDOC1 expression using transwell assay with spread matrige glue.6. Establishing nude mice xenografted model by using subcutaneous injection of stably infected TPC-1 cells to further observe the effect of tumorigenic ability of LDOC1 on papillary thyroid carcinoma cells in vivo.Results1. With the help of recombinant lentiviral vector, we infect TPC-1 cells with exogenous h LDOC1 c DNA successfully. Under the fluorescence microscope, we saw more than 90% of the cells expressing green fluorescent; the transfection efficiency was above 95% detected by flow cytometry; the results of q RT-PCR and western blot showed that LDOC1 in group Lv-LDOC1 was higher than that of UNT and Lv-NC group, the difference was statistically significant(P < 0.05), UNT and Lv-NC groups had no difference(P > 0.05).2. LDOC1 inhibits the clone formation ability of TPC-1 cells. Compared with UNT and Lv-NC group, the number of colony formation in Lv-LDOC1 group was the lowest, the difference was statistically significant(P < 0.01), UNT and Lv-NC between groups had no difference(P > 0.05).3. The apoptosis rate of UNT, Lv-NC and Lv-LDOC1 groups was respectively(9.07 ± 1.45)% and(10.33 ± 0.91) and(16.80 ± 2.46)% detected by TUNEL.Compared with the other groups, the apoptosis rate in Lv-LDOC1 group was the highest(F = 17.29, P < 0.01), the apoptosis rate of UNT and Lv-NC groups had no difference(P > 0.05).4. The migration ability of the cells in Lv-LDOC1 decreases significantly.According to the monolayer cell scratch test, after doing cell culture without serumfor 48 h, the distance of scratch reduced greatly in UNT and Lv-NC groups and the scratch was nearing healing, while in Lv-LDOC1 group, the distance of scratch was wider than that in the other two groups(all P < 0.01).5. LDOC1 has no obvious effect on cell invasion and metastasis of TPC-1. The results of transwell invasion assay showed that compared with the UNT and Lv-NC group, the cell number of polycarbonate membrane under-chambers surface in Lv-LDOC1 group did not decrease significantly(P > 0.05), after cultured for 24 hours and 48 hours in the transwell.6. The final weight and final volume of Lv-LDOC1 was less than UNT and Lv-NC groups, the difference was significant(F = 7.538, P < 0.05/F = 59.830, P <0.001); the UNT group and Lv-NC group no significant difference(P > 0.05); The nude mice transplanted tumor tissue sections stained with hematoxylin and eosin(HE)showed that tumor cell nucleolus was hyperchromatic, clear, visible mitoses, tumor tissue surrounded by few fibrous cells in UNT and Lv-NC groups, while a great deal of fibrous tissue and few tumor cells in Lv-LDOC1 group. The apoptosis of tumor cells in group Lv-LDOC1 was higher than that of UNT and Lv-NC group, the difference was statistically significant(P < 0.05).Conclusion1. LDOC1 gene can inhibit the proliferation and migration of papillary thyroid carcinoma cells TPC-1, promote cell apoptosis, but not effect the cell invasion ability obviously.2. LDOC1 gene can inhibit TPC-1 cells growth in nude mice.Part three LDOC1 promotes papillary thyroid carcinoma cells apoptosis by regulating NF-κB activity ObjectiveTo explore the possible mechanism of LDOC1 in promoting TPC-1 cells apoptosis, to provide experimental and theoretical basis for LDOC1 as a therapeutic target.Methods1. Seeding the same number of UNT, Lv-NC and Lv-LDOC1 cells(1000 / pore)into 96 pore plates, using the CCK-8 method to detect the number of cells in three groups daily for a week.2. After synchronizing Lv-NC and Lv-LDOC1 cells, analyze the cell cycle with flow cytometry.3. Detecting the apoptotic rate with flow cytometry after staining the Lv-LDOC1 and Lv-NC cells with APC-Annexin V and 7-AAD; Analyzing the expression of apoptosis related gene in the three groups UNT, Lv-NC and Lv-LDOC1; Detecting level of Bax, c-myc and Bcl-xl m RNA with q RT-PCR; Detecting the protein level of expression of Bax, cl-PARP, cl-Caspase3, c-Myc and Bcl-xl with western blot.4. Dropping in 10 ng/m L of TNFα to stimulate Lv-LDOC1 and Lv-NC cells for30 minutes, and detecting distribution of NF-κB p65 protein in the cytoplasm and nucleus with western blot and immunofluorescence; Stimulating Lv-LDOC1 and Lv-NC cells for 24 hours with 10 ng/m L of TNFα, and using p NF-κB-Luc dual luciferase reporter genetic experiment further verify the transactivation ability of NF-κB.5. To stimulate the Lv-LDOC1 and Lv-NC cells 5 days with different doses of TNFα(0~20 ng/m L) and use CCK-8 assaying cell viability; To stimulate the Lv-LDOC1 and Lv-NC cells with 10ng/m L TNFα for 30 minutes or 24 hours(IκBα),and assay Bcl-xl, Bax, c-Myc, IκBα protein levels with western blot.6. Dropping r TGF-β1 5 ng/m L in Lv-NC and Lv-LDOC1 cells for 24 h or 48 h.Assaying nuclear p65 and total IκBα protein with western blot; Assaying the changes of cell cycle with flow cytometry after dropping r TGF-β1 5 ng/m L for 48 h; Testing cell viability with CCK-8 after dropping r TGF-β1 5 ng/m L in Lv-NC and Lv-LDOC1 cells for 12 h, 24 h, 48 h.Results1. LDOC1 can inhibit the growth of TPC-1 cells. The number of cells in Lv-LDOC1 group was significantly lower than that in UNT and Lv-NC group sincethe third day. The difference was statistically significant(P < 0.05), and there was no significant difference between UNT and Lv-NC group.2. The proportion of Sub G1 phase cells in Lv-LDOC1 was higher than that in group Lv-NC, and the two groups were compared with P < 0.01. LDOC1 might inhibit cell proliferation by promoting TPC-1 cells to Sub G1 phase transition.3. LDOC1 promotes apoptosis of TPC-1 cells. The apoptosis rate of cells in Lv-LDOC1 groups(17.1%) was lower than that in Lv-NC group(11.8%), the difference was statistically significant(P < 0.05); Compared with UNT and Lv-NC groups, the expression of promoting apoptosis gene Bax m RNA was up-regulated in Lv-LDOC1 group by q RT-PCR, while the anti-apoptosis gene c-Myc m RNA and Bcl-xl m RNA levels decreased; in LDOC1 transfection group Bax, cl-PARP,cl-Caspase3 protein levels increased and c-Myc, Bcl-xl protein levels reduced by western blot, the differences were statistically significant(P < 0.05).4. LDOC1 inhibites the basal and stimulated NF-κB activation by TNFα.Western blot showed that TNFα could stimulate the activation of NF-κB, promote the increase of nuclear p65, the expression of LDOC1 reduced the basal and inducing p65 stimulated by TNFα in the nucleus, the difference was statistically significant(P <0.05). Although, LDOC1 could reduce the expression of the total number of p65, it still not reach the statistical standards(P > 0.05); Immunofluorescence experiments also showed that LDOC1 lowered the amount of nuclear p65; Finally, the p NF-κB-Luc luciferase reporter gene experiment further verified that the basal and inducing NF-κB activity stimulated by TNFα could be significantly inhibited by LDOC1(P < 0.05).5. LDOC1 enhances the apoptotic effect induced by TNFα. CCK-8 assay results showed that under the action of any dose TNFα for 5 days, the cell activity in LDOC1 group was lower than group Lv-NC, and LDOC1 reduced cell viability in TPC-1;Western blot analysis revealed that LDOC1 made IκBα increased, up regulation of the expression of apoptotic protein Bax and down regulation the anti-apoptotic protein c-myc level, all P < 0.056. Over expression of LDOC1 could promote the response of TPC-1 cells to TGF-β1 inhibitory signal. Western blot results showed that under the function ofTGF-β1 5 ng/m L, the amount of nuclear p65 in group Lv-LDOC1 was lower than that in group Lv-NC at 0 h, 24 h, and 48 h, the difference was statistically significant(P <0.05); Cell cycle analysis showed that LDOC1 promoted TPC-1 cells to the sub G1 phase transition. CCK-8 assay results showed that LDOC1 reduced the cell viability under the function of the r TGF-β1 5 ng/m L at different time points, all P < 0.05.Conclusion1. Overexpression of LDOC1 inhibits TPC-1 cell proliferation, promotes apoptosis and the expression of pro-apoptosis related protein. Its mechanism may achieved through the inhibition of basal and inducing NF-κB activity stimulated by TNFα.2. LDOC1 improves the reactivity of TPC-1 cells to TGF-β1 inhibitory signal by inhibiting the activation of NF-κB.