The Effecct Of SMP30on Development Of Hepatocellular Carcinoma

Objectives1. To observe the effect of cell passage on the protein expression of SMP30, and to discuss the secretary characteristics of SMP30.2. To observe the trend of SMP30changes in development of hepatocellular carcinoma (HCC), confirm the relationship of SMP30expression level with the progress of HCC.3. To study the effect of DNA methylation and siRNA on the expression of SMP30during the occurrence and development of hepatocellular carcinoma.4. To study the effect of SMP30on the biological characteristics of hepatocellular carcinoma cell (apoptosis, proliferation, invasion and cell cycle).Methods1. The bioinformatics methods were utilized to predict the secretary characteristics of SMP30. Hep G2cell was cultured with DMEM, and gathered every each2generations to extract total cellular protein, membrane protein, cytosol protein, nuclear protein; at the same time,the cell culture supernatant was gathered and concentrated with ultra filtration techniques. The content of SMP30was detected by western blotting.2. Hep G2cells were injected into subcutaneous tissue of nude mice with injector to establish the implantation tumor model in nude mice. The nude mice were divided into three groups randomly; every group had10nude mice. The other10nude mice without implantation tumor were selected as control group. According to different drawing materials time, blood was taken from the orbital veins to separate serum, detecting SMP30protein with western blot; the mice were sacrificed and the tumor tissue was taken at different time points, one part of them were made into paraffin block, detecting SMP30protein with immunofluorescence histochemistry. Another part were storaged in refrigerator to extract total RNA and genome DNA, total RNA were used for reverse transcription and fluorescence quantitative PCR; genome DNA was modified by sodium bisulfate for methylation-specific PCR.3.40cases of fresh hepatocellular carcinoma tissue, tumor-adjacent tissues and normal liver tissue far from the tumor margin of more than5cm were surgically resected from patients with hepatocellular carcinoma who underwent hepatectomy in The First Affiliated Hospital of Guangxi Medical University from2012February to2012February. Extracted total RNA and genome DNA, total RNA were used for reverse transcription and fluorescence quantitative PCR; genome DNA was modified by sodium bisulfate for methylation-specific PCR.4. SMP30gene sequence was retrieved in the GENBANK, three small interfering RNA (siRNA) were designed and then synthesized after homology search without error. Hep G2cells were transfected by means of Xfect transfector, FAM green fluorescent was used to detect transfection situation. Then FQ-PCR and Western-blot were used to detect silence efficiency of each group, and then screened out the highest silence efficiency group. Experiment group and negative control group were transfected with optimal MOI and set up blank control group. FQ-PCR and Western-blot were used to detect SMP30mRNA and protein expression in Hep G2cells after SMP30gene was silenced, MTT method and absorbance were used to draw Hep G2cell growth curve, and detect inhibition effect on proliferation of Hep G2; Transwell chamber was used to detect cancer cell migration and invasion ability; Cell cycle distribution and apoptosis were quantified by flow cytometry; Western-blot were used to detect Bax/Bcl-2expression in Hep G2cells after SMP30gene was silenced.Results1. SMP30was a non classical pathway secretary protein by the prediction of Secretome2.0.2. The expression level of SMP30was constant during the cell passage (P>0.05); SMP30protein was found in cell culture supernatant.3. All the Hep G2cells injected into nude mice changed into tumor, according to the histological examination, they had the character of hepatocellular carcinoma. Immunofiuorescence staining revealed that SMP30expression were significantly higher in two-week group than that in the other two groups (P＜0.05); there was no difference between four-week group and six-week group (P＞0.05).4. The SMP30expression in serum of nude mice were significantly higher in two-week group than the other two groups, There was no SMP30expression in serum of nude mice without tumor. 5. The relative expression of SMP30mRNA were calculated with2-△△Ct, SMP30mRNA level were significantly higher in two-week group than that in the other two groups with significant difference (P＜0.05).6. The methylation rate of SMP30gene in human hepatocellular carcinomal transplanted in nude mice were37.74%(two-week group),57.78%(four-week group) and58.14%(six-week group). Two-week group was lower than the other two groups (P＜0.05), there was no different in four-week group and six-week group (P＞0.05). The methylation rate of SMP30gene fresh hepatocellular carcinoma tissue (57.5%) was higher than tumor-adjacent tissues (17.5%) and normal liver tissue (12.5%).7. The interference efficiency of RGN-homo-1681on SMP30mRNA (65.58±12.69%) and SMP30protein (68.62±13.77%) were the highest, so selected as the siRNA in experiment group.8. Hep G2cell proliferation and growth curve was made, there was no difference in blank group and control group (P＞0.05), The level of cell growth in experiment group was higher than the other two (P＜0.05).9. The invaded cell number in blank group, control group and experimental group were54.7±5.5,60.5±10.6and120.8±15.8respectively; comparing with blank group and control group, the invaded cell number in experimental group significantly increased, difference between them was significant (P＜0.05). The invaded cell number in blank group and control group had no significant difference (P＞0.05). 10. The percent of apoptosis in three groups had no difference (P＞0.05); after UV irradiation, the percent of apoptosis in blank group and control group were (25.54±4.33)%and (26.45±5.37)%, they had no significant difference (P＞0.05), the percent of apoptosis in experimental group was(45.45±11.36)%, compared with the other two groups, the difference was obvious(P＜0.05).11. The Bcl-2in Hep G2was detected after UV irradiation. The relative expression of Bcl-2in blank group and control group were44.67±2.76and47.25±5.45, they had no significant difference (P＞0.05), The relative expression of Bcl-2in experimental group was24.33±2.53, compared with the other two groups, the difference is obvious(P＜0.05).12. The Bax in Hep G2was detected after UV irradiation. The relative expression of Bax in blank group and control group were45.13±3.26and46.25±5.45, they had no significant difference (P＞0.05), the relative expression of Bax in experimental group was65.38±5.46, compared with the other two groups, and the difference was obvious (P＜0.05).13. The cell proliferation index in blank group, control group and experimental group were26.25±5.64%、25.45±3.89%and44.38±5.28%respectively, compared with blank group and the control group, the cell proliferation index in experimental group significantly increased, difference between them was significant (P＜0.05). The cell proliferation index in blank group and control group had no significant difference (P＞0.05).Conclusions1. SMP30is a non classical pathway secretary protein. 2. The protein expression level of SMP30is constant during the cell passage.3. The expression level of SMP30is closely related to the development of hepatocellular carcinoma.4. The methylation rate of SMP30gene in HCC can decrease the expression of SMP30protein; there is no correlation between the gene methylation of SMP30and the protein expression of expression of AFP.5. SMP30can inhibit the invasion capacity of Hep G2.6. SMP30can inhibit the apoptosis of Hep G2caused by a UV irradiation by decreasing the ratio of Bax/Bcl-2.