| Over the last decade, more than 100 Forkhead box ?FOX? class members have been identified,with key roles in development,differentiation,proliferation,apoptosis,stress resistance and metabolism. Forkhead box O ?FOXO? subfamily consists of transcription factors ?FOXO1,FOX03,FOXO4,and FOX06? that regulate expression of target genes involved in DNA damage repair response, apoptosis,metabolism, cellular proliferation, stress tolerance, and longevity. FOXO1 plays an important role in regulation of metabolism. Dephosphorylation of FOXO1 was an activator for the transcription of the target gene. In recent years, several in-vitro studies have found that FoxO1 dephosphorylation, from the cytoplasm into the nucleus, start down stream target gene transcription, is the key factor in the occurrence of insulin resistance,and may affect the liver cholesterol secretion. FoxO1 are considered critical effectors of the pathway regulating hepatic glucose production.Research result showed that FoxO1 increased triglyceride accumulation and decreased fatty acid oxidation. Epidemiologic studies defined there was no positive correlation between hypercholesteremia and cholelithiasis, but positive correlation between hypercholesteremia and hypertriglyceridaemia. The overexpression of FoxO1 in the liver may cause the cholesterol gallstones. In patients with metabolic syndrome, the lipid metabolism and glycometabolism were influenced by the nuclear receptors FXR as the dephosphorylation of FOXO1. Normal concentrations of insulin inhibit FoxOl and then promoted the transcription of CYP7A1. According to this background, we assess the relationship between gallstone disease and FoxO1 expression by the means of up regulate and down regulate the FoxO1 in vitro.Construct the mice model of up regulate or down regulate FoxO1 in vivo through the recombinant vector or small interferenceadenovirus RNA to expound the molecular mechanism between FoxO1 and cholesterol gallstones.Objective:The stable over expression and low expression of FOXO1 HepG2 cell line were constructed by using lentivirus infection technique and RNA interference technique.To study the genes related to lipid metabolism and transportation.To construct mouse FoxO1 gene shRNA recombinant adenovirus vector and mouse FoxO1 gene overexpressing recombinant adenovirus vector, inject these adenovirus into C57BL/6 mouse through tail vein. To study the role of FoxO1 gene in cholesterol gallstone formation.Methods:1. Study on the regulation of gene transcription by FOXO1 in human hepatocyte cell1) FOXO1 gene cloning.The FOXO1 gene was cloned from human liver tissue by PCR. The lentivirus vector pEZLv203 was used to construct the expression vector. The result was identified by sequencing.2) Design and synthesis of ShRNA interference target sequences. Three pairs of shRNA interference sequences target to the human gene FOXO1 were designed. These sequences were synthesized by Shanghai Gene Pharma Co.Ltd. The results were identified by sequencing.3) Establishment of the FOXO1 overexpressing HepG2 cell lines?1? Construction of human FOXO1 gene overexpressing recombinant lentivirus vector. The cloned FOXO1 vector was sent to Guangzhou FulenGen company to synthesis, packaging, purification and got the pEZ-FOXO1 lentivirus.?2? Identification and selection of HepG2 cell line with stable overexpressing FOXO1.a. FOXO1 overexpressing recombinant lentivirus was used to infect HepG2 cells. Cells were harvested respectively after 72 hours and 96 hours culture. FOXO1 mRNA was detected using real-time quantitative PCR and FOXO1 protein was detected using Western Blot.b. The lentivirus-infected HepG2 cell line was cultured with 1.0 ?g/mL puromycin, and harvested after 96 hours culture. FOXO1 mRNA was detected using real-time quantitative PCR and FOXO1 protein was detected using Western Blot.c. Culture and collect the HepG2 stable cell lines, extract total RNA and protein. mRNA of the lipid metabolism and transcription genes were detected using real-time quantitative PCR and the protein was detected using Western Blot if there was a difference detected in mRNA level.4) Establishment of the FOXO1 low expression HepG2 cell line?1? Construction of human FOXO1 gene ShRNA recombinant lentivirus vector. Three pairs of shRNA interference sequences target to the human gene FOXO1 were designed and synthesized. Send them to Gene Pharma Biotech ?Shanghai? Co, Ltd. to synthesize, packing, purification.?2? Identification and selection of stable low expression of FOXO1 HepG2 cell linea. FOXO1 shRNA interference Lentivirus was used to infect HepG2 cells and harvested respectively after 72 hours and 96 hours culture.FOXO1 mRNA was detected using real-time quantitative PCR and FOXO1 protein was detected using Western Blot.b. The lentivirus-infected HepG2 cell line was cultured with 1.0 ?g/mL puromycin, and harvested after 96 hours culture. FOXO1 mRNA was detected using real-time quantitative PCR and FOXO1 protein was detected using Western Blot.c. Culture and collect the HepG2 stable cell line, extract total RNA and protein. mRNA of the lipid metabolism and transcription genes were detected using real-time quantitative PCR and the protein was detected using Western Blot if there was a difference detected in mRNA level.5) Real-time fluorescence quantitative PCR ?qRT-PCR?. The total RNA was extracted following the PAXgene RNA kit operation instruction. Agarose gel electrophoresis or the Agilent 2100 assay was used to analysis the RNA integrity. RNA was reverse transcribed to cDNA according to the ABI RT kit manual. Gene expression was detected on ABI7900 according to the ABI lifetech Taqman Gene Expression Analysis System. The results were analyzed by 2-??Ct method.6) Western Blot. Cell lysis buffer was added into the harvested cell,and cells were disrupted by sonication. The protein concentration was measured by BCA method and diluted to the same concentration before loading. 5.0%concentrated gel electrophoresis 80V 30 min, then 10% separation gel continued electrophoresis 120V 60 min. 400 mA current was used to transfer film 120 min, and then 5% skim milk closed for two hours. Add the first antibody and incubation over nigh. Add the secondary antibody and incubation 2h before took photo. The results were analyzed using Image J analysis software.2. Study of the relationship of between the FoxO1 and cholesterol gallstone in vivoa) FoxO1 gene Cloning. The FoxO1 gene was cloned from mouse liver tissue by PCR. The shuttle plasmid M3Flag-Fox01 was constructed using PDC316-MCS2 adenovirus vector. The results were identified by sequencing and Western Blot.b) Design and Synthesis of ShRNA interference target sequences. Three pairs of shRNA interference sequences target to the gene FoxO1 were designed and synthesized. These sequences were inserted into the PGMAV-S1 adenovirus vector. The results were identified by sequencing and Western Blot.c) Produce adenovirus. The AdMax system was used to produce adenovirus,and the CsCl density gradient centrifugation and dialysis were used to purify the adenovirus.Virus titer was determined by gradient dilution method.d) Animal grouping and handling. Fourty 4-week-old to 6-week-old C57BL/6 male mice were obtained from Shanghai Research Center For Model Organisms ?Shanghai,China?. Animals were housed in humidity and temperature controlled room with reverse cycle lighting. At 8-10 weeks of age, the mice were randomly divided into four groups. The four groups were intravenously injectied with recombinant adenovirus FOXO1 over-expression vector/control and recombinant adenovirus FOXO1 RNAi expression vectors/control. All mice were maintained with water and lithogenic diet contains 1.5% cholesterol and 0.5% cholic acid ?Trophic Animal Feed High-tech Co.Ltd, China?. Surgery was performed on mice after fasted over 12h with free access to water.e) Collection of the sample. Mice were anaesthetized with anintraperitoneal injection of pentobarbital ?4.5mg/100g body weight?, blood samples were collected from the retro-orbital venous plexus of the mice and the abdominal cavity was exposed through aventral incision, the cystic duct was ligated and the GB removed. Mice were then killed and the liver removed. The GB was examined visually for the presence of stones or sediment. Bile was aspirated by puncturing the GB with a fine needle and liver were then stored at -80?for further lipid analysis.f Measure the level of cholesterol, phospholipids and bile in bile. Take 20?L bile and add 180 ?L physiology saline diluted 10 times, Roche Modular P automatic biochemical was used to analysis. Using the Carey table to calculate the cholesterol saturation index ?CSI?.g) Fluorescence quantitative PCR. qRT-PCR was used to detect the expression of lipid metabolism related genes in C57BL/6 mice, and its molecular mechanism was studied.Western Blot was used to detect the protein level if there was a difference detected in mRNA level.Results:1. One cell line with overexpressing FOXO1 and one with low expression FOXO1 were obtained1) LV3-FOXO1-shRNA2 was identified as the best interference of FOXO1.The mRNA expression level relative blank was 0.17 at 72 hour and 0.19 at 96 hour.The protein relative to blank were 0.899 at 72 hour and 0.848 at 96 hour.While the overexpression lentivirus pEX-FOXO1 mRNA level of FOXO1 relative to the blank were 8.94 at 72 hour and 6.17 at 96 hour. The protein level relative to the blank were 0.027 at 72 hour and 1.400 at 96 hour.2) After 96 hours culture with 1.0 ?g/mL puromycin, the level of LV3-FOXO1-shRNA2 mRNA relative to blank was 0.527 ?n=3, P<0.05 ? , the protein level relative to blank was 2.01 ?n=3, P<0.05?. The level of pEX-FOXO1mRNA relative to blank was 105.10 ?n=3, P<0.05?, the protein level relative to blank was 0.62 ?n=3, P<0.05?.2. Effects of FOXO1 on CYP7A1 and FXR gene expression. When the FOXO1 expression decreased, the CYP7A1 gene level increased up to around 4 times?t=21.03, P<0.01?, FXR gene level increased up to around 1.4 times ?t=3.047, P<0.05?.While the FOXO1 expression increased, CYP7A1 gene decreased around 4 times?t=21.85, P<0.01?,and FXR gene decreased to around 0.8 times ?t=12.62, P<0.05?.3. FoxO1 overexpressing adenovirus vector and lowexpressing adenovirus vector were constructed successfully1) FoxO1 overexpressing adenovirus vector was transfected into 293T cells.Total protein was extracted after cultured for 48h. Western Blot results showed that the vector could be expressed in 293T cells, suggesting that the vector was constructed successfully.2) Three FoxO1 shRNAs adenovirus were transfected into murine ST2 cells.Total Protein was extracted after cultured for 72h. Base on the Western blot result, the FoxO1-shRNA2 with the best interference effect was selected for follow-up experiments.3) Amplification of constructed adenovirus titer results. FoxO1 overexpression ?FoxO1-OE? and FoxO1 RNA interference ?FoxO1-shRNA?adenovirus were obtained with titer of 1X1011 PFU/mL.4. Results of in vivo1) FoxO1 overexpressing group and its control group, feeding with lithogenic diet for 4 weeks, observe the gallstones in gallbladder, collect the specimen.The control group of FoxO1 over expression, 4 mice with gallstone ?4/10?,stone rate of 40%. FoxO1 over expression group, 8 mice with gallstone ?8/10?,stone rate of 80%. FoxO1 interference group and its control group, feeding the lithogenic diet for 6 weeks, observe the gallstones in gallbladder, collect the specimen. FoxO1 interference control group, 10 mice with gallstone ?10/10?,stone rate of 100%. FoxO1 interference group, 2 mice with gallstone ?2/10?,stone rate of 20%.2) Comparing with the control group, the cholestasis of gallbladder bile was significantly decreased in FoxO1 RNA interference group, the total bile acid was significantly increased, CSI decreased significantly, which was statistically significant. Phospholipid changes were not statistically significant but showed a decreasing trend. While the FoxO1 overexpression group had the opposite trend.3) The serum levels of CHOL, LDLC and LDL/HDL in FoxO1 RNA interference group were decreased comparing with the control group, and the difference was statistically significant. TG, GLU and HDLC were not showed significantly difference. Comparing with the control group, the levels of serum CHOL, LDLC and LDL/HDL were increased in FoxO1 overexpression group, the difference was statistically significant. TG and GLU were not showed significantly difference.4) The mRNA level of Cyp7al and Fxr in the FoxO1 RNA interference group was significantly higher than that in the control group. While the FoxO1 overexpression group showed the opposite trend. We found that the mRNA level of Cyp7b1, Cyp8bl and Cyp27al were increased comparing with the control group, Cyp8bl showed statistically significant.5) Comparing with the control group, the protein level of Cyp7al and Fxr in FoxO1 interference group increased significantly. While the FoxO1 overexpression group showed the opposite trend. These results were consistent with the results of mRNA level.Conclusions:1. FOXO1 low expression and high expression of lentivirus vectors were constructed successfully. The stable HepG2 cell lines of low expression of FOXO1 and high expression of FOXO1 were constructed successfully.2. The mRNA level of CYP7A1 and FXR gene changed following with the mRNA level of FOXO1 gene change. While the lipid transport genes such as ABCG5,ABCG8, ABCB4 and ABCB11 did not show change accordingly, suggesting that FOXO1 gene may be involved in the formation of cholesterol stones by affecting the metabolism of cholesterol and bile acids.3. The adenovirus vectors with FoxO1 high expression and low expression were successfully constructed. C57BL/6 mice were injected with these adenovirus through the tail vein. The mice model with low FoxO1 and high FoxO1 were constructed successfully.4. FoxO1 over expression in mice, have a certain role in promoting gallstone formation. While FoxO1 RNA interference has a significant anti-stone effect in mice experiment. Suggesting the feasibility of FoxO1 as a target for the prevention and treatment of cholesterol gallstones.5. Cyp7al and Fxr gene expression changes following the FoxO1 gene expression changes. Abcg5, Abcg8, Abcbll and other lipid transport genes did not show change accordingly. Suggesting that FoxO1 gene may be involved in the formation of cholesterol stones by affecting the metabolism of cholesterol and bile acids. When FoxO1 low expression, Cyp8b1 expression increased, suggesting that inhibition of FoxO1 may increase the proportion of cholic acid and deoxycholic acid to promote the dissolution of cholesterol, which may be one of its anti-stone mechanism. |
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