The Epigenetic Regulation Mechanism Of Caveolin-1 Gene And Its Effect On The Activation Of Epidermal Growth Factor 2 In The Proximal Gastric Cancer

Gastric cancer(GC) is a common malignant tumor of digestive tract. The patients often has no specific symptoms in early stage and most of them have been in the middle and late stage when they to see the doctor. Due to the poor treatment effect, GC has become a major health concern in China. The studies indicated that the distal gastric cancer(DGC) had shown declining incidence rate and the proximal gastric cancer(PGC) had shown increasing rate in recent years, especially in Taihang mountain areas of North China. Epidemiological studies indicate that there was a strong tendency toward familial aggregation of proximal gastric cancer. Although a lot of work implemented to prevent the incident of gastric cancer in high-risk areas, but the incidence and mortality remained at a higher level.Epigenetic alterations, specifically, DNA methylation of promoter Cp G islands(CGIs), play an important role in regulating gene transcription. Loss of Cav-1 expression with aberrant promoter methylation was observed in severval human cancers, including breast, prostate carcinoma, colorectal cancer and hepatocellular carcinoma. However, the methylation frequency of this gene and the effect of aberrant methylation on gene transcription were very different. In recent years, some studies have shown that methylation frequency of each Cp G site may differ and some CG sites may have a close relationship with the inhibition of transcription which called critical Cp G sites, whether it resides within Cp G islands or non-Cp G islands. The hypermethylation of critical Cp G sites could be hindered the transcription factors and reduce or completely inhibited the transcription level. UCSC genome browser view of Cav-1 gene and Methyprimer-Software predicted that there was a big Cp G island in Cav-1 gene promoter. This study intends to examine the effects of the methylation of Cav-1 gene and the critical Cp G site of this gene.Human epidermal growth factor receptor(EGFR) is a tyrosine kinase active receptor. There are four structure similarly receptor molecules in EGFR family and epidermal growth factor receptor 2 is one of the main members of this family. Her-2 could formed the dimer with other members of the family, and lead to activate of intracellular a series of signal transduction through combined with EGFR ligands, such as MAPK, PI3 K / Akt, stat, PLC pathway. Previous studies have confirmed that Cav-1 can be directly connected with EGFR, which can regulate the activity of tyrosine kinase, and affected the proliferation, migration and invasion of tumor cells. The effect of Cav-1 on the activation of Her-2 in gastric cancer has not been reported.Based on the high-risk areas of the digestive tract tumor in Taihang Mountains of Hebei Province, we collected a large number of tumor and corresponding adjacent noncancerous tissues of proximal gastric cancer patients, who had a family aggregation or not, and combined with cytological experiments to achieve the following purposes(1) The regulated mechanism of Cav-1 expression and the critical Cp G regions of Cav-1 methylation.(2) It was detected that the role of Cav-1 in gastric cancer and the effect of Cav-1 on activation of HER-2 to clear the precise molecular mechanisms of the development and progression of GC, which would benefit to improve the patient’s survival. The study is divided into three Parts listed below: Part One The impacts of Cav-1 on biological behaviour of GC cells and the mechanism of regulated expressionMethods:1 Cell culture and drug treatmentThe human gastric cancer cell lines BGC-823,MKN45,NCI-N87 were cultured in regular medium. Cells were seeded at a low density and treated with 5μmol/L DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine(5-Aza-d C) for 72 h and 0.3μmol/L histone deacetylase(HDAC) inhibitor trichostatin A(TSA) for 24 h. The medium was changed every 24 h. DNA and RNA were isolated from these cells.2 The m RNA expression of Cav-1 gene in different GC cells were detected by Transcription-Polymerase Chain Reaction(RT-PCR) methodThe human gastric cancer cell lines BGC-823,MKN45,NCI-N87 were cultured in regular medium and the m RNA expression of Cav-1 gene was detected before and after treated with 5-Aza-d C/ TSA.3 The methylation status of Cav-1 gene in different GC cells were detected by methylation-Specific Polymerase Chain Reaction(MSP) methodThe human gastric cancer cell lines BGC-823,MKN45,NCI-N87 were cultured in regular medium and the methylation status of Cav-1 gene was detected before and after treated with 5-Aza-d C/ TSA.4 Construction of stably transfected cell linesNCI-N87 cells were stably transfected with plasmids expressing human full Cav-1 gene or empty plasmid and selected with G418, resulting in N87/Cav-1 and N87/pc DNA3.1 cell lines.5 MTT assay was used to examine the proliferation abilities of GC cell linesN87/pc DNA3.1, N87/Cav-1 and NCI-N87 cells treated with 5-Aza-Dc were seeded in 96-well plates, respectively. MTT assay was used to measure the OD values and examine the proliferation ability cell lines.6 Wound healing was used to examine the immigration abilities of GC cell linesN87/pc DNA3.1, N87/Cav-1 and NCI-N87 cells treated with 5-Aza-Dc were seeded in 24-well plates, respectively, and then scratched with a 10μl pipette tip. Images of cells at the different time were taken until closure of the scratch.Results:1 The m RNA expression of Cav-1 gene in different GC cells before or after treated with 5-Aza-d C/ TSA.RT-PCR method was applied to examine the m RNA expression in BGC-823,MKN45,NCI-N87 cell lines. The expression of Cav-1 gene m RNA are detected in MKN45,BGC-823 cell lines and undetected in NCI-N87 cell line. After treated with 5-aza-d C(a demethylation agent), the expression level of Cav-1 m RNA in NCI-N87 cell line was obviously increased and there was no change in MKN45,BGC-823 cell lines. The level of Cav-1 m RNA expression was no any change in BGC-823,MKN45,NCI-N87 cell lines treated with trichostatin A(TSA).2 The methylation status of Cav-1 gene in different GC cells before or after treated with 5-Aza-d C/ TSAMSP method was applied to examine the different regions methylation of the Cav-1 gene in MKN45、BGC-823 and NCI-N87 cell lines. The results showed that it can be amplified methylated bands in four regions of Cav-1 in NCI-N87 cell line, and the methylated bands all disappeared after treated with 5-aza-Dc; MKKN45 and BGC-823 cell lines were no any methylated bands amplified before and after treatment.3 The effect of over expression of Cav-1 gene or treated with 5-aza-Dc on proliferation abilities of cell linesMTT assay was used to examine the proliferation abilities of GC cell lines. The results showed that over expression of Cav-1 gene or treated with 5-aza-Dc inhibited the proliferation abilities of NCI-N87.4 The effect of over expression of Cav-1 gene or treated with 5-aza-Dc on immigration abilities of cell linesWound healing was used to examine the immigration abilities of cell lines. The results showed that over expression of Cav-1 gene or treated with 5-aza-Dc inhibited the immigration abilities of NCI-N87. Part Two The expression and methylation of Cav-1 on proximal gastric cancer tissuesMethods:1 Patients and Specimens One hundred and seventy-two paired tumor tissue samples and corresponding adjacent non-cancerous tissues of proximal gastric cancer patients were collected from the Fourth Affiliated Hospital, Hebei Medical University between the years of 2006 and 2009. All study subjects were residents of High incidence of upper digestive tract cancer in Hebei Province. The tumor epicenters located between 1 cm proximal and 2 cm distal of the EGJ. The adjacent non-cancerous tissues(normal tissues or hyperplasia tissues) were about 5 cm from tumor tissues and confirmed by microscope examination. The study was approved by the local ethics committee, and informed consent was obtained from all of the patients.2 RT-PCR and IHC methods were applied to examine the m RNA and protein expression of Cav-1 in tumor and corresponding adjacent tissues of 172 patients3 MSP method was applied to examine the methylation of Cav-1 different regions in tumor and corresponding adjacent tissues of 172 patients4 Bisulfite Genomic Sequencing(BGS) method was applied to examine the methylation frequency of Cav-1 gene Cp G sites in 5 pairs tumor and corresponding adjacent tissues.Results:1 the m RNA and protein expression of Cav-1 in tumor and corresponding adjacent tissues of 172 patientsRT-PCR method was applied to examine the m RNA expression of Cav-1 in tumor and corresponding adjacent tissues. The results indicate that the relative quantitive values of Cav-1 m RNA was 0.418±0.143 in tumor tissues, which was significantly lower than that in adjacent tissues(0.684±0.219, t=-14.486, P=0.000).IHC method was applied to examine the protein expression of Cav-1 in tumor and corresponding adjacent tissues. The results indicate that frequency of Cav-1 protein was 39.5%(68/172), was significantly lower than that in adjacent tissues(77.3%, 133/104,χ2=50.565, P=0.000).When stratified for clinicopathologic characteristics, Cav-1 m RNA and protein expression were all associated with UGIC family history(P<0.05), but not with age, gender, histological grade, clinical stage and Lymph node metastasis(P>0.05).2 The methylation of Cav-1 different regions in tumor and corresponding adjacent tissues of 172 patientsThe methylation analysis of four primer sets was successfully performed by MSP method in all specimens. Of primary PGC and corresponding non-cancerous tissues, hypermethylation was observed in 51.7%(89/172) and 27.3%(47/172) at MSP1, 23.8%(41/172) and 1.2%(2/172) at MSP2, 33.1%(57/172) and 26.2%(45/172) at MSP3, 45.3%(78/172) and 37.8%(65/172) at MSP4, respectively. The methylation frequency of Cav-1 in the regions of MSP1-4 were all significantly higher than those in corresponding adjacent tissues, whereas only MSP1 and MSP2 showed the statistically different(P<0.001).BGS assay was further used to verify the methylation status of each Cp G site in the MSP1 and MSP2 regions. We analyzed two segments from-590 bp to-143 bp and from-286 to 498 bp, which contains 52 Cp G dinucleotides. The results showed that the tumor tissues with low Cav-1 level showed frequent hypermethylation of Gp C site than that in corresponding adjacent tissues.When stratified for clinicopathologic characteristics, the methylation status of MSP1-region was associated with clinical stage, LN metastasis and UGIC family history, MSP2-region was associated with clinical stage, LN metastasis and UGIC family history. However, no significant correlation was observed between MSP3/4 regions hypermethylation and clinicopathological parameters(P>0.05).3 Association between expression and different regions methylation status of Cav-1 geneThe m RNA and protein expression of Cav-1 in tumor tissues where Cp G island shore(MSP1) and near the transcription start site(MSP2) regions was methylated of this gene were significantly reduced compared to those in tumor tissues without methylation of these regions(P<0.05). The m RNA and protein expression of Cav-1 were associated with methylation of MSP1 and MSP2 regions, but not with MSP3 and MSP4 regions of this gene.4 Survival Analysis of Cav-1In the PGC tumors, Cav-1 protein expression was not correlated with GCA patients’ survival(χ2=2.698, P=0.100, Log-rank test). The 5-year OS were 40.1% and 31.2% in MSP1-unmethylated group and MSP2-unmethylated group, respectively, which higher than those in methylation groups. Cav-1 MSP1 and MSP2 regions methylation were inversely correlated with PGC patients’ survival(χ2MSP1=41.679,PMSP1<0.001;χ2MSP2=34.927,PMSP2<0.001;Log-rank test). The PGC patients with simultaneous methylation of MSP1 and MSP2-regions showed worst prognosis(χ2=53.167,P<0.001, Log-rank test). PGC patients in stage III / IV or with positive UGIC family history, and with MSP1/MSP2-region methylation showed poor prognosis. To determine whether Cav-1 expression or methylation was an independent predictor of GCA patients’ survival, a multivariate analysis was performed using COX proportional hazard regression model. The results demonstrated that Cav-1 MSP1-region methylation, TNM stage and UGIC family history were independently associated with PGC patients’ survival(P<0.05). Part Three The impacts of Cav-1 on activation of Her-2 in GC cellsMethods:1 Protein expression of Cav-1in different gastric cancer cells by western blotThe human gastric cancer cell lines AGS 、 MKN28 、 SGC7901 、BGC823、MKN45、NCI-N87 were cultured in regular medium. The protein expression of Cav-1 and Her-2 were detected by western blot in different cell lines.2 Construction of stably transfected cell linesStably transfected cell lines were selected the cell lines which negative expression of Cav-1 and positive expression of Her-2. The expression efficiency of Cav-1 was measured by Western blot.3 MTT assay was used to examine the proliferation abilities of GC cell linesN87/pc DNA3.1 and N87/Cav-1 cells were seeded in 96-well plates, respectively. After stimulating with different concentrations of EGF(0n M, 4n M, 20 n M, 100 n M) for 48 hours, MTT assay was used to measure the OD values and examine the proliferation ability of cell lines.4 Wound healing was used to examine the immigration abilities of GC cell linesN87/pc DNA3.1 and N87/Cav-1 cells were seeded in 24-well plates, respectively. Cells were serum-starved for 4 hours and then scratched with a 10μl pipette tip. Cells were divided into two groups: EGF stimulated group and no EGF group. Images of cells at the different time were taken until closure of the scratch.5 Colony formation assay was used to evaluate the effect of over expresson of Cav-1 in anchorage-dependent cell growthN87/pc DNA3.1 and N87/Cav-1 cells were seeded in 35 mm dishes(800 cells/dish) and incubated in medium with or without EGF(20n M). The cell colonies were stained with hematoxylin and counted under a microscope.Results:1 The protein expression of Cav-1 and Her-2 were detected by western blot in AGS、MKN28、SGC7901、BGC823、MKN45、NCI-N87 cell lines. The results showed that the protein expression of Cav-1 was strong in MKN28 and SGC7901, but weak in BGC823, MKN45, AGS and NCI-N87; The Her-2 expression was positive in NCI-N87 and negative or weak in other five cell lines.2 It was selected that the cell lines which weak expression of Cav-1 and positive expression of Her-2 to construct the stably transfected cell lines. The effect was verified by Western blot.3 Effects of Cav-1 protein on the biological behavior of NCI-N87 cells induced by ligandMTT assay was used to examine the proliferation abilities of GC cell lines. After stimulating by EGF(0n M, 4n M, 20 n M, 100 n M), OD values of N87/Cav-1 group were significantly lower compare with N87/pc DNA3.1group(t0n M=13.718, P<0.001; t4 n M=20.777, P<0.001; t0 n M=12.623, P<0.001; t4 n M=9.788, P<0.001).Colony formation assay was used to evaluate the effect of over expresson of Cav-1 in anchorage-dependent cell growth. Cells were incubated in medium containing 10% fetal bovine serum(FBS) or 1% FBS +20n M EGF for two weeks. N87/Cav-1 cells group exhibited a lower rate of colony formation than the N87/pc DNA3.1 cells group(t10%FBS=3.434, P<0.001; tEGF=3.794, P<0.001).Wound healing was used to examine the immigration abilities of GC cell lines. The results showed that the ability of migration in N87/Cav-1 cells was significantly weaker than N87/pc DNA3.1 cells treated or untreated with EGF.4 Effects of Cav-1 protein on activation of HER-2 induced by ligandThe serum-starved N87/pc DNA3.1 and N87/Cav-1 cells were stimulating with different concentrations of EGF(0n M, 4n M, 20 n M, 100 n M), respectively.Western blot was used to examine the protein expression of Cav-1, Her-2, PHer-2, 4G10. The results showed that Cav-1 protein inhibited the Tyrosine phosphorylation level of Her-2.5 Effects of Cav-1 protein on MAPK and PI3K/Akt signaling pathway induced by ligandThe protein expression of ERK, P-ERK, Akt and P-Akt proteins in N87/Cav-1 and N87/pc DNA3.1 cells were detected by Western blot after stimulating by EGF(20n M) for 0min, 5min, 10 min and 30 min. The results showed that phosphorylation level of P-ERK and P-Akt were significant lower in N87/Cav-1 group than N87/pc DNA3.1 group(P<0.01).Conclusion:1 The Cp G island shore and near transcription start site region were frequently methylated and had a close association with reduced expression of Cav-1.2 The methylation of Cav-1 Cp G island shore(MSP1 region) may serve as a prognostic methylation biomarker for PGC patients.3 Cav-1 may inhibit the proliferation and immigration abilities of GC cell lines via weakening the activation(phosphorylation) of Her-2 and downstream signal transduction.