The Relationship Between The HHIP, Patched Gene Expression、Methylation Levels And Clinical Feature In Gastric Carcinoma

Background and objects：Gastric cancer is one of the most common cancers with serious hazard to humanhealth，accounting for the second most frequent malignancy and the third cause ofcancer-related death. Now radical surgery is still the main treatment and the only mean tocure gastric cancer. But most gastric cancer patients are usually diagnosed in the laterstages of the disease in clinical and has very poor prognosis due to patients withunresectable therapeutic options. Therefore, there is clinical significance to extensivelyinvestigate the molecular mechanisms of gastric carcinogenesis, to Provide the basis for theearly diagnosis of gastric cancer, and also provide an effective target in the treatment ofclinical treatment.In recent years, studies have shown that the hedgehog pathway plays an importantrole in gastral embryonic development and carcinogenesis. Gastric cancer development hasbeen intimate associated with hedgehog signaling pathways. To aim pathway-specifictargeted therapy may become a effective new measure in the clinical treatment ofgastric cancer. The HH pathway mainly includes HH ligand，membrane receptor ofPatched (Ptch)、Smoothened (Smo) and downstream of the transcription factor Gli. Humanhedgehog-interacting protein (HHIP) has been discovered recently. HHIP binds all threeHh proteins with an affinity equal to that of Patched, and functions to negatively regulatethe hedgehog pathway. Indeed, both HHIP and Patched genes, two negative regulators ofhedgehog signaling, can directly inhibit the pathway, has a very important significance.Therefore this topics chosen HHIP and Patched gene in-depth study.Epigenetics refers to the sequence of DNA does not change, but the gene expressionwas heritable changed, which were changed to the genetic information in cells other than genetic material change, namely the phenotype changed but genotype has not changed, andthis change can propagate stably in development and cell proliferation.DNA methylation，histone modification, non coding RNA were included inepigenetic modification, and also a research hotspot. The current study found: widehypomethylation of the genome is a common feature of the tumor cells; hypermethylationof the tumor suppressor gene promoter region silencing of tumor suppressor genes, canlead to reduction or loss of the inactivation induced expression product. DNA methylationis closely related with gastric cancer, the study found methylation existed in each stage ofgastric cancer，even precancerous lesions. The article willdetect and analyze the expressionand methylation levels of HHIP、Patched gene in gastric carcinoma, and through thefollow-up, to observe the correlation between the expression of HHIP、Patched gene andthree year overall survival rate, trying to find HHIP、Patched gene mechanism in gastriccarcinogenesis、development and their relationship with prognosis of gastric cancer.It was said Patched gene promoter methylation in gastric cancer. However, thesestudies focus on gastric cancer cell lines，no study has precisely analyzed the methylationof Patched and HHIP gene promoters in gastric cancer to date. In this study, we focus onHHIP and Patched gene，detect the expression of HHIP and Patched in gastric cancertissues and adjacent normal tissues，and then detect the promoter methylation status ingastric cancer，analyze its relationship with clinical pathological features. Throughfollow-up, to observe the correlation between the expression of HHIP、Patched gene andthree year overall survival rate. Then to discover the HHIPmRNA expression differencesand the change of CpG island methylation status before and after demethylation drug5-aza-dc intervention. To build a stable HHIP overexpression of lentivirus vector,transfected into gastric cancer cell lines AGS, then observe the difference about mRNAexpression and methylation status of CpG islands in promoter region before and aftertransfection. To reveal the development mechanism of HHIP, Patched gene in gastriccarcinogenesis, and their relationship with prognosis of gastric cancer. Which can providenew ideas for the early diagnosis、prognosis and targeted therapy of gastric.Methods and material： 1. Research material Surgical specimens from60patients with gastric cancer andadjacent normal tissues were collected from the Department of Surgery, Zhangjiagang FirstHospital between June2009and December2013，without preoperative chemotherapy andradiotherapy, pathologic examination to determine tissue types are gastric adenocarcinoma.All surgically resected tissue specimens were snap frozen in liquid nitrogen until use.Patients included34males and26females with an age range from36to72years (mean60.82years). According to the TNM staging system,40cases were stage II patients and20cases were stage III patients.32cases were well and moderately differentiated, while28were poorly differentiated.24cases had lymph node metastasis, but36cases were withoutlymph node metastasis. The gastric cancer AGS cell line was purchased from ShanghaiInstitute of Cell Bank, which come from a resected gastric tumor fragments untreatedadeno-carcinoma cell, have the capability of the local Baptist passage of transfer.2. Research Methods: mainly focus on gastric carcinoma and adjacent normaltissues，focus on gastric cancer cell line AGS.2.1Research about gastric tissues2.1.1reverse transcription-PCR RNA was isolated from the gastric cancer andnormal tissues by Trizol agent. cDNA was then converted from RNA using a reversetranscription kit. Detect the expression levels of HHIP, Patched mRNA by RT-PCRmethod.2.1.2Immunohistochemistry Take paraffin-embedded tissue sections to detectthe HHIP and Patched protein according with APC method.2.1.3bisulfite sequencing PCR（BSP） DNA was extracted per our standardprotocol. After bisulfite treatment, to analyse HHIP and Patched promoter CpG islandmethylation status of each locus by detecting and sequencing with BSP method.2.1.4methylation-specific PCR (MSP) DNA was extracted per our standardprotocol. After bisulfite treatment, to design MSP primers by Methyl Primer Express v1.0software, then to detect HHIP promoter CpG island methylation status by MSP.2.1.5follow up Statistics of three year overall survival rate by Kaplan-Meiermethod.2.2Research about gastric cancer cell lineAGS2.2.1To interfere AGS cell line by demethylation drug5-aza-dC, undertake relatedresearch. 2.2.1.1MTT assay AGS cell line treated with0、0.5、1.0、2.0、5.0、10uM5-aza-dcafter24h,48h,72h, to detect AGS cell growth on different concentrations using the MTTassay.2.2.1.2Annexin V/PI-flow cytometry assay AGS cell line treated with0、0.5、1.0、2.0、5.0、10uM5-aza-dc, then detect apoptosis by flow cytometry.2.2.1.3reverse transcription-PCR RNAwas isolated from AGS cell line by Trizolagent before and after intervention. cDNA was then converted from RNA using a reversetranscription kit, then to detect the expression differences of HHIP mRNA by RT-PCRmethod.2.2.1.4BSP method AGS cell line DNA was extracted per our standard protocol.After bisulfite treatment, to analyse HHIP promoter CpG island methylation status of eachlocus by detecting and sequencing with BSP method.2.2.1.5MSP method AGS cell line DNAwas extracted per our standard protocol.After bisulfite treatment, to detect HHIP promoter CpG island methylation status by MSPbefore and after intervention.2.2.2To build a stable HHIP overexpression of lentivirus vector, to transfect AGS,undertake related research.2.2.2.1To build a stable HHIP overexpression of lentivirus vector.2.2.2.2MSP method AGS cell line DNAwas extracted per our standard protocol.After bisulfite treatment, to detect HHIP promoter CpG island methylation status by MSPbefore and after intervention.2.2.2.3To observe the effect overexpression HHIP gene onAGS cell line.3. Statistical Analysis All of the measurement data were expressed as mean±SD,count data are expressed as a percentage (%) by SPSS16.0software. Apply X2test, t test,Kaplan-Meier mean，Spearman correlation analysis, et al. A value of P<0.05wasconsidered statistically significant.Results1. Research about gastric tissues1.1reverse transcription-PCR HHIP and Patched express in gastric cancer andadjacent normal tissues. HHIP mRNA intensity of expression0.8181±0.380in gastriccancer lower than1.6033±0.263in adjacent normal tissues. PatchedmRNA intensity ofexpression1.7537±1.150in gastric cancer lower than2.7993±1.458in adjacent normal tissues. HHIP, Patched mRNA expression have no statistically significant differences withclinical pathological features: gender, age, TNM stage, lymph node metastasis. ButPatched mRNA in poorly differentiated gastric carcinoma expression was significantlylower than in highly differentiated, difference of HHIP mRNA expression and the degreeof differentiation was observed in the control.1.2Immunohistochemistry HHIP protein positive rate was18/60(30%) ingastric cancer, while36/60(60%) in adjacent normal tissues, the positive rate ofPatched protein in gastric cancer20/60(33.33%), adjacent tissues was48/60(80%). HHIP,Patched protein expression have no statistically significant differences with clinicalpathological features: gender, age, TNM stage, degree of differentiation, lymph nodemetastasis.1.3BSP60cases of gastric cancer tissues showed HHIP and Patched CpG sitescompletely or partially methylated, while unmethylated or minority methylation inadjacent tissues. Through Spearman correlation analysis we found the HHIP and PatchedmRNAexpression was negatively correlated with the degree of methylation.1.4MSP There were28cases HHIP promoter CpG island methylation in60casesof gastric cancer, the methylation rate of46.67%, while methylation rate of20%inadjacent tissues, HHIP gene promoter CpG island methylation was significantly higherthan the corresponding adjacent tissues (p <0.05).1.5follow up Through follow-up, found that the patients of positive expression ofHHIP and Patched in the three year overall survival rate surpass the negative expression, soHHIP and Patched can be used as a predictor of prognosis of gastric cancer.2. Research about gastric cancer cell lineAGS2.1. To interfere AGS cell line by demethylation drug5-aza-dC, undertake relatedresearch.2.1.1MTT assay The effect of proliferation inhibition was gradually increasewith the increase of the concentration of the drug and the extension of time.,which was atime-and dose-dependent manner.2.1.2Annexin V/PI-flow cytometry assay In a certain dose range rate ofapoptosis increased with the increase of the concentration of5-aza-dc. But after reached acertain concentration, the apoptotic rate decreased with the increase of the concentration.2.1.3reverse transcription-PCR After5-aza-dc intervention, HHIP gene is activated. The expression of HHIP was significantly higher.2.1.4BSP method After5-aza-dc intervention, HHIP gene promoter CpG sitesmethylation was significantly reduced.2.1.5MSP method The methylation degree in gastric cancer cell line AGS was99.7%±0.67%. After5-aza-dc intervention. the degree of methylation was significantlydecreased, and even showed unmethylation.2.2. To build a stable HHIP overexpression of lentivirus vector, to transfect AGS,undertake related research.2.2.1Astable HHIP overexpression of lentivirus vector was build successful.2.2.2The methylation degree in gastric cancer cell line AGS was99.67%. Aftertransfect by overexpression of lentivirus vector, the degree of methylation was significantlydecreased, and even showed unmethylation.2.2.3Overexpression HHIP gene can inhibit the growth and proliferation of gastriccancer cell line.Conclusion:1、There were HHIP、Patched mRNA and protein expression，the intensity ofexpression in gastric cancer lower than in adjacent normal tissues.2、HHIP、Patched mRNA and protein expression have no statistically significantdifferences with clinical pathological features: gender, age, TNM stage, lymph nodemetastasis. But Patched mRNA expression was significantly lower in poorly differentiatedgastric carcinoma than in highly differentiated, difference of HHIP mRNA expression ofthe degree of differentiation was observed in the control.3、HHIP and Patched can be used as a predictor of prognosis of gastric cancer.4、There were HHIP and Patched gene low expression and promoter CpG islandmethylation in gastric cancer, which was a molecular event distinguished gastric cancerfrom normal gastric tissue, maybe concerned with the incidence of gastric cancer. It maybecome a tumor marker for early diagnosis of gastric cancer.5、5-aza-dc can induce apoptosis, Inhibit tumor proliferation,in a certain concentrationrange it appeared In a time-and dose-dependent manner. Demethylation may become atumor therapeutic approach.6、Overexpression HHIP gene can inhibit the growth and proliferation of gastric cancer cell line, may be a new biological marker in gastric cancer, the drug targets HHIPformation, may become a new way to the treatment of gastric cancer.