| Esophageal cancer is one of the most common malignant worldwide. InChina, esophageal cancer represents the fourth most frequent cause ofcancer-related deaths. Ci county in Hebei province, one of the high-risk areasfor esophageal cancer in northern China, has the highest incidence andmortality rate of esophageal cancer among all cancers. Esophageal cancerseverely threatens the lives and health of the population in this region.Many environmental and genetic factors interplay to cause complexdiseases including esophageal cancer. In Ci county of Hebei province, thefamily aggregation of esophageal cancer suggests that genetic backgroundmay play an important role in esophageal carcinogenesis. Candidate geneapproaches have also identified some single nucleotide polymorphisms (SNP)associated with esophageal cancer in this region. However, the exact geneticbasis of susceptibility to esophageal cancer is not well defined. Genome-wideassociation study (GWAS), an approach to investigate the geneticdeterminants of complex diseases without a prior hypothesis based on genefunction or disease pathway, may represent an unbiased yet fairlycomprehensive approach with high-throughput genotyping technology.Recently, three GWAS successively indicated significant associations ofPLCε1gene rs2274223A/G SNP with esophageal squamous call carcinama(ESCC) risk in Chinese population. Subsequently, the association between thisSNP and ESCC susceptibility was validated in a population with a much lowerESCC incidence in eastern China. However, to date, this finding has not beenconfirmed in other independent studies in populations from high-incidenceregions of northern China. Additionally, PLCε1gene rs11599672T/G SNP issituated in the transcription factor binding site, and the substitution of T to Gmay lead to the alteration of transcriptional activity and the expression level of PLCε1. This SNP was reported to be related to the risk of non-oropharyngealsquamous cell carcinoma of the head and neck associated with tobacco andalcohol exposure in a non-Hispanic white population. Whether rs11599672T/G SNP affects the susceptibility to ESCC in population from northern Chinahas not been studied.As a direct downstream effector of Ras proto-oncogene, the role ofPLCε1in the tumor has been studied widely. PLCε1has stimulative orsuppressive effect on the development of tumor. PLCε1played a crucial rolein chemical carcinogen-induced skin carcinogenesis in mice. Subsequent studydemonstrated that the tumor promotion role might be ascribed to theaugmentation of inflammatory responses. PLCε1also promoted intestinaltumorigenesis of APCMin/+mice through the augmentation of inflammation andangiogenesis. However, PLCε1might suppress the development of colorectalcancer. PLCε1also plays critical roles in the progression of cancer. Theactivation of RhoA-PLCε1stimulated inositol1,4,5-triphosphate production,intracellular Ca2+mobilization, and the up-regulation of Ca2+calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of thecytoskeletal protein, filamin. The phosphorylation of filamin reduced itsinteraction with filamentous actin, promoting tumor cell migration. SilencingPLCε1by RNA interference technique might decrease the invasive power ofbladder cancer cells. PLCε1has an impact on development and progression ofcancer.As for the role of PLCε1in development and progression of ESCC, Onestudy showed that more cells contained PLCε1in ESCC tissues than in normaltissues. Expression of PLCε1significantly increased in tumor tissuescompared with normal esophageal tissues, up-regulation of PLCε1wassignificantly correlated with advanced tumor-node-metastasis stages andlymph node metastasis. However, how PLCε1affects the biological behaviorof esophageal cancer cells and its mechanism have not been reported.Firstly, this study would explore the association between PLCε1geners2274223A/G and rs11599672T/G SNPs and susceptibility to ESCC in order to find genetic markers predicting the individuals with high risk for ESCC,actually realize early detection and early diagnosis of ESCC, improve thepatients’ survival rate, and provide a basis for genetic research of ESCC.Secondly, this study would silence PLCε1gene by RNA interferencetechnique, observe the alteration of ESCC cells’ biological behavior, measurethe expression of proliferation, apoptosis, and invasion related gene, anddetermine the inherent mechanism how PLCε1affects the biological behaviorof esophageal cancer cells. The research would ascertain the effect of PLCε1on progression of esophageal cancer, and provide experimental data for genetherapy of esophageal cancer.Part I The association between PLCε1gene polymorphisms andsusceptibility to esophageal squamous cell carcinomaObjects: To explore the association of PLCε1gene rs2274223A/G SNPand rs11599672T/G SNP with susceptibility to esophageal cancer in apopulation of Ci county high incidence region in Hebei province.Methods: The study included527ESCC patients and527healthycontrols. Five milliliters of venous blood from each subject was drawn inVacutainer tubes containing ethylene diamine tetraacetic acid and stored at4℃. The information on age, sex, smoking habit and family history of UGICfrom cancer patients and healthy controls was obtained by two professionalinterviewers directly after blood sampling. Genomic DNA was extractedwithin1week by proteinase K digestion, followed by a salting out procedureaccording to the method published by Miller et al. The genotypes of PLCε1gene rs2274223A/G SNP and rs11599672T/G SNP were determined by theShanghai Generay Biotech Co., Ltd. using the polymerase chainreaction-ligase detection reaction (PCR-LDR) method. The correlationbetween these two SNPs and the risk of ESCC were analyzed.Results:1The frequency of positive family history of UGIC in ESCC patientswas48.6%, significantly higher than that of the healthy controls (39.3%)(χ2=9.25, P=0.002). Therefore, UGIC family history increased the risk of ESCC (age and gender-adjusted OR=1.47,95%CI=1.15~1.87).2The genotype distributions of the PLCε1gene rs2274223A/G SNPin the healthy controls were consistent with the Hardy-Weinberg equilibrium(χ2=0.21, P=0.65). The AA, AG and GG genotype frequencies of the ESCCpatients and the healthy controls were48.0%,43.9%,8.1%and57.1%,37.5%,5.4%. Compared with AA genotype, AG, GG and AG/GG genotypeenhanced the risk of ESCC. Age, gender, smoking status, and UGIC familyhistory-adjusted OR were1.41(95%CI=1.09~1.83),1.71(95%CI=1.03~2.86) and1.45(95%CI=1.13~1.85), respectively. AG/GGgenotype carriers with UGIC family history and AG/GG genotype carrierswithout UGIC family history had a higher susceptibility to ESCC than AAgenotype carriers without UGIC family history (OR=1.41and2.10，95%CI=1.02~1.97and1.46~3.01).3The genotype distributions of the PLCε1gene rs11599672T/G SNPin the healthy controls did not deviate from the Hardy-Weinberg equilibrium(χ2=0.46, P=0.50). There was no significant difference in the frequency ofthe genotype and allele between the ESCC cases and the controls (P>0.05).UGIC family history enhanced the risk of ESCC in subjects with the TTgenotype (OR=1.59,95%CI=1.13~2.24).4PLCε1gene rs2274223A/G SNP and rs11599672T/G SNP werecombined to analyze using2LD software. The results showed that there wasno linkage disequilibrium between these two SNPs (D’=0.11). The AThaplotype was the most frequent, representing55.2%of the healthy controls.The haplotype frequency of AG, GT and GG were20.7%,17.5%and6.6%,respectively. The GT haplotype significantly increased the risk of ESCC(OR=1.36,95%CI=1.08~1.71).Conclusions:1UGIC family history might increase the risk of ESCC, whichsuggested that genetic background played an important role in esophagealcarcinogenesis.2For the first time, this study reported that PLCε1gene rs2274223 A/G SNP might serve as marker predicting genetic susceptibility to ESCCfor the population from Ci county high incidence region in Hebei province.The subjects with UGIC family history, the AG or GG genotype carriers andespecially the AG or GG genotype carriers with UGIC family historysimultaneously had higher risk of ESCC. These subjects should receiveperiodic upper gastrointestinal fiber tests for the sake of early detection andearly treatment of ESCC.3PLCε1gene rs11599672T/G SNP was not associated with the risk ofESCC in the population from Ci county high incidence region. When UGICfamily history and rs11599672T/G SNP were combined to analyze, theresults indicated that UGIC family history increased the risk of ESCC forthe TT genotype carriers.4Compared with AT haplotype, the GT haplotype significantlyenhanced the risk of ESCC.Part II The effect of shRNA interference silencing PLCε1gene onproliferation of esophageal carcinoma Eca109cells and itsmechanismObjects: To explore the impact of silencing PLCε1gene on proliferationof esophageal carcinoma Eca109cells and its mechanism.Methods: According to small hairpin RNA (shRNA) design principles,three plasmid expression vectors (PLCε11, PLCε12and PLCε13) wereconstructed to silence PLCε1gene. A negative control plasmid expressionvector (HK) was constructed at the same time to serve as control. The plasmidexpression vectors were transfected into esophageal carcinoma Eca109cellsby cation liposome. PLCε11was used to determine the transfection conditionand transfection efficiency. The plasmid expression vector with the bestinterference effect (PLCε12) was chosen. RT-PCR and Western blot were usedto measure PLCε1mRNA and protein expression of Eca109cells at24h,48hand72h after transfection. The interference effect of PLCε12at different timewere observed. Cell viability of Eca109cells were evaluated at48h,72h and96h after transfection by MTT assay. The cell cycles were detected by FCM at 24h after transfection. The mRNA expression of p16and CyclinD1weremeasured by RT-PCR. The impact of silencing PLCε1gene on proliferation ofesophageal carcinoma Eca109cells and its mechanism were analyzed.Results:1When the ratio of plasmid expression vector and the cation liposomewas2μg and6μl, the transfection had the highest efficiency (averagetransfection efficiency72.6%). PLCε12had the best interference effect forPLCε1gene. The mRNA expression of PLCε1began to decrease at24hafter transfection, and was the lowest at48h, and recovered at72h. Thechange of PLCε1protein expression was consistent with that of PLCε1mRNA expression.2MTT results showed that the cell viability of Eca109cells in PLCε12group were80.73%and75.88%at48h and72h after transfection, whichwere significantly lower than that of Eca109cells in HK group (P<0.05).There was no significant difference of cell viability at96h after transfectionbetween the HK group and the PLCε12group Eca109cells.3The percentage of S phase Eca109cells in Eca109group, HK groupand PLCε12group at24h after transfection were19.53%,17.20%, and4.80%, respectively. The percentage of S phase Eca109cells in Eca109group was similar to that of Eca109cells in HK group (P>0.05). Thepercentage of S phase Eca109cells in PLCε12group was lower than that ofEca109cells in HK group (P<0.05), the cell cycle of PLCε12group Eca109cells arrested in G0/G1phase.4The relative optical density of p16in Eca109cells of HK group andPLCε12group at48h after transfection were0.38and0.58. The p16mRNAexpression of PLCε12group Eca109cells was higher than that of HK groupEca109cells (P<0.05). The CyclinD1mRNA expression of PLCε12groupEca109cells was similar to that of HK group Eca109cells (P>0.05).Conclusions:Silencing PLCε1gene might inhibit the proliferation of Eca109cellsand arrest the cell cycle in G0/G1phase. Silencing PLCε1gene led to up-regulation of p16mRNA expression. p16protein might suppress theactivity of CDK4, phosphorylation of Ser and Tyr residue of Rb combinedwith CyclinD1, and the release of transcription factor E2F. The transcriptionof E2F dependent genes were inhibited, which block the transition from G1to S phase. Therefore, the proliferation of Eca109cells was inhibited.Part III The effect of shRNA interference silencing PLCε1gene onapoptosis and invasion of esophageal carcinoma Eca109cellsand its mechanismObjects: To explore the impact of silencing PLCε1gene on apoptosisand invasion of esophageal carcinoma Eca109cells and its mechanism.Methods: Apoptosis rate of Eca109cells at48h after transfection weredetermined by FCM. Invasion of Eca109cells at48h after transfection wereexamined by Transwell chamber invasion assay. RT-PCR was used to evaluatethe mRNA expression of Fas, FasL, CD44, MMP9and VEGF. The impact ofsilencing PLCε1gene on apoptosis and invasion of esophageal carcinomaEca109cells and its mechanism were analyzed.Results:1The apoptosis rate of Eca109cells in Eca109group, HK group andPLCε12group at48h after transfection were26.22%,22.06%and30.27%,respectively. There was no significant difference of apoptosis rate betweenthe Eca109group and HK group Eca109cells (P>0.05). The apoptosis rateof Eca109cells in PLCε12group was higher than that of HK group Eca109cells (P<0.05).2The relative optical density of Fas in Eca109cells of HK group andPLCε12group at48h after transfection were0.43and0.53. The relativeoptical density of FasL were0.42and0.33. The mRNA expression of Fasand FasL of Eca109cells in PLCε12group were different from that ofEca109cells in HK group (P<0.05).3The number of Eca109cells across the polycarbonate membrane oftranswell chamber in Eca109group, HK group and PLCε12group at48hafter transfection were85.00,82.00and62.67, respectively. There was no significant difference of the number of Eca109cells across thepolycarbonate membrane between the Eca109group and the HK group(P>0.05). The number of Eca109cells across the polycarbonate membranein PLCε12group was lower than that of the HK group (P<0.05).4The relative optical density of MMP9were0.60and0.53. Therelative optical density of VEGF were0.28and0.17. The mRNA expressionof MMP9and VEGF of Eca109cells in PLCε12group were different fromthat of Eca109cells in HK group (P<0.05). There was no significantdifference of CD44mRNA expression between the HK group and PLCε12group Eca109cells (P>0.05).Conclusions:1Silencing PLCε1gene might promote the apoptosis of Eca109cells.Up-regulation of Fas mRNA expression and down-regulation of FasLmRNA expression inhibited immune escape and immune counterattack andpromoted the apoptosis of Eca109cells.2Silencing PLCε1gene might suppress the invasion of Eca109cells.The possible mechanism might be that silencing PLCε1gene decreased themRNA expression of MMP9and VEGF, decreased the ability of Eca109cells to degradate extracellular matrix and to promote angiogenesis andsuppressed the invasion of Eca109cells. |
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