The Role Of MiR-133b In Human Esophageal Squamous Cell Het-1A Injury Induced By HCl

Background:GERD should be defined as symptoms or complicationsresulting from the refl ux of gastric contents into theesophagus or beyond,into the oral cavity(including larynx)or lung.GERD is a frequent disease worldwide.Even in Asia,it is known to have a lower rate of incidence,the prevalence of GERD has been reported as more than 5%.Despite the high morbidity rate,the number of GERD patients has increased in these few decades.Reflux esophagitis(RE)is a common digestive system disease and one of GERD.RE have a number of complications,such as esophageal stricture,Barrett's esophagus,and esophageal adenocarcinoma.The pathogenesis of RE remains poorly understood,while age,sex,unhealthy diet,Helicobacter pylori infection and central obesity have been identifed as risk factors of RE.More important,excessive exposure to gastric contents is a directly main cause of RE,that hydrogen ion in gastric contents diffuses into the mucosa,and leads to tissue necrotic damage.Thus,p H control has been considered as important RE management,and alleviates the effects of hydrogen ion on mucosa can be a novel direction to prevent and treat this disease.Heartburn and regurgitation are the most typical symptoms of GERD.Heartburn is defined as retrosternal burning sensation.Regurgitation is the feeling of the flow of the contents of the stomach into the pharynx or the mouth.Chest pain,abdominal pain,epigastric burning sensation,belching are atypical symptomsof GERD.Coughing,pharynx and larynx,asthma,and dental erosion are GERD's external symptoms of the esophagus.At present,the treatment of RE is mainly through four aspects,including alleviating reflux of gastric esophagus,reducing acidity of reflux contents,promoting pumping of esophagus and protecting the esophageal mucosa.Common measures for RE are basic treatment,drug therapy,endoscopic therapy and surgical treatment.Basic treatment mainly refers to improve the way of life,including reducing body weight,raising bed head,giving up smoking and drinking,avoiding eating before going to bed,and avoiding eating foods that may induce regurgitation symptoms,such as coffee,chocolate,spicy or sour food and high-fat diet.Drug therapy is the most commonly method to treat RE,common drugs include acid resistant drugs,gastric acid secretion suppression drugs and gastric power promoter drugs.PPI is the first choice for the treatment of GERD.A double dose of PPI can be used while single doseis not effective.Another PPI can be usedwhile one kind of PPI is not effective.70%-80% patients with reflux esophagitis and 60% of non erosive reflux patients receive complete remission after 8 weeks of PPI treatment.So the PPI course is at least 8 weeks.For GERD patients with hiatal hernia and patients with severe esophagitis,the dose of PPI usually needs to be doubled.The endoscopic treatment of GERD is mainly divided into radiofrequency,injection or implantation,and endoscopic gastroesophagoplasty.However,endoscopic treatment is short and the number of the treatment population is limited.It's long-term effectiveness needs further confirmation.Surgical treatment is mainly used for patients who are effective but long-term medication of PPI.The most commonly used of anti reflux surgery is laparoscopic fundus folding.Surgical treatment is not recommended in non acid reflux patients.The study shows that laparoscopic fundoplication can improve the reflux of acid and weak acid,and has a high symptom relief rate after operation,but the weak alkali reflux increased after the operation.Micro RNA(miRNA)is a noncoding small RNA molecule that is widely existed in plants and animals,with a length of about 20 to 24 nucleotides.It can combine with the 3' untranslated regions(UTR)of the target m RNA to degrade target genes at the transcription level and inhibit the production of proteins at the translation level.The main function of miRNA is to regulate the expression of gene in the process of growth and development.The previous studies found that in tumor tissues,the expression level of miRNA was up-regulated or down-regulated and it indicated that miRNA was related to the development of tumor.Multiple lines of evidence suggest that the expression profiles of miRNAs in cancer may serve as a biomarker for cancer diagnosis and prognosis.In addition,miRNA can also participate in regulating the differentiation,proliferation and apoptosis of intestinal cells and esophageal squamous cells.miR-133 b is located in P12.1 of human chromosome 6,and miR-133 b has been reported as a tumor suppressor in numerous types of cancers,such as colorectal,gastric,bladder and lung cancer.However,to the best of our knowledge,the role of miR-133 b in RE has not yet clear.Objects:The main purpose of this experiment was to explore the role of miR-133 b in Het-1A cell injury induced by HCl.Firstly,Het-1A cells were exposed in HCl and we aimed to explore the effect of HCl on cell viability and expression levels of miR-133 b in cells.Then,the expression of miR-133 b was up-regulated or down-regulated by cell transfection technology.We investigated the effects of expression abnormality of miR-133 b on cell viability,apoptosis and apoptosis related factors.Finally,we studied on the effects of miR-133 b on extracelliar signal-regulated kinase(ERK)and phosphalyl-3 kinase(PI3K)/protein kinase B(AKT)signaling pathways.Methods:The effect of HCl on Het-1A cell viability and expression level of miR-133b: Firstly,Het-1A cells were exposed to p H 4.0 for 0 min,5 min,15 min and 30 min,and then cell viability was measured by Methyl Thiazolyl Tetrazolium(MTT)analysis,respectively.Finally,Quantitative Real-time PCR(q RT-PCR)was utilized to monitor the expression level of miR-133 b in cells,after 30 min of HCl exposure.The role of miR-133 b in Het-1A cells: Firstly,Het-1A cells were transfected with miR-133 b mimic,miR-133 b inhibitor and their negative control by cell transfection technology.Then,quantitative Real-time PCR(q RT-PCR)was utilized to detect transfection effciency.Further,cell viability,apoptosis and the expression levels of apoptosis related factors,including Caspase-3,Fas and p53 were respectively determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide(MTT),flow cytometry and Western blot.The effect of miR-133 b on PI3K/AKT and ERK1/2 signaling pathway: Het-1A cells were treated with HCl for 30 min.miR-133 b mimic or/and miR-133 b inhibitor were transfected into Het-1A cells by transfection technology.Afterward,the expression levels of ERK and PI3K/AKT pathway proteins were detected by Western blot.Results:The effect of HCl on Het-1A cell viability and expression level of miR-133b: The experimental results showed that the exposure of HCl reduced cell viability in a time dependent manner.In 0-30 min,cell viability was decreased with the increasing of time.Moreover,cell viability was significantly decreased after cells were treated with HCl for 30 min(P< 0.05);the miRNA expression level of miR-133 b in cells which were treated with HCl for 30 min was detected by q RT-PCR.We found that HCl signifcantly increased the expression level of miR-133b(P< 0.001).The role of miR-133 b in Het-1A cells: The results showed that,the expression of miR-133 b was significantly up-regulated after miR-133 b mimic transfection(P< 0.01).Conversely,the expression of miR-133 b was significantly down-regulated after miR-133 b inhibitor transfection(P< 0.001).Cell viability experiments showed that miR-133 b overexpression significantly inhibited cell viability after cells were cultivated for 3 days and 4 days(P< 0.01).However,miR-133 b suppression significantly strengthened the cell viability(P< 0.01).Cell apoptosis experiments showed that the up-regulation of miR-133 b induced apoptosis(P< 0.001)and the down-regulation of miR-133 b inhibited apoptosis(P< 0.05).Furthermore,the expressions of apoptosis related factors in transfected cells were detected.Results showed that the levels of Cleaved-caspase-3,Fas and p53 were signifcantly up-regulated by miR-133 b overexpression(Cleaved-caspase-3 and Fas: P< 0.01;p53: P<0.001),whereas were down-regulated by miR-133 b suppression(Cleavedcaspase-3 and Fas: P< 0.05;p53: P< 0.01).The effect of miR-133 b on PI3K/AKT and ERK1/2 signaling pathway: As results showed that down-regulations of p-ERK1/2,p-AKT and p-PI3 K were found in cells which were treated with HCl.The same effects were observed in cells transfected with miR-133 b mimic.The protein expression levels of p-ERK1/2,p-AKT and p-PI3 K were increased after the simultaneous transfection of miR-133 b mimic + inhibitor.Statistical results found down-regulations of p-ERK1/2(P< 0.01),p-AKT(P< 0.001 or P< 0.01)and p-PI3K(P< 0.05 or P< 0.01)were found in cells which were treated with HCl or were transfected with miR-133 b mimic.Surprisingly,miR-133 b inhibitor signifcantly recovered the regulatory effects of miR-133 b mimic on these factors(P< 0.001 or P< 0.01).Conclusions:In this paper,we mainly study the role of miR-133 b in HCl induced Het-1A damage in esophageal squamous cells.We can draw the following conclusions from three aspects:1.HCl could inhibit cell viability and significantly increased the expression level of miR-133 b in cells.2.Down-regulated of miR-133 b strengthened the Het-1A cell viability and inhibited apoptosis and the expressions of apoptosis related factors.However,up-regulated of miR-133 b displayed the reversed impacts.3.miR-133 b mimic and HCl can inhibit the expression of p-ERK1/2,p-AKT and p-PI3 K and miR-133 b inhibitor can alleviate these effects.In this study,we successfully constructed the model of esophageal squamous cell injury by HCl and found that the abnormal expression of miR-133 b.We temporarily speculated that miR-133 b might be a pivotal regulator in Het-1A cells viability and apoptosis and inferred that,down-regulation of miR-133 b alleviated HCl induced injury might be via regulating ERK and PI3K/AKT pathways.This also lays a foundation for us to study the mechanism of miR-133 b in Het-1A cells,and also provides a new direction for our treatment of reflux esophagitis.