| Background and objectiveGastric cancer(GC)is one of the most commonly diagnosed malignancy,with about 952,000 new cases diagnosed in 2012,the morbidity and mortality of Gastric cancer are higher of other malignant tumors in our country.Although the global rate of GC has been declining every year,the total number of new patients has been rising for various reasons.So far,it is commonly believed that many facters are related with the occurrence and progression of GC,especillay the aberrant expreesion of oncogenes.Both the aberrant activation of proto-oncogenes and inactivation of tumor suppressor genes(TSGs)are critical for GC occurrence and progression.Therefore,the intensive study on cancer genes is essential to discover new targets for cancer treatment.Early in the 1960s,Britten came up with the concept of activator RNA;nonetheless,this idea was controversial.Later,Li and Janowski both discovered and named RNAa and saRNA:small double-stranded RNA(dsRNA)can activate gene expression at the transcriptional level;the dsRNA was named small activating RNA(saRNA),and this phenomenon was called RNA activation(RNAa).Subsequently,various studies on the principle and function mechanism were reported.All these further strengthened the feasibility of RNAa.p21WAF1/CIP1(p21),a tumor suppressor gene located on chromosome 6p,was marked by broad-acting cyclin-dependent kinase inhibitor and belonged to the Cip/Kip family of cyclin-dependent kinase inhibitor.p21 is induced by wild-type p53 reacting to DNA damage and helped to cause G1 cell cycle arrest primarily through inhibiting the activity of cyclin/cdk2 complexes.The efficient saRNA for p21WAF1/CIP1(p21),dsP21-322,has been proved in many cancer cells,such as liver cancer cell and lung cancer cell,but there has been no report on gastric cancer cells so far.Therefor,in this study,we planed to explore the reactivation effect of p21 gene in GC cells through RNAa,then found out the optimum condition for the transfection of GC cells with saRNA and the essential factors for reactivation.At last,we would explore how this up-regulation effects the cell proliferation,migration and invasion,to provide the theoretical basis for gene therapy of GC.In the previous study,we had verified the mechanism of saRNA in GC cells.In order to study the biological mechanism of RNAa,we selected VEZT which was discovered by our group initially as the target of saRNA.Vezatin(VEZT),a novel putative TSG located on chromosome 12q22,consists of 12 exons and 11 introns,and encodes a plasma membrane protein.The protein mainly includes three parts:the long intracellular domain interacted with myosin ?A,short extracellular domain and the transmembrane domain.The previous studies shows that the up-regulation of VEZT gene can inhibit the proliferation,invasion and migration of GC cells;besides,the expreesion of VEZT was under the regulation of promoter methylation and microRNA,what is more,this regulation was connected with the mechanism of saRNA.To further confirmed this effect,and explore the design principle and mechanism of saRNA,we designed several saRNAs for VEZT promoter through biology software.Subsequently,Western Blot and qRT-PCR were used to select the effective saRNA that could up-regulate the expression of VEZT protein and mRNA.Then the selected saRNA was transfected to GC cells in order to conduct related experiments.At the same time,pGPU6/GFP/Neo-shRNA expression vector was synthesized to eliminate the off-target effect and further explore the effect that induced by saRNA on GC cells proliferation,migration and invasion.MethodsThe exact sequence of saRNA for p21WAFT/CIP1(p21),dsP21-322,was found in the previous studies,so did the double stranded control RNA(dsControl)that acted as negative control;then both were synthesized.The GC cell lines SGC-7901 and M-28 were transfected with dsP21-322 of different concentration.Western Blot was applied to select the most suitable concentration?Then the cells were transfected with the selected concentration for different time.Western Blot was applied to select the most suitable time for transfection again.According to the selection of concentration and time,both cell lines were transfected with dsP21-322 or dsControl to act as the experimental group or negative control group,respectively;and the mock group was just treated with the Lipofectamine2000 under certain conditions.The proliferation of transfected cells was assessed by CCK-8 assay.The invasion and migration of transfected cells were determined by Transwell chamber assay.We designed several saRNAs for VEZT promoter through biology software and synthesized them.The GC cells,SGC-7901 and M-28,were transfected with saRNA,then Western Blot and qRT-PCR were used to select the effective saRNA that could up-regulate the expression of VEZT protein and mRNA.Then the selected saRNA was transfected to GC cells in order to conduct related experiments;at the same time,pGPU6/GFP/Neo-shRNA expression vector was synthesized and acted as negative control to eliminate the off-target effect.The proliferation of transfected cells was assessed by CCK-8 assay either.The invasion and migration of transfected cells were determined by Transwell chamber assay either.Results1.dsP21-322 induced the reactivation of p21WAF1/CIP1(p21)gene in GC cells,and the reactivation were time-course and dose-dependent.2.The reactivation of dsP21-322 achieved maximum efficiency with 50nM dsP21-322 for 72h.3.dsP21-322 effectively inhibited the proliferation,migration and invasion of GC cells through the reactivation of p21WAF1/CIP1(p21)gene in GC cells.4.Up-regulation of VEZT by small activating RNA inhibits the proliferation,invasion and migration of gastric cancer cells.ConclusionIn this study,we found that dsP21-322,a saRNA,could up-regulate the expression of p21WAF1/CIPI(p21),that is to say the level of p21 mRNA and protein was improved,in that the proliferation,migration and invasion of GC cells were inhibited.Besides,the reactivation were time-course and dose-dependent.The reactivation of dsP21-322 achieved maximum efficiency with 50nM dsP21-322 for 72h.dsVEZT-94,an effective saRNA for VEZT,could effectively inhibited the proliferation,migration and invasion of GC cells through the reactivation of VEZT gene in GC cells.The up-regulation of TSGs by saRNA could effectively inhibited the proliferation,migration and invasion of GC cells.Therefore,the study of the mechanism would provide the theoretical basis and/or the potential means for gene therapy of GC. |
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