| Part one Intervention effect of Huazhuojiedu decoction on precancerous lesions of gastric cancer and screening of related long non-coding RNAObjective: To observe the intervention effect of Huazhuojiedu decoction on precancerous lesions of gastric cancer(PLGC),screen the differentially expressed long non-coding RNAs(lncRNAs),thus exploring the possible target of Huazhuojiedu decoction.Methods: PLGC rat model was prepared and randomly divided into Huazhuojiedu decoction group(HG),vatacoenayme group(VG),model group(MG),and compared with normal group(CG).Each group was given corresponding concentration of drugs and distilled water for 10 weeks.The pathological changes of gastric mucosa were observed by hematoxylin-eosin staining.Identification of differentially expressed lncRNAs in the HG group,MG group and CG group were performed via high-throughput sequencing and validated using real-time quantitative polymerase chain reaction(RT-q PCR).Rat and human homology informations were obtained from the University of California,Santa Cruz(UCSC)human genome browser.Results:1.General condition of rats in each group Rats in CG group had moderate body size,good posture,white fur and teeth,and pale pink color of claws,auricles and limbs.Rats in the MG group were small in size,listless in spirit,with yellow fur and teeth,and white claws,auricles and limbs.During the treatment stage,the condition of rats improved,especially in the HG group.2.Morphological comparison of gastric mucosa in each group In CG group,the posterior gastric mucosa was dark red,shiny,and the folds were arranged regularly,and the anterior gastric mucosa was light pink,smooth,and the folds were arranged neatly.In the MG group,the posterior gastric mucosa was pale red,with poor gloss,and the folds were reduced,and the anterior gastric mucosa was thickened,grainy,with granular eminence,and the folds disappeared.Gastric mucosa was improved in the administration group,especially in the HG group.3.Histological analysis of gastric mucosa Gastric mucosa in CG group were intact,mucosal thickness was normal,glands were arranged neatly,and the number of glands was moderate,without congestion,edema,or mild.In the MG group,the gastric mucosa was thinned,inflammatory cells were infiltrated in the mucosa and submucosa,the inherent glands were reduced and arranged disorderly,often back-to-back,and the nuclei in the mucosal basement were enlarged and hyperstained,even cancerous lesions were found.Gastric mucosal pathology was improved in both HG and VG groups,and the improvement was most obvious in HG group.4.Expression profiles of lncRNAs Compared with CG group,there were 699 differentially expressed lncRNAs in MG group,of which 293 were up-regulated and 406 were down-regulated(P < 0.05).Compared with MG group,there were 860 differentially expressed lncRNAs in HG,of which 434 were up-regulated and426 were down-regulated(P < 0.05).Compared with CG group,91 lncRNAs up-regulated in MG group were restored and down-regulated in HG group(P< 0.05);Among the down-regulated lncRNAs in the MG group,115 lncRNAs were restored and up-regulated in the HG group(P < 0.05).5.Verification of lncRNAs by RT-q PCR Among the screened differential lncRNAs,6 were selected for RT-q PCR detection to verify the sequencing results.Compared with CG group,the expression of NONRATT006284.2,NONRATT006744.2,ENSRNOT00000079699 in MG group was up-regulated(P < 0.05),and all above genes in HG group were down-regulated after Chinese medicine intervention(P < 0.05).The expressions of NONRATT006503.2,NONRATT015614.2,and NONRAT T001596.2 were down-regulated,and the above genes were up-regulated in HG group after Chinese medicine intervention(P < 0.05).The results were consistent with the sequencing results.6.Differential lncRNA homology between rats and humans Homology information was obtained by UCSC database,and the differentially genomic rat lncRNA ENSRNOT00000079699 was homologous to human lncRNA ENST00000517368(lnc 517368).Part two The role of lncRNA ENST00000517368 in Huazhuojiedu decoction in the intervention of precancerous lesions of gastric cancerObjective: To confirm the induction of lncRNA ENST00000517368 on the proliferation and inhibition of apoptosis of precancerous lesions of gastric cancer cells as well as the effect of Huazhuojiedu decoction on them.That is to explore the possible mechanism of Huazhuojiedu decoction in the treatment of PLGC.Methods: Human gastric mucosal epithelial cells(GES-1)were used to prepare precancerous lesions of gastric cancer cells(MC).Meanwhile,Huazhuojiedu decoction drug-containing serum was prepared.Lnc 517368 overexpressed plasmid and small interfering RNA(siRNA)were constructed and transfected into cells.The experimental group was divided into the control group(GES-1),model group(MC),model + Huazhuojiedu decoction group(HZJD),model + lnc 517368 overexpression group(pc DNA3.1 lnc 517368),model + no-load plasmid group(pc DNA3.1),model + lnc 517368 silence group(si lnc 517368),model + empty transfection group(si NC).The effects of lnc 517368 knockdown or overexpression on MC cell growth and apoptosis were evaluated using RT-qPCR,CCK-8,and flow cytometry.Results:1.Morphological changes of cells in each group GES-1 group was characterized by regular cell morphology,spindle- shaped,orderly arrangement and scattered growth.In MC group,some cells were irregular in shape,mostly trilateral,polygonal or fusiform,accompanied by pseudopod formation,and some cells died.The remaining cells were vague in shape,lost their polarity,disordered in arrangement,and overlapping at the edge of the cells.At the same time,the cells gathered and fell off the bottle wall.The cell morphology of pc DNA3.1 lnc 517368 was more severe and advanced to malignancy.There was no significant difference between pc DNA3.1 group,si NC group and MC group.The cells of HZJD group and si lnc 517368 group were arranged neatly,and a few cells were triangular,polygonal or spindle-shaped.2.Expression of lnc 517368 in each group Compared with GES-1 group,the expression of lnc 517368 in MC group was increased(P < 0.05).Compared with MC group,the expression of lnc517368 in HZJD group was decreased(P < 0.05);Compared with pc DNA3.1,the expression of lnc 517368 in pc DNA3.1 lnc 517368 group was significantly increased(P < 0.05).Compared with si NC group,the expression of lnc517368 in si lnc 517368 group was significantly decreased(P < 0.05).3.Cell proliferation in each group Compared with GES-1 group,the cell proliferation ability of MC group was increased(P < 0.05).Compared with MC group,the proliferation ability of HZJD group was decreased(P < 0.05).Compared with pc DNA3.1 group,cell proliferation of pc DNA3.1 lnc 517368 group was increased(P < 0.05).Compared with si NC group,the cell proliferation ability of si lnc 517368 group was decreased(P < 0.05).4.Apoptosis of cells in each group Compared with GES-1 group,the cell apoptosis rate of MC group was decreased.Compared with MC group,the cell apoptosis rate of HZJD group was increased.Compared with pc DNA3.1,the cell apoptosis rate of pc DNA3.1 lnc 517368 group was significantly decreased.Compared with si NC group,the cell apoptosis rate of si lnc 517368 group was significantly increased.Part three LncRNA ENST00000517368 targeted miR-203b-3p to regulate SMAD2 expressionObjective: To confirm that lnc 517368 targeted miR-203b-3p to regulate the expression of SMAD2,and further explore the mechanism of lnc 517368 affecting the function of precancerous lesions of gastric cancer cells.Methods: The information of miR-203b-3p and SMAD2 related target genes of lnc 517368 was retrieved from the database,and the expression of them in GES-1 and MC cells was detected by RT-q PCR.The 3’UTR wild-type and mutated dual luciferase reporter vectors of miR-203b-3p and SMAD2 were constructed,as well as the overexpression and silencing vectors of lnc517368,miR-203b-3p,and the negative control vectors.The fluorescence activity of the cells was determined after co-transfection.Results:1.Screening of miRNA and m RNA data miR-203b-3p and SMAD2 were finally screened out by DIANALnc Base V2 database and Targetscan database according to the binding score,number of binding sites,degree of freedom of nodes,and combined with literature for subsequent studies.2.Expression of miR-203b-3p and SMAD2 in GES-1 and MC cells Compared with GES-1 cells,the expression of miR-203b-3p in MC cells was significantly decreased(P < 0.05),while the expression of SMAD2 was significantly increased(P < 0.05).3.Results of double luciferase activity Overexpression of lnc 517368 significantly decreased the luciferase activity of miR-203b-3p wild-type(P < 0.05),and silencing of lnc 517368 significantly increased the luciferase activity(P < 0.05).Overexpression of miR-203b-3p significantly decreased luciferase activity in the wild type of SMAD2(P < 0.05),and silencing of miR-203b-3p significantly increased luciferase activity(P < 0.05),but there was no significant difference in the mutant type(P > 0.05).Conclusions:1.Huazhuojiedu decoction could improve the state of gastric mucosa,prevent further development,and has a good therapeutic effect on PLGC.2.Lnc 517368 promoted the proliferation of precancerous lesions of gastric cancer cells and inhibited apoptosis,and participated in the occurrence and development of PLGC.3.Huazhuojiedu decoction could improve the statement of proliferation and apoptosis of precancerous lesions of gastric cancer cells,and its effect may partly depend on the down-regulation the lnc 517368.4.Lnc 517368 can target miR-203b-3p to regulate the expression of SMAD2,which may be one of the mechanisms on which lnc 517368 acts. |
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