| ObjectiveOur clinical research demonstrates that Yangzheng Sanjie Decoction(YZSJD)relieves the precancerous lesion of gastric cancer(PLGC)by downregulating the expression of EGF and EGFR,additionaly,our previous research also shows that the dysregulation of microRNA-7(miR-7)could prevent or turnover the genesis and development of PLGC.On the base of the results,we made a hypothesis that miR-7 could involve in the process of PLGC developing into GC by targeting EGFR,which is the mechanism YZSJD functions on MKN-45 cells,and sets an example for research and treating GC by using Chinese compound formula.Methods1.Clinical research11 GC tissue of postoperative patients were collected,and three specimens from the same patient were marked as normal tissue,adjacent tissue and GC tissue.The tissues were stained with hematoxylin and eosin(HE)routinely to confirm the exact property,and the expression of microRNA-7 in each tissue was screened by hybridization in situ(ISH).2.Research in vitro2.1 Preparation for the YZSJD containing serum(YCS)YZSJD was frozen into ice,then herbal extract powder was prepared.Additionally,the quality of YZSJD was tested with high performance liquid chromatography(HPLC).In order to prepare for YCS,20 Sprague Dawley rats of male were abtained,and were seperated into two groups randomly including Chinese Medicine(CM)and Contral(CON).The effective dose equivalent was calculated according to the rat surface skin and weight,continuous intragastric administration with 10 ml/kg of YZSJD or saline for 7 days.Blood colloction from the abdominal aorta,centrifugaed,serum remaining,sterilized and stored in the fridge.2.2 Proliferation of MKN-45 cells mediated by YCS MKN-45 cells were separated into two groups including CM and CON,in which(10%,15%and 20%)YCS and culture medium were added into CM while according concentration of rat serum of CON and culture medium were added into CON,after cultured for 48 hours,Cell Counting Kit-8(CCK-8)was added to mesure the Optical Density(OD)with Multimode Reader.10%was the most properconcentration for anylizing the results and was chose to use for the rest of the experiments in this research.Dilute the MKN-45 cells of logarithmic phase into 500/ml,approximately 1000 cells were added into each plate,then 200 μl YCS was added into CM,while with the same amount of rat serum of CON was added into CON.Shakened and cultured for 2 weeks,the cells were stained with methylene violet.Finally,the clone number were observed.2.3 Apoptosis and cell cycle of MKN-45 cells mediated by YCSMKN-45 cells in logarithmic phase were seeded into six well plates,with the same method as CCK-8 to treat the cells,48 hours later,the cells were stained with Annexin V and PI according to the company’s protocol,thenscreened with Flow Cytometer.Similarly,the cells treated with YCS or saline were stained as the cell cycle kit instruction of Beyotime,finally,the DNA content of cells were measured with Flow Cytometer.2.4 Apoptosis of MKN-45 stained with Hoechst33258 mediated by YCSFixed with methanol after the MKN-45 cells were treated,stained with Hoechst33258,kept in dark place for 5 minutes,wash with phosphate buffer(PBS),then the cell morphology was obsverved in the fluorescence microscope.2.5 qRT-PCR tested the expression of miR-7 in different groupsExtract the RNA from the MKN-45 cells of different groups,stored in the-80℃ deep freezer,measurement for the concentration of totle RNA to make sure the number of OD260/OD280 was between 1.8 and 2.1.According to the instruction of the Prime ScriptTM RT reagent kit and Bulge-LoopTM miRNA qRT-PCR Primer,the reverse transcription and qRT-PCR were performed.2.6 Western Blot Analysis the expression of EGFRProtein was extracted from MKN-45 cells reference to the manufacturer’ s protocol.Then,western blot was performed with EGF Receptor XPO Rabbit mAb and anti-β-actin antibodies.Signals were detected with BeyoECL Plus according to the manufacturer’s instructions.Results1.1 Clinical parameters6 of 11 included GC patients were youger than 60,8 patients were male,7 patients were-distal.For the histological pattern,9 were intestinal,the invasion of GC were mostly between T1 and T2,6 of which were with no lymph node metastasis,9 of which were with low differentiation,and 9 of which with the TNM stage between Ⅰ and Ⅱ.1.2 The expression of miR-7 in the postoperative GC tissuesAfter assuring the exact property of the according tissue with HE,ISH was performed.The results show that the expression of miR-7 downregulated in the GC tissue compared with the normal tissue,and the expression of miR-7 in paracancerous ones were between the both.Statistical significance exists among them(P<0.05).1.3 The results of HPLCThe picture for YZSJD of HPLC shows the reasonable condition to be analyzed,with stability and repeatability,and laid a solid foundation for the next experiment.1.4 Proliferation results of MKN-45 mediated by YCSWith CCK-8 mesurement for the OD of cells in different groups,the growth inhibition ratio(GIR)of CM was 22.57%,and statistical significance exists between CM and CON(P<0.05).Additionally,YCS inhibits colony formation ability of MKN-45 compared to the CON group(P<0.05).1.5 Apoptosis results of MKN-45 mediated by YCSStained for the cells with the method of AnnexinV-FITC/PI,tested by flow cytometry,the results show that the percentage of apoptosis cells of CM was higher than that of CON(P<0.05).Furthermore,most cells of CM displayed typical changes including condensed and fragmented nucleus in the group of CM,whereas cells in CON showed distribution of the stain and round homogeneous nuclei.1.6 Cell cycle results of MKN-45 mediated by YCSFollowing the manual for cell cycle kit of Beyotime,tested the DNA content of cells in different groups,the results showed that the percentage of cells in synthesis(S)phase of CM increased than that of CON(P<0.05).1.7 Results of qRT-PCR and Western blotThe result of real-time PCR assay demonstrates that YCS treatment upregulated the expression of miR-7 in MKN-45 cells compared to that in CON(P<0.05).Conversely,the protein level of EGFR in the group of CM was lower than that of CON by Western blot(P<0.05).Conclusion1.The expression of miR-7 was downregulated in GC tissue compared with the normal and adjacent ones(P<0.05),which shows that miR-7 can be a tumor suppressor and involved in the process of precancerous lesions of GC(PLGC)progression to GC.2.YCS inhibits the cell proliferation of MKN-45,induces apoptosis and cell cycle arrest in S phase(P<0.05).Based on the results of qRT-PCR,Western blot and the published results,it shows that YCS functions on GC cells by dysregulation of miR-7 targeting EGFR,which might be the possible novel therapeutic program for PLGC and GC by combination of CM and western medicine. |
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