| BackgroundCancer-associated fibroblasts(CAFs)are the most principal cells of depositing and remodeling extracellular matrix(ECM)within solid tumors.Both CAFs and ECM have been demonstrated to play critical roles in tumor development.However,the functional roles of CAFs-associated ECM or ECM remodeling in the pathogenesis of gastric cancer remain unclear.At present,few drugs have been found to effectively intervene the invasion and metastasis of gastric cancer cells affected by CAFs.Danggui Beimu Kushen pill is a traditional Chinese medicine prescription which has been proved to have a significant effect on the treatment of tumors.To explore the role of CAFs-related ECM remodeling in the pathogenesis of gastric cancer,as well as the molecular mechanism of Danggui Beimu Kushen pill intervention in CAFs affecting the invasion and metastasis of gastric cancer cells,can be a good supplement for the prevention and treatment strategies of gastric cancer.MethodsBioinformatics-analysis of the differentially expressed genes between CAFs and corresponding normal fibroblasts(NFs)in gastric cancer was performed.The clinical relevance of hyaluronan and proteoglycan link protein 1(HAPLN1)was investigated using TCGA data and human gastric cancer specimens.Spheroid cell invasion assay and nude mouse xenograft model were introduced to assay cell invasion.Second harmonic generation(SHG)was used to image and analyze the changes of collagen fibers in ECM.RT-qPCR,Western blot,H&E,IHC and IF were used to study the expression of HAPLN1 and the molecular mechanism of HAPLN1 inhibition by Danggui Beimu Kushen pill.Results(1)GEO data analysis results showed that HAPLN1 was the most significantly up-regulated gene of gastric CAFs compared with NFs.Analysis of clinical data from patients with gastric cancer using TCGA and GTEx databases showed that higher HAPLN1 levels were associated with shorter overall survival of gastric cancer.IHC showed a positive correlation with tumor T stage(P<0.0001),lymph node metastasis(P=0.0006)and TNM stage(P=0.0063).qPCR detection suggested that HAPLN1 mRNA level in moderately differentiated(Grade 2)(P<0.01)or poorly differentiated(Grade 3)(P<0.05)gastric cancer was higher than that in highly differentiated(Grade 1)gastric cancer.HAPLN1 was up-regulated in gastric cancer tissues compared with normal gastric tissues,and HAPLN1 was mainly produced by CAFs.The mechanism is related to the up-regulation of HAPLN1 expression in gastric cancer cells by stimulating TGF-β1/Smad2/3 signaling pathway.TGF-β1 promotes the up-regulation of Smad signaling pathway protein and HAPLN1 expression in a time-dependent manner,and is significantly inhibited by S8144(Smad3 inhibitor).The results of ChIP-PCR showed that the DNA binding ability of Smad2/3 was significantly higher than that of IgG,indicating that Smad2/3 was a transcription factor of HAPLN1 expression.(2)Cell wound healing,Transwell and 3D spheroid invasion assay showed that CAFs-derived HAPLN1 significantly promoted the migration and invasion of MKN45 cells(P<0.05).The primary CAFs cell line with HAPLN1 knockdown expression(CAFs-sh HAPLN1)was constructed,and it was found that CAFs increased the migration and invasion ability of MKN45 cells(P<0.05).However,HAPLN1 knockdown significantly reduced the promotion of CAFs-mediated invasion of MKN45 cell migration(P<0.05),and this inhibition could be recovered by adding rHAPLNl(P<0.05).In mouse gastric cancer xenotransplantation model,tumor volume in MKN45/CAFs-sh HAPLN1 group was smaller than that in MKN45/CAFs group(P<0.001),and rHAPLN1 addition significantly offset this effect(P<0.01).SHG analysis of a mouse gastric cancer xenograft model showed that HAPLN1 knockdown increased the number,density,width,and length of fibers in ECM,but significantly reduced fiber alignment.In contrast,rHAPLN1 treatment significantly offset these effects on ECM caused by HAPLN1 knockdown.The number,density,width,and length of fibers in the SHG analysis ECM performed on human samples decreased significantly from Grade 1 to 3,but the fiber alignment increased.(3)Danggui Beimu Kushen pill inhibited the migration and invasion of gastric cancer cells in mouse xenotransplantation model.The mechanism was that oxymatrine inhibited the down-regulation of HAPLN1 expression in fibroblasts by regulating TGF-β1/Smad2/3 signaling pathway.With the increase of intervention time,The expression of FAP,α-SMA,HAPLN1,p-Smad2,p-Smad3,Smad2/3 and Smad4 were down-regulated simultaneously.At the same time,oxymatrine significantly reduced the promotion of CAFs-mediated invasion of MKN45 cell migration(P<0.05),and this inhibition could be recovered by adding rHAPLN1(P<0.05).SHG analysis of a mouse gastric cancer xenograft model showed that oxymatrine increased the number,density,width,and length of fibers in ECM,but significantly decreased their alignment.In contrast,rHAPLN1 treatment significantly offset these effects on ECM caused by oxymatrine.ConclusionsTGF-β1 mediated CAFs expression of HAPLN1,and was associated with the progression of gastric cancer.HAPLN1 expressed by CAFs remodeled extracellular matrix of gastric cancer and promoted tumor invasion.The mechanism of Danggui Beimu Kushen pill inhibiting gastric cancer invasion is related to oxymatrine,which regulates the expression of HAPLN1 in CAFs and inhibits the invasion and metastasis of gastric cancer.In our study,we elucidate the role of CAFs-derived HAPLN1 in the pathogenesis of gastric cancer,and explore the molecular mechanism of Danggui Beimu Kushen pill inhibiting the invasion of gastric cancer.The active ingredient oxymatrine down-regulated the expression of HAPLN1 in CAFs and inhibited the migration and invasion of gastric cancer. |
PDF Full Text Request