| Objective Hepatocellular carcinoma(HCC)is one of the most common malignant tumor in our country,the mortality rate remains high,exploring the pathogenesis of HCC can help improving the clinical treatment effect and the quality of life for patients.Tumor microenvironment(TME)provides a new idea on the studing of the pathogenesis of tumor,and it has become a focus of cancer research in recent years.This study plansrealizing the effects of sulfhydryl oxidase 1(QSOX1)over-expression on the function of HCC cells by means of testing the biological function changings of the SMMC-7721 HCC cells,and exploring the HCC development mechanism of the tumor associated macrophage exocrine protein in the tumor microenvironment.Methods The lentivirus withQSOX1 over-expression genes infect the human acute monocytic leukemia cell(THP-1),and selecting stable THP 1 cells of QSOX1 over-expression,regard it as over-expression group(OE group);At the same time,the empty virus transfection THP 1 cells was the negative control group(NC group)and the blank control group(BC group)do not make any deal with THP 1 cells.Use PMA(Phorbol-12-myristate-13-acetate)to induce THP 1cells into undifferentiated macrophages(UM0),culturingit in serum-free medium and collecting UM0 conditioned medium.Using real-time quantitative PCR(RT-PCR)and Western blot detect QSOX1 relative expression of RNA and protein expression,respectively.After culturingSMMC-7721 HCC cells with conditioned medium of three macrophage groups.CCK-8experiment tested the changes of proliferation of HCC cells.The clone formation experiment detected clone forming ability and independent survivalability of HCC cells.Transwell assay tested invasion and metastasis ability of HCC cells.Scratch experiment tested the migrate function of SMMC-7721 HCC cells.Use SPSS21.0 for statistical data analysis.Results RT-PCR results showed QSOX1 genes had over expressed in THP-1 cell;Western blot found QSOX1 protein has two subtype,they are QSOX1-S and QSOX1-L,the quantity of protein expression of OE group was obviously higher than negative control group and blank control group.THP-1cell was successfully induced to macrophage(M0)by using PMA.The clone formation experiment found that HCC cellcloning number of OE group was82.67±9.07 on average,higher than that of BC group(48.33±7.50 on average)and NC group(44.67±9.29on average),the difference was statistically significant(P<0.05).CCK-8 experiment have shown that cell proliferation abilityin QSOX1 over-expression group in 72h point was higher than the other two groups(P<0.05),QSOX1 can promote cell proliferation of HCC.Cell scratch experiment show that the HCC cell migration area of OE group in 12h and 24h was 0.613±0.613mm~2 and 1.477±0.160 mm~2on average,respectively.The migration area were significantly higher than that of the other two groups at the same time,the difference was statistically significant(P<0.05).Transwell assay shows,the number of metastasiscells in OE group was 157±16.66 on average,the BC group was 105±14.68 and the NC group was 116±11.98,the metastasis cells of QSOX1 over-expression group were obviously higher than that other two groups,the difference was statistically significant(P<0.001).Transwell invasion experiment showed,the number of invasion HCC cells of three groups has no significant difference(P>0.05).Conclusion THP-1 cell can be induced to tumor associated macrophageM0in tumor microenvironment by PMA.QSOX1 protein has two subtypes,they areQSOX1-S(67kd)and QSOX1-L(83kd).QSOX1 protein over-expression can promote proliferation,migration and metastasis ability of SMMC-7721 HCC cells.QSOX1 has regulating effect for the development of HCC,at the same time,this study also provide the evidence for the researching of the relationship between tumor associated macrophages secretory protein and the hepatocellular carcinoma. |
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