The Mechanism Study About Inhibitory Effect Of Parvovirus H-1 Nonstructural Protein 1 On Gastrointestinal Tumor Cells

Background:Gastrointestinal cancer is one of the most common malignant tumors in the world.Incidence rate and mortality of it are high in the world.The nonstructural protein 1(NS1)gene of parvovirus H-1 can inhibit the proliferation of some tumors.CD44 is one of the surface markers of tumor stem cells,including standard form CD44(CD44s)and CD44 variant(CD44v),which is related to the formation and progress of tumor.The rise of reactive oxygen species(ROS)is one of the important mechanisms of radiotherapy and chemotherapy.Objective:To investigate the effect of NS1 on the expression of CD44 whether through CD44-xCT-ROS signal pathway to inhibit tumor and its molecular mechanism.Methods:1.Construction of recombinant plasmid pFlag-NS1 with protein tag;2.Expression of NS1 in gastrointestinal cancer cells by x-tremegene HP liposome transfection technology;3.The expression of NS1 in gastrointestinal cancer cells was detected by Western blot;4.The effect of NS1 on the proliferation of gastrointestinal cancer cells was observed by CCK-8 cell proliferation experiment;5.The effect of NS1on the migration of gastrointestinal cancer cells was detected by cell scratch experiment;6.The effect of NS1 on the expression of CD44 and ROS level of gastrointestinal cancer cell line was analyzed by flow cytometry;7.The effect of NS1 on CD44s and CD44v expression of HT29 cell line was detected by qRT-PCR.Results:1.The results of CCK-8 cell proliferation experiment showed that there was no significant difference in cell proliferation ability between CD44~-highly differentiated gastric cancer cell MKN28 recombinant plasmid pFlag-NS1 group and empty plasmid/blank group(P>0.05);the proliferation ability of CD44~+moderately differentiated gastric cancer cell SGC7901,CD44~+poorly differentiated gastric cancer cell MKN45,and CD44~+HT29 colon cancer cell recombinant plasmid pFlag-NS1group decreased(P<0.05).2.The results of scratch test showed that there was no significant difference in cell migration ability of MKN28 between pFlag-NS1 group and empty plasmid/blank group(P>0.05);the migration ability of SGC7901,MKN45 and HT29 cell in pFlag-NS1 group decreased in different degrees(P<0.05).3.The results of flow cytometry showed that there was no significant difference in CD44 expression of MKN28 cell between pFlag-NS1 group and empty plasmid group(P>0.05),and CD44was negative,no expression;compared with empty plasmid group,the CD44 expression of SGC7901,MKN45 and HT29 cell in pFlag-NS1 group decreased in different degrees(P<0.05).4.The results of flow cytometry showed that there was no significant difference(P>0.05)in ROS level of MKN28 cell between pFlag-NS1 group and empty plasmid group,and the ROS level was low;the ROS level of SGC7901,MKN45 and HT29 cell in pFlag-NS1 group increased significantly(P<0.05),and ROS increased significantly in 6-24h,and began to decrease in different degrees after 24h.5.The expression of CD44s and CD44v in HT29 cells detected by qRT-PCR showed that compared with the empty plasmid group,CD44s in pFlag-NS1 group increased(P<0.05),and CD44v decreased in different degrees,among which CD44v3-CD44v6and CD44v9-CD44v10 decreased significantly(P<0.05),and CD44v6 decreased most obviously.Conclusion:1.NS1 can inhibit the migration and proliferation of CD44~+gastrointestinal tumor cells.It has a good inhibitory effect on the moderately and poorly differentiated gastric cancer cells.2.NS1 may increase ROS in gastrointestinal tumor cells by down regulating CD44,that is to say,it plays an anti-tumor role through CD44v-xCT-ROS signal pathway.3.NS1 plays an anti-tumor effect on colon cancer mainly by up regulating CD44s,down regulating CD44v,and CD44v6 is the main one.