| Objective: The expressions of mRNA and protein of Piwil4,Piwil2,Mael and Ddx4 genes in PIWI/piRNA pathway were detected in rat liver cancer model and liver cancer cells.The Piwil4 gene was knocked out from the hepatoma cell line to further investigate the effect of PIWIL4 on the proliferation of hepatoma cells.Methoeds:(1)Preparation of rat liver cancer animal model and application of enzyme-linked immunosorbent assay(ELISA)to detect changes in the expression level of tumor markers(HSP,AFP,ASMA)in the model serum.(2)qRT-PCR and Western-blot and immunohistochemistry were used to detect the changes of mRNA transcription levels and protein expression level of PIWI/piRNA pathway-related genes Piwil2,Piwil4,Mael and Ddx4 in liver cancer animal models..(3)Rat liver cancer cells(CBRH-7919)and rat normal liver cells(BRL)were cultured in vitro,and mRNA expression levels and protein expression of Piwil2,Piwil4,Mael and Ddx4 were detected by qRT-PCR and Western-blot.(4)The liver cancer cell line knocked out of piwil4 gene in CBRH-7919 cells was established by CRISPR/Cas9 technology.The expression of Piwil4 and cell proliferation were detected by Western-blot and CCK-8.Results:(1)Elisa method showed that compared with normal liver tissue,the tumor markers HSP HSP(27.43±3.18)、AFP(6.70±3.17)、ASMA(62.63±2.65)were significantly increased in the liver cancer model group,and the differences were statistically significant(P<0.05).(2)qRT-PCR results showed that the relative expression levels of Piwil2(51.61±22.98),Piwil4(114.8±15.74),Mael(5.66±2.02),Ddx4(15.07±6.17)mRNA in liver cancer model tissues were up-regulated compared with the normal group.Western-blot examination showed that the expression levels of Piwil2(1.16±0.34),Piwil4(2.23±0.49),Mael(0.14±0.02),and Ddx4(1.36±0.12)were up-regulated compared with the normal group,IHC examination showed up-regulated expression of Piwil2(807.4±7.58),Piwil4(879.9±12.64),Mael(904.6±8.73),and Ddx4(874.8±7.95),which was significant different.(3)qRT-PCR results showed that the relative expression levels of Piwil2,Piwil4,Mael and Ddx4 mRNA in CBRH-7919 were(5.33±0.54、6.62±1.39、1.05±0.19、5.17± 0.73),mRNA in BRL of normal hepatocytes.Compared with BRL(0.92±0.18 、 2.69±0.82 、0.26±0.11、1.02 ± 0.17),The relative expression levels of Piwil2,Piwil4,Mael and Ddx4 were higher.Western-blot analysis showed that the expression levels of of Piwil2,Piwil4,Mael and Ddx4 in CBRH-7919 were(1.12 ± 0.19 、 1.35±0.14 、0.84±0.10 、 0.79±0.11)higher than that(0.50±0.10 、 0.56±0.11 、 0.48±0.18 、0.34±0.19).(4)Using the CRISPR/Cas9 technology to construct a hepatoma cell line that knocked out the Piwil4 gene,and explore the effect on cell proliferation with knocking Piwil4 gene out.The results of Western-blot showed Piwil4 in KO cells was not expressed.CCK-8 assay showed that the cell proliferation activity of KO cells was significantly lower than that of no-load groups.The results of FCM showed that the apoptosis rate of KO cells was significantly higher than that of no-load groups.Conclusion:(1)the serum concentration of tumor markers in rat liver cancer animal model increased.(2)PIWI/piRNA pathway may play an important role in the occurrence and development of liver cancer.(3)cell proliferation ability was decreased and apoptosis rate was increased in KO group.These results indicated that Piwil4 could promote cell proliferation and inhibit cell apoptosis. |
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