The Expression Of PYCR1 And Its Function Research In Hepatocellular Carcinoma

Objective:Pyrroline-5-carboxylate reductase(P5CR)is an enzyme,which plays an important role in cell metabolism,and its function is to catalyze the conversion of pyrroline-5-carboxylic acid to proline.PYCR1 is one of the coding genes of P5CR.A large number of studies have found that PYCR1 shows high expression levels in various tumors,but it has not been reported in Hepatocellular Carcinoma(HCC).We intend to study the expression status of PYCR1 in HCC tissues,and to learn about the effects of down-regulated PYCR1 on the proliferation,migration,invasion and subcutaneous tumorigenic ability of HCC cells.On this basis,we explore the mechanism of PYCR1 on HCC cell-related functions,and seek new treatment methods for HCC diagnosis and prognosis in the future.Methods:1.The relevant transcriptome data for liver cancer were downloaded using the Online Database of The Cancer Genome Atlas(TCGA,https://xena.ucsc.edu/public).After screening of the data strictly,the PYCR1 expression analysis,clinical pathological characteristics correlation analysis and survival analysis were performed.2.After the collection clinical samples,the tissue RNA and protein were extracted.The expression of PYCR1 in HCC and paracancerous tissues were confirmed by immunohistochemistry(IHC),real-time quantitative PCR(RT-qPCR)and Western blot(WB).3.The lentiviral PYCR1 stably silenced cell line Huh7-shPYCRl and lentiviral negative control Huh7-NC were constructed by knocking down the expression of Huh7 HCC cells with short hairpin RNA(shRNA).After forming a stable cell line,the expression of PYCR1 mRNA and protein was detected by RT-qPCR and WB,respectively.4.As the PYCR1 was knockeddown,the proliferation ability of HCC cells were detected by Cell Count Kit-8 method.Moreover,the migration ability and the invasiveness of HCC cells were detected by cell scratch test and,transwell chamber test respectively.5.Established a HCC nude mice model and studied the influence of PYCR1 in the pathogenicity of hepatocarcinoma cells.Results:1.The TCGA data was rigorously screened(excluding 10 non-HCC sample data),and a total of 363 HCC samples and 50 normal samples datas were included in the study.The results showed that the mRNA expression of PYCR1 was significantly increase in HCC tissues and multiple clinical indicators related to differential expression of PYCR1.2.In tissues of patients with liver cancer,the results of IHC,RT-qPCR and WB showed that PYCR1 had a high expression level in HCC tissues(P<0.05).3.This study successfully packaged and synthesized PYCR1 silenced lentiviral solution,and constructed a stable silencing Huh7-shPYCR1 cell line to provide a research object for the next functional experiment.4.The results of WB and RT-qPCR showed that the expression of Huh7-shPYCRl protein and mRNA was lower than the no-load control group(NC).5.After knocking down PYCR1 in Huh7 cells,the proliferation,migration and invasion ability of HCC cells were decreased,and the size and weight of murine cells in Huh7-shPYCRl experimental group were lower than those in Huh7-NC control group(P<0.05).Conclusions:PYCR1 showed high expression levels in HCC through the data analysis of liver cancer on public database,and this result was consistent with the actual HCC patient sample results.2.Survival analysis revealed that low survival time in patients was associated with high expression of PYCR1 in HCC survival analysis and the results were statistically significant.3.The PYCR1 gene silencing in Huh7 was successfully constructed,and the stable cell line Huh7-shPYCR1 roughly analyzed the biological function of PYCR1 in HCC.In addition,down-regulation of PYCR1 in HCC cells would reduce the proliferation,metastasis and invasion ability of HCC cells.4.Established subcutaneous tumor formation model in nude mice,the results show that down-regulation of PYCR1 can inhibit the growth of subcutaneous tumor formation in nude mice.