Analysis Of Differentially Expressed Genes In Human Hepatoma Cells Induced By Hypoxia And The Effect Of SLC6A8 Gene On The Proliferation And Invasion Ability Of Hepatoma Cells

Objective:Using gene chip technology and information biology analysis methods to study the differentially expressed genes related to hypoxic response in human hepatoma cells,and to screen and explore the up-regulated and significantly differentially expressed factors--SLC6A8 to regulate the proliferation,apoptosis,invasion,The mechanism of migration.Methods :Affymetrix’s Gene Chip Clariom S Array chip was used to detect the gene expression profiles of hypoxia-induced and normoxia-cultured Huh-7 and Hep3 B human hepatoma cells,and the results were analyzed using edge R software to study human hepatoma cells.The differentially expressed genes related to hypoxic response were screened for the differentially expressed genes whose expression was up-regulated and had the largest variation.Establish human Huh-7 and Hep3 B liver cancer cell line culture system.Lentiviral vectors were constructed to silence the expression of the SLC6A8 gene.There are control group(Con),negative control group(NC),siRNA-1(KD1)experimental group and siRNA-2(KD2)experimental group.Using Q-PCR to test the m RNA expression of SLC6A8 gene,Western blot detection of HIF-1α and SLC6A8 protein levels;MTT detection of cell proliferation,TUNEL detection of cell apoptosis,scratch test to detect cell migration,Transwell The cell detects the cell’s ability to invade.A comparative study of the effects of SLC6A8 silencing on the proliferation,apoptosis,invasion and migration of liver cancer cells.Results:The two differentially expressed genes of the two cell lines totaled 177,127 were up-regulated,and 50 were down-regulated.After intersection analysis of the KEGG pathway,the five genes whose expressions were up-regulated and increased the most were selected for information query,TXNIP,ATF3,SLC6A8,TIPARP,and PFKFB4.The results showed that TXNIP,ATF3,SLC6A8,and PFKFB4 were all related to the occurrence of liver cancer;TCGA database analysis found that the expression of SLC6A8 gene in liver cancer tissues increased significantly,and its high expression was associated with histological typing.the expression level of SLC6A8 m RNA in Huh7 cells was significantly lower in the KD1 and KD2 groups(0.23 ± 0.10,0.61 ± 0.06)than in the blank control group(1 ±0.10)and the negative control group(1.29 ± 0.11)(P<0.05).The expression levels of SLC6A8 protein in experimental KD1 and KD2 groups(0.246 ± 0.003;0.0261 ±0.003)were lower than those in blank control group(0.606 ± 0.001)and negative control group(0.547 ± 0.004)(P<0.05);HIF1α protein expression levels The experimental KD1 and KD2 groups(0.176 ± 0.001;0.250 ± 0.031)were lower than the blank control group(0.435 ± 0.003)and the negative control group(0.492 ± 0.003)(P <0.05).the relative expression levels of SLC6A8 m RNA in Hep3 B cells were significantly lower in the KD1 and KD2 experimental groups(0.47 ± 0.05,0.75 ±0.12)than in the blank control group(1 ± 0.07)and the negative control group(1.36 ±0.05)(P<0.05).The expression levels of SLC6A8 protein in experimental KD1 and KD2 groups(0.299 ± 0.008;0.159 ± 0.005)were lower than those in blank control group(0.818 ± 0.007)and negative control group(0.692 ± 0.004)(P<0.05);HIF1αprotein expression The experimental KD1 and KD2 groups(0.228 ± 0.010;0.134 ±0.003)were lower than those in the blank control group(0.556 ± 0.002)and the negative control group(0.516 ± 0.006)(P<0.05).In the two groups of cell experiments,after SLC6A8 was knocked out,the cell proliferation was detected by MTT technology,which showed that the cell proliferation and cell activity of the KD1 and KD2 groups were significantly reduced.TUNEL staining showed dark brown in both KD1 and KD2 groups,suggesting increased apoptosis.In Hep3 B cells,both KD1 and KD2 inhibited cell migration with the extension of the virus infection time(P<0.05).Compared with phase KD2,the inhibitory effect of KD1 was more significant(P<0.05);The cell migration rate increased in Huh-7 cells 24 hours after infection.Both KD1 and KD2 inhibited cell migration 48 hours after transfecting(P<0.05).However,there was no significant difference between KD1 and KD2(P﹥ 0.05).SLC6A8 gene knockout reduced the invasion ability of Huh-7 and Hep3 B cells(P<0.05),but there was no significant difference between KD1 and KD2(P﹥0.05).Conclusion : After culturing human liver cancer Huh-7 and Hep 3B cells under hypoxia,a large number of cytokines are abnormally expressed;TXNIP,ATF3,SLC6A8,TIPARP and PFKFB4 are the five differentially expressed genes with the highest up-regulation and the highest increase.The differential genes may be related to the occurrence and development of liver cancer;bioinformatics analysis showed that the expression of SLC6A8 gene in liver cancer tissues increased significantly,and its high expression was related to histological typing.SLC6A8 is highly expressed in hepatocellular carcinoma cell lines.SLC6A8,which is low in expression,can inhibit cell proliferation,invasion,migration,and induce apoptosis.The protein expression level of HIF-1α decreased after knocking down SLC6A8,suggesting that SLC6A8 has a positive network regulation relationship with HIF-1α.SLC6A8 may be an upstream driver of HIF-1α,and mediates the biology of liver cancer cells through HIF-1α Learn to change.