miR155 Enhances Autophagy by Reducing MBD5 and METTL3 in Human Liver Cancer Stem Cells

Background: miR-155 is a widely reported carcinogenic miRNA, with up-regulated expression in a variety of human cancers. Methods: Liver cancer stem cells were isolated from Huh7 cells; gene infection, RT-PCR, Western blotting and tumorigenesis test in vitro and in vivo were performed to analyze the signaling pathway. Results: we demonstrate that miR-155 inhibits the expression of MBD5 and METTL3 , reducing the expression of DNMT1. In particular, miR155 inhibits the interaction between MBD5 and DNMT1, thereby inhibiting the binding capacity among MBD5, DNMT1 and IGFII (H19 ICR, IGFII DMR, IGFII Enhancer) DNA, inhibiting IGFII DNA methylation modication. Importantly, miR-155 promotes the formation of DNA loops in the IGFII DNA region (H19 ICR-IGFII Enhancer, IGFII DMR-IGFII Enhancer). Therefore, miR155 promotes the binding ability of H19-pre miR675 to histone methyltransferases SUV39h2 dependent on the H19 ICR-IGFII enhancer DNA loop and enhances the methylation modication of histone H3 on the ninth lysine . Then, the binding ability of RNA polII to P300 was enhanced by the formation of H3K9me2-RNA polII-P300 complex. Furthermore, miR155 promotes the binding ability of the RNA PolII-P300 complex to the IGFII promoter dependent on the IGFII DMR-IGFII enhancer DNA loop and promotes the expression of IGFII. Further research shows that miR155 promotes the binding of IGFII to stemness factors and the loading of stemness factors into the promoter region of HGF, which promotes the expression of hepatocyte growth factor HGF. In particular, miR155 promotes the expression of albumin gene (ALB) via HGF-c-MET, and then promotes the Sirt1 expression through ALB. Strikingly, miR155 promotes the autophagy dependent on acetylase Sirt1,including that miR155 promotes LC3 activation dependent on acetylase sirt1 and enhances binding capacity of LC3 to TP53INP2 / DOR, ATG4, ATG3 and ATG7, and the binding ability of the interaction between ATG5, ATG12, ATG6L1, ATG9. More meaningfully, miR155 enhances the ability of H-Ras to bind to the autophagosomes Beclin1, vps34, vps5, Uvrage, bif1, Lamp1, Lamp2, Lamp3, Rubicon, Rab24 dependent on autophagy, thereby the expression of H-Ras. interaction between MBD5 and DNMT1 in liver cancer stem cells, thereby The binding ability between MBD5, DNMT1 and the three DNA methylation binding regions of IGFII (H19 ICR, IGFII DMR, IGFII Enhancer). (4) The results of specic methylation PCR and DpnI methylation PCR showed miR-155 inhibition the methylation modication levels of the three DNA methylation binding regions of IGFII (H19 ICR, IGFII DMR, IGFII Enhancer) are shown. Studies have shown that MBD5 may contribute to the formation or function of heterochromatin In particular, our results also conrm that miR-155 can reduce the interaction between MBD5 and DNMT1. miR-155 reduced the expression of DNA methyltransferase DNMT1 and the interaction between MBD5 and DNMT1 in human liver cancer stem cells by inhibiting METTL3.LINP1 promotes the progression of cervical cancer by scaffolding EZH2, LSD1 and DNMT1 These results suggest that miR-155 inhibits the binding ability of MBD5 or DNMT1 to the three DNA methylation binding sequences (H19 ICR, IGFII DMR, IGFII Enhancer) in liver cancer stem cells. Our results suggest that miR155 enhances IGFII expression through the IGFII DNA loop in human liver stem cells. Insulin-like growth several that miR-155 the of autophagy Our indicate that miR-155 activates and promotes the of autophagy. First of all, our study found that miR-155 promoted the binding of IGFII to stemness factors and loaded the stemness factors into the HGF promoter region of hepatocyte growth factor, which enhanced the activity of HGF and promoted the expression of HGF. miR-155 signicantly enhances the transcription and expression ability of HGF denpendent on IGFII. Further research shows that miR-155 promotes HGF-MET-mediated albumin gene (ALB) transcription and translation, and signicantly promotes Sirt1 transcription and expression through ALB. After Sirt1 expression increases, miR-155 relies on Sirt1 to promote The deacetylation of LC3 in the nucleus enhanced the binding of LC3 to TP53INP2/DOR, resulting in LC3 nucleation. Our results also show that miR-155 promotes the binding of LC3 and ATG4 in the cytoplasm, which causes LC3 to form LC3-I under the results revealing the new function of autophagy, further study. Our results show that in human liver cancer stem cells, miR-155 stabilizes the MAPK signaling pathway by activating H-Ras. The main molecular mechanisms involved are as follows: (1) miR-155-dependent cell autophagy enhances H in human liver cancer stem cells -Ras expression; (2) miR-155 enhanced the binding ability of GTP-Ras and Raf1, and after adding autophagy inhibitors, the binding ability of GTP-Ras and Raf1 did not increase signicantly, suggesting that miR-155 dependent cell self Phagocytosis enhanced the binding ability of GTP-Ras to Raf1, indicating that miR-155 activated H-Ras; (3) overexpression of miR-155 also signicantly increased pRaf1, pMEK1/2, pERK1/2, pElK, pJak, Jun, pAKT, pmTOR, P70S6K, 4E-BP1, SGK1, C-myc levels, and after transfer into the H-Ras interference plasmid, pRaf1, pMEK1/2, pERK1/2, pElK, pJak, Jun, pAKT, pmTOR, P70S6K, 4E-BP1, SGK1, C-myc levels did not change signicantly, suggesting that miR-155 relies on H-Ras to enhance pRaf1, pMEK1/2, pERK1/2, pElK, pJak, Jun, pAKT, pmTOR, The levels of P70S6K, 4E-BP1, SGK1, and C-myc indicate that miR-155 enhances the function of H-Ras and relies on H-Ras to activate the MAPK signaling pathway. Oncogene super-RNA-EMSA C. pEZX-MT-DNMT1 3’UTR-Luc luciferase reporter gene activity was tested. E. RT-PCR to detect the transcriptional ability of DNMT1, β-actin as an internal reference gene. Western blotting was used to detect the translation ability of DNMT1, and β-actin was used as an internal reference gene. G. RT-PCR to detect the transcriptional ability of DNMT1, β-actin as an internal reference gene. H. Western was used to detect the translation ability of DNMT1, and β-actin was used as the internal reference gene. The co-immunoprecipitation with anti-MBD5 and Western with anti-DNMT1. IgG co-precipitation was used as a negative control. The samples before co-precipitation were subjected to Western blotting with anti-MBD5 as INPUT. immunoprecipitation (CHIP) using ICR-IGFII Enhancer chromosomal method. The cells were cross-linked with formaldehyde and then chromosomal conguration capture (3C) -chromatin immunoprecipitation (CHIP) with anti-CTCF. Using the DNA isolated and puried from the CHIP-3C precipitate as a template, polymerase chain reaction (PCR) amplication was performed using a pair of mixed primers designed according to H9 ICR and IGFII Enhancer. IgG CHIP-3C was used as a negative control; the DNA retained before chromatin immunoprecipitation was used as a template, and the products amplied by independent primers designed by H9 ICR and IGFII Enhancer were used as internal reference (INPUT). CTCF and IGFII DMR-IGFII Enhancer cells chromosomal conguration capture (3C) -chromatin immunoprecipitation (CHIP). into the IGFII DMR-IGFII Enhancer loop by chromosomal conguration capture -chromatin immunoprecipitation Chromosome conguration capture -chromatin immunoprecipitation using gene activity detected. gene activity detected detect the transcription ability of IGFII as an reference gene. detect the transcription ability of gene .β-actin gene. gene.

Introduction miR-155 is also a widely reported carcinogenic miRNA, with up-regulated expression in a variety of human cancers (1). Moreover, miR-155 modulates hepatic stellate cell proliferation (2) and exosome mediated miR-155 delivery confers cisplatin chemoresistance (3). Furthemore, miR-155 inhibits apoptosis(4) and PD-L1 expression (5). In particular, miR-155 promotes glioma progression (6) and the inhibition of miR-155 rejuvenates aged mesenchymal stem cells (7). So far, miR-155 has played a role in Page 5/44 chloroform extraction and ethanol precipitation. PCR products were ampli ed with AccuPrime Tag High Fidelity DNA Polymerase (Invitrogen) Colony formation ability assay. cells were plated in 10 cm dish and incubated in a humidi ed atmosphere of 5% CO 2 incubator at 37ºC for 10 days. For visualization, colonies were stained with 0. 5% Crystal Violet (sigma) in 50% methanol and 10% glacial acetic acid. Colonies were counted using a dissecting microscope by MacBiophotonics Image J.
Tumorigenesis test in vivo Four-weeks male athymic Balb/C mice were maintained in the Tongji animal facilities. Balb/C mice were injected at the armpit area subcutaneously with cells in 100 µl of phosphate buffered saline. The mice were observed over 4 weeks for tumor formation. The mice were then sacri ced and the tumors recovered. The wet weight of each tumor was determined for each mouse. A portion of each tumor was xed in 4% paraformaldehyde and embedded in para n for histological examination and anti-PCNA immunohistochemical staining.
Although the transcription level of METTL3 was signi cantly altered in the four groups (Fig. 3I). However, the translation level of METTL3 was signi cantly reduced in the rLV-miR-155 group compared to the rLV group and signi cantly increased in rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 3J). Collectively, these results suggest that miR-155 targets MBD5 and METTL3, and inhibits the expression of MBD5 and METTL3. miR-155 reduced the expression of DNA methyltransferase DNMT1 and the interaction between MBD5 and DNMT1 in human liver cancer stem cells by inhibiting METTL3 Given that miR-155 inhibits the expression of METTL3, we will adress whether miR-155 reduces the mRNA methylation modi cation of DNA methyltransferase DNMT1 in human liver cancer stem cells by inhibiting METTL3. The binding of METTL3 to DNMT1 mRNA was signi cantly reduced in rLV-miR-155 group compared to the rLV group and signi cantly increased in rLV-Cas9-miR-155 group compared to rLV-Cas9 group (Fig. 4A). The binding of METTL3 to DNMT1 mRNA probes was signi cantly reduced in rLV-miR-155 group compared to the rLV group and signi cantly increased in rLV-Cas9-miR-155 group compared to rLV-Cas9 group ( Fig. 4Ba & b).The methylation modi cation of DNMT1 mRNA was signi cantly reduced in rLV-miR-155 group compared to the rLV group and signi cantly increased in rLV-Cas9-miR-155 group compared to rLV-Cas9 group ( Fig. 4C).pEZX-MT-DNMT1 3'-UTR-Luc luciferase reporter gene activity was signi cantly reduced in rLV-miR-155 group compared to the rLV group (88456.89 ± 9928.304 vs 8756.12 ± 1905.79, P = 0.0022279 < 0.01) and signi cantly increased in rLV-Cas9-miR-155 group compared to rLV-Cas9 group (86132.303 ± 14771.81 vs 218093.41 ± 18504.61, P = 0.0072004 < 0.01) (Fig. 4D).The expression of DNMT1 was signi cantly reduced in rLV-miR-155 group compared to the rLV group and signi cantly increased in rLV-Cas9-miR-155 group compared to rLV-Cas9 group ( Fig. 4E & F). Compared with hLCSCs, a control stable cell line infected with rLV, the expression of DNMT1 was signi cantly reduced in rLV-miR-155 group compared to the rLV group. However, the expression of DNMT1 was signi cantly not altered in the rLV-miR-155 + rLV-METTL3 group ( Fig. 4G & H). The binding of MBD5 to DNMT1 was signi cantly reduced in rLV-miR-155 group compared to the rLV group and signi cantly increased in rLV-Cas9-miR-155 group compared to rLV-Cas9 group (Fig. 4I).
Collectively,these results suggest that miR-155 inhibits the expression ability of DNMT1 gene and the interaction between MBD5 and DNMT1 dependent on METTL3. miR155 inhibits the binding ability between MBD5 or DNMT1 and IGFII DNA methylation binding region (H19 ICR, IGFII DMR, IGFII Enhancer) Since miR-155 inhibits the interaction between MBD5 and DNMT1, it will be considered whether miR-155 inhibits the three DNA methylation binding sequences (H19 ICR, IGFII DMR, IGFII Enhancer) regions of IGFII in liver cancer stem cells.The interaction between MBD5 and H19 ICR probe was signi cantly reduced in the the rLV-miR-155 group compared with the rLV group and was signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5A).The interaction between MBD5 and the IGFII DMR probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5B). The interaction between MBD5 and the IGFII Enhancer probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5C). The interaction between MBD5 and H19 ICR probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5D). The interaction between MBD5 and IGFII DMR probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5E). The interaction between MBD5 and IGFII Enhancer probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5F). The interaction between the three DNA methylation binding region (H19 ICR, IGFII DMR, IGFII Enhancer) and MBD5 was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group ( Fig. 5G).The interaction between DNMT1 and H19 ICR probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5H). The interaction between DNMT1 and IGFII DMR probe w was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5I). The interaction between DNMT1 and IGFII Enhancer probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5J). The interaction between DNMT1 and H19 ICR probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5K).The interaction between DNMT1 and IGFII DMR probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5L). The interaction between DNMT1 and IGFII Enhancer probe was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 5M). The three DNA methylation binding region (H19 ICR, IGFII DMR, IGFII Enhancer) and DNMT1 was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group ( Fig. 5N). Collectively,these results suggest that miR-155 inhibits the binding ability of MBD5 or DNMT1 to the three DNA methylation binding sequences (H19 ICR, IGFII DMR, IGFII Enhancer) in liver cancer stem cells. miR155 inhibits DNA methylation modi cation of the three DNA regions on IGFII (H19 ICR, IGFII DMR, IGFII Enhancer) To investigate the DNA methylation modi cation of three DNA sequences on IGFII (H19 ICR, IGFII DMR, IGFII Enhancer) ,the speci c methylation PCR and Dot blot were used to detect this methylation modi cation in liver cancer stem cells. H19 ICR DNA methylation modi cation was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 6A). However, when DNMT1 was overexpressed or the DNA methylation inhibitor 5-Aza-2 was added in the cells, these effects were completely abolished (Fig. 6A).
The DNA methylation modi cation of IGFII DMR was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 6B). The methylation modi cation of IGFII Enhancer DNA was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and signi cantly increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 6B). The methylation modi cations of H19 ICR, IGFII DMR, IGFII Enhancer DNA were signi cantly reduced in the rLV-miR-155 group compared with the rLV group and increased in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 6C&D). Collectively,these results suggest that miR-155 inhibits the methylation modi cation of three DNA methylation binding sequences (H19 ICR, IGFII DMR, IGFII Enhancer) on IGFII in liver cancer stem cells.
miR-155 promotes the formation of IGFII DNA loops (H19 ICR-IGFII Enhancer, IGFII DMR-IGFII Enhancer) In order to investigate whether miR-155 affects the formation of DNA loops on the IGFII DNA region, we rst analyzed the binding ability of CTCF to the three DNA methylation binding sequences (H19 ICR, IGFII DMR, IGFII Enhancer) in liver cancer stem cells. The interaction between CTCF and H19 ICR probe was signi cantly enhanced in the rLV-miR-155 group compared with the rLV group and signi cantly reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 7A).The interaction between CTCF and IGFII DMR probe was signi cantly increased in the rLV-miR-155 group compared with the rLV group and signi cantly reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 7B). The interaction between CTCF and IGFII Enhancer probe was signi cantly increased in the rLV-miR-155 group compared with the rLV group and signi cantly reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 7C). The interaction between CTCF and H19 ICR probe was signi cantly increased in the rLV-miR-155 group compared with the rLV group and signi cantly reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 7D). The interaction between CTCF and IGFII DMR probe was signi cantly increased in the rLV-miR-155 group compared with the rLV group and signi cantly reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 7E). The interaction between CTCF and IGFII Enhancer probe was signi cantly increased in the rLV-miR-155 group compared with the rLV group and signi cantly reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 7F). However, it was signi cantly altered in IGFII in rLV-miR-155 + rLV-MBD5 group, rLV-miR-155 + rLV-METTL2 group, rLV-miR-155 + rLV-DNMT1 group (Fig. 7G). The interaction between CTCF and the three DNA methylation binding sequences (H19 ICR, IGFII DMR, IGFII Enhancer) was signi cantly enhanced in the rLV-miR-155 group compared with the rLV group and signi cantly reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group ( Fig. 7H). However, it was signi cantly altered in rLV-miR-155 + rLV-DNMT1 group ( Fig. 7I).The formation of H19 ICR-IGFII Enhancer DNA loops was increased signi cantly in the rLV-miR-155 group compared with the rLV group and signi cantly reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 7J). The formation of IGFII-DMR-IGFII Enhancer DNA loops was signi cantly increased in the rLV-miR-155 group compared with the rLV group and signi cantly reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group ( Fig. 7K). Collectively, these observations suggest that miR-155 enhances the formation of IGFII DNA loops (H19 ICR-IGFII Enhancer, IGFII DMR-IGFII Enhancer) in liver cancer stem cells.

miR155 enhances IGFII expression through the IGFII DNA loop
Given that miR155 reduces the methylation modi cation of H19 ICR DNA and promotes the formation of H19 ICR-IGFII Enhancer DNA loop, we will rst analyze whether miR155 increases H19-pre miR675 (intragene gene, miR675 precursor is located in H19 Exons) transcription. The transcription level of H19pre miR675 was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8A). The ability of H19-pre miR675 probe to bind to histone methyltransferase SUV39h2 was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8B). The binding ability of the H19-pre miR675 probe to histone methyltransferase SUV39h2 was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8C).The ability of H19-pre miR675 to bind to histone methyltransferases SUV39h2 or HistoneH3 was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8D).
The binding ability of SUV39h2 to Histone H3 was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8E). However, it was signi cantly altered in the rLV-miR-155 + pGFP-V-RS-H19 group compared to rLV group (Fig. 8F). The H3K9me2 was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8G). However, it was signi cantly altered in the rLV-miR-155 + pGFP-V-RS-H19 group compared with the rLV group (Fig. 8H).The binding ability of H3K9me2 to RNApolII or P300 was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8I). However, it was signi cantly not altered in the rLV-miR-155 + pGFP-V-RS-H19 group compared to rLV group (Fig. 8J). Moreover, the binding ability of RNApolII to P300 was signi cantly altered in the rLV-miR-155 + rLV-JMJD2A (JMJD2A can inhibit H3K9me2) group compared with the rLV group (Fig. 8KCa&b). The ability of RNA PolII to enter the IGFII DMR-IGFII Enhancer loop was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8L). The ability of P300 to enter the IGFII DMR-IGFII Enhancer loop was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8M). However, The ability of RNA PolII to enter the IGFII DMR-IGFII Enhancer loop was signi cantly altered in the rLV-miR-155 + rLV-MBD5 and rLV-miR-155 + rLV-DNMT1 groups compared to rLV group (Fig. 8N). The ability of P300 to enter the IGFII DMR-IGFII Enhancer loop was signi cantly altered in the rLV-miR-155 + rLV-MBD5 and rLV-miR-155 + rLV-DNMT1 groups compared to rLV group (Fig. 8O). The binding ability of RNApolII or P300 to the IGFII promoter was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 8P). However, it ws not signi cantly altered in rLV-miR-155 + rLV-MBD5 and rLV-miR-155 + rLV-DNMT1 (Fig. 8Q). pEZX-MT-IGFII promoter -Luc activity was signi cantly increased signi cantly in the rLV-miR-155 group compared with the rLV group (352455.02 ± 37889.29 vs 2670666.56 ± 321009.37, P = 003626 < 0.01) and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group(340699.19 ± 51915.15 vs 73593.6 ± 15545.73, P = 0.0096644 < 0.01) (Fig. 8R). However, it was signi cantly altered in rLV-miR-155 + rLV-MBD5 group, rLV-miR-155 + rLV-DNMT1 group (253084. 27 (Fig. 8S). The expression of IGFII was signi cantly increased (352455.02 ± 37889.29 vs 2670666.56 ± 321009.37, P = 003626 < 0.01) (Fig. 8T&U). However, it was signi cantly not altered in rLV-miR-155 + rLV-MBD5 group and rLV-miR-155 + rLV-DNMT1 group compared to rLV group (Fig. 8V&W). Collectively,these results suggest that miR-155 enhances the expression of IGFII human liver cancer. miR155 promotes the expression of hepatocyte growth factor HGF dependent on IGFII Because Oct4, Nanog, Sox2, and C-myc are expressed in liver cancer stem cells, we will analyze the relationship between IGFII and these stem factors, thereby revealing whether miR155 plays a role by affecting these stem factors through IGFII in liver cancer stem cells. The binding ability of IGFII to Oct4, Nanog, Sox2, and C-myc was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 9A). The binding ability of Oct4, Nanog, Sox2, C-myc to HGF promoter was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 9B). The binding ability of the HGF promoter probe to RNApolII was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 9C). The binding ability of RNApolII to the HGF promoter was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 9D). The HGF promoter luciferase reporter gene activity was signi cantly increased in the rLV-miR-155 group compared with the rLV group (244182.22 ± 26755.99, vs 991690.64 ± 96119.204, P = 0.00273075 < 0.01) and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (234102.91 ± 36007.75 vs 47444.93 ± 8591.94, P = 0.007357251 < 0.01) (Fig. 9E). The expression of HGF was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 9F & G). The binding ability of Oct4, Nanog, Sox2, C-myc to HGF promoter was signi cantly enhanced in the rLV-miR-155 group compared with the rLV group. However, it wasn signi cantly altered in the rLV-miR-155 + pGFP-V-RS-IGFII group (Fig. 9H). The binding ability of the HGF promoter probe to RNApolII was signi cantly not increased in the rLV-miR-155 group compared with the rLV group. However, it wasn signi cantly altered in the rLV-miR-155 + pGFP-V-RS-IGFII group (Fig. 9I). The binding ability of RNApolII to the HGF promoter was signi cantly enhanced in the rLV-miR-155 group compared with the rLV group. However, it was signi cantly not altered in the rLV-miR-155 + pGFP-V-RS-IGFII group (Fig. 9J). The HGF promoter luciferase reporter gene activity was signi cantly increased in the rLV-miR-155 group compared with the rLV group (145241.67 ± 10458.73 vs 772462.65 ± 100729.75, P = 0.0035029 < 0.01). However, it was signi cantly not altered in the rLV-miR-155 + pGFP-V-RS-IGFII group (145241.67 ± 10458.73vs 134490.03 ± 27214.11, P = 0.3080063 < 0.01) (Fig. 9K). The expression of HGF was signi cantly increased in the rLV-miR-155 group compared with the rLV group. However, it was signi cantly not altered in the rLV-miR-155 + pGFP-V-RS-IGFII group (Fig. 9L & M). Collectively, these results suggest that miR-155 enhances the expression of HGF dependent on IGFII t in human liver cancer stem cells. miR155 promotes the expression of deacetylase Sirt1 dependent on HGF-MET Because albumin ALB regulates the expression of many genes and miR155 promotes the expression of HGF in liver cancer stem cells, we will analyze whether miR155 affects the expression of ALB through HGF.The binding ability of HGF to c-MET was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 10A). The expression level of ALB was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group ( Fig. 10B & C). The expression level of ALB was signi cantly increased in the rLV-miR-155 group compared with the rLV group. However, it was signi cantly not altered in the rLV-miR-155 + pGFP-V-RS-HGF group and rLV-miR-155 + pGFP-V-RS-c-MET group ( Fig. 10 & E). The binding ability of Sirt1 promoter probe to ALB was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 10F). The ability of Sirt1 promoter to bind to ALB was signi cantly enhanced in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 10G).The Sirt1 promoter luciferase reporter gene activity was signi cantly increased in the rLV-miR-155 group compared with the rLV group (98765.34 ± 9580.63 vs 314101.303 ± 37418.49, P = 0.0065 < 0.01) and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (81727.97 ± 15696.44 vs 15048.53 ± 2206.36, P = 0.0081916 < 0.01) (Fig. 10H). The binding ability of ALB to Sirt1 promoter was signi cantly enhanced in the rLV-miR-155 group compared with the rLV group. However, it was signi cantly not altered in the rLV-miR-155 + pGFP-V-RS-HGF group, rLV-miR-155 + pGFP-V-RS-c-MET group ( Fig. 10I). The Sirt1 promoter luciferase reporter gene activity was signi cantly increased in the rLV-miR-155 group compared 89668.56 ± 22722.66, P = 0.07928 > 0.05) (Fig. 10J). The expression of Sirt1 was signi cantly enhanced in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared with the rLV-Cas9 group (Fig. 10K & L). However, it was signi cantly not altered in the rLV-miR-155 + pGFP-V-RS-HGF group, rLV-miR-155 + pGFP-V-RS-c-MET group (Fig. 10M& N). Collectively, these results suggest that miR155 promotes Sirt1 expression dependent on HGF-c-MET in human liver cancer stem cells. miR155 promotes autophagy dependent on Sirt1 in human liver cancer stem cells Because miR-155 promotes the expression of Sirt1 which can cause the deacetylation of the cellular autophagy structural protein LC3, thereby activating LC3, we will analyze whether miR155 affects the activation of LC3 through Sirt1 in liver cancer stem cells. The binding ability of Sirt1 to LC3 was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 11A). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group compared to the rLV-Cas9 group (Fig. 11B). The level of acetylation modi cation of LC3 was signi cantly reduced in the rLV-miR-155 group compared with the rLV group and increased in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 11C). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group compared with the rLV group (Fig. 11D). The binding ability of LC3 to TP53INP2/DOR was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 11E). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group and rLV-miR-155 + sirtinol group compared with the rLV group (Fig. 11F). The binding ability of LC3 and ATG4 was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 11G). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group, rLV-miR-155 + sirtinol group (Fig. 11H). The binding ability of LC3 to ATG3 or ATG7 was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 11I). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group and rLV-miR-155 + sirtinol group compared with the rLV group (Fig. 11J). The levels of LCI and LC3II (active type) was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 11K). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group and the rLV-miR-155 + sirtinol group i group compared with the rLV group (Fig. 11L). The interaction between ATG5, ATG12, ATG6L1, and ATG9 was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 11M). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group and rLV-miR-155 + sirtinol group compared with the rLV group (Fig. 11N). The expression of beclin1 was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (Fig. 11O). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group and the rLV-miR-155 + sirtinol group did not change signi cantly (Fig. 11P). The incidence of autophagy as signi cantly increased in the rLV-miR-155 group compared with the rLV group(7.63 ± 1.82% vs 35.91 ± 4.36%, P = 0.0042416 < 0.01) and reduced in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (8.32 ± 1.067% vs 0.977 ± 0.21%, P = 0.0049583 < 0.01) (Fig. 11Qa & b). The incidence of autophagy as signi cantly increased in the rLV-miR-155 group compared with the rLV group (9.04 ± 1.67% vs 26.51 ± 3.68%, P = 0.00268 < 0.01). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group and rLV-miR-155 + sirtinol group (9.04 ± 1.67% vs 8.27 ± 1.01%,, P = 0.33331 > 0.05; 9.04 ± 1.67% vs 8.71 ± 1.32%%,, P = 0.431059 > 0.05) (Fig. 11Ra & b). The autophagy ow value was signi cantly decreased in the rLV-miR-155 group compared with the rLV group (183.97 ± 29.21 vs 29.85 ± 4.72, P = 0.007876 < 0.01) and increased in the rLV-Cas9-miR-155 group compared to the rLV-Cas9 group (172.84 ± 17.62 vs 402.82 ± 50.11, P = 0.00851 < 0.01) (Fig. 11S). The autophagy ow value was signi cantly decreased in the rLV-miR-155 group compared with the rLV group (141.45 ± 21.48 vs 16.46 ± 4.53, P = 0.004659 < 0.01). However, it was signi cantly not altered in the rLV-miR-155 + NAMPT-NAD + inhibitor group, the rLV-miR-155 + sirtinol group (141. 45 (Fig. 11T). Collectively, these results suggest that miR-155 enhances the occurrence of autophagy through Sirt1 to in liver cancer stem cells.

miR-155 promotes the expression of oncogene H-Ras dependent on autophagy
Given that miR-155 enhances the occurrence of autophagy in liver cancer stem cells, we will analyze whether miR-155 promotes the expression of oncogene H-Ras dependent on autophagy in human liver cancer stem cells.The binding ability of H-Ras to Beclin1, vps34, vps5, Uvrage, bif1, Lamp1, Lamp2, Lamp3, Rubicon, Rab24 was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group comoared to the rLV-Cas9 group (Fig. 12A).The expression level of H-Ras was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group comoared to the rLV-Cas9 group (Fig. 12B). However, it was signi cantly not altered in the rLV-miR-155 + 3-MA group compared with the rLV group (Fig. 12C). The expression level of H-Ras was signi cantly reduced in the rLV-Cas9-miR-155 group compared with the Rlv-Cas9 group. However, it was signi cantly not altered in the rLV-Cas9-miR-155 + Thapsigargin group compared with the rLV group (Fig. 12D). The binding ability of GTP-Ras or Raf was signi cantly increased in the rLV-miR-155 group compared with the rLV group and reduced in the rLV-Cas9-miR-155 group comoared to the rLV-Cas9 group (Fig. 12E).However, it was signi cantly not altered in the rLV-miR-155 + 3-MA group (Fig. 12F).

Discussion
At present, a large number of studies have clari ed that miRNA is closely related to tumorigenesis. Our results clearly con rmed that miR-155 is a miRNA closely related to the occurrence and development of liver cancer. In particular, miR-155 promotes the stemness of liver cancer stem cells and the malignant transformation of liver-like cells. Moreover, miR-155 mainly plays a role by triggering autophagy (Fig. 15).
A study show that LncRNA MEG3 regulates ALG9 by sponging miR-155(26) and miR-155 regulates the pathogenesis of heart failure (27). These results are consistent with our experimental results. However, miR-155 expression is down-regulated in plasma cells from patients with multiple myeloma, and miR-155 may induce anti-multiple myeloma activity by inhibiting the proteasome (28), indicating that miR-155 is not always play a positive role in carcinogenesis.
Our results have con rmed that miR-155 can target the 3′-UTR of methyltransferase METTL3 and inhibits the expression of METTL3, reducing the binding ability of METTL3 to DNMT1, thereby reducing the m6A methylation modi cation and stability of the DNMT1 mRNA, resulted in the suppression of DNMT1 transcription and expression, suggesting miR-155 suppressed the expression of DNMT1 dependent on METTL3. More and more studies have shown that METTL3 is involved in the malignant proliferation of tumors by relying on m6A methylation.A heterozygous MBD5 frameshift mutation was found in a family with intellectual disability and epilepsy (29). single-nucleotide variants in MBD5 was associated with autism spectrum disorders and schizophrenia phenotypes (30). Tomato MBD5 interacts with UV-damaged DNA binding protein-1 (31). m6A RNA methylation regulators correlate with malignant progression (32) and METTL3 inhibits PI3K/Akt signaling pathway (33). At present, there are few reports about miR-155 regulating the m6A methylation modi cation of DNMT1 mRNA through METTL3, and its speci c mechanism still needs to be further clari ed. shown. Studies have shown that MBD5 may contribute to the formation or function of heterochromatin (34). In particular, our results also con rm that miR-155 can reduce the interaction between MBD5 and DNMT1. miR-155 reduced the expression of DNA methyltransferase DNMT1 and the interaction between MBD5 and DNMT1 in human liver cancer stem cells by inhibiting METTL3.LINP1 promotes the progression of cervical cancer by scaffolding EZH2, LSD1 and DNMT1 (35). These results suggest that miR-155 inhibits the binding ability of MBD5 or DNMT1 to the three DNA methylation binding sequences (H19 ICR, IGFII DMR, IGFII Enhancer) in liver cancer stem cells.
Our results suggest that miR155 enhances IGFII expression through the IGFII DNA loop in human liver cancer stem cells. Insulin-like growth factor II (IGFII) acts as a potent mitogen for several tumor types. Matrix metalloproteinase-9 interplays with the IGFBP2-IGFII complex to promote cell growth and motility (36). EphB4 phosphodegron was regulated by the autocrine IGFII in malignant mesothelioma (37). Long noncoding RNA HULC accelerates the growth of human liver cancer stem cells by miR675-PKM2 pathway (38). Histone 3 lysine 9 dimethylation (H3K9me2) orchestrates inheritance of spatial positioning of peripheral heterochromatin through mitosis (39). Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin (40).
Notably, our ndings show that miR155 promotes the expression of hepatocyte growth factor HGF dependent on IGFII in human liver cancer stem cells. These results suggest that miR155 promotes Sirt1 expression dependent on HGF-c-MET. HGF regulates myoblast migration (41).Selective MET inhibitors inhitits non-small cell lung cancer with MET exon 14 skipping (42). HGF-PARP-1 signaling promotes invasion of ovarian cancer cells (43). Anti-tumor activity of Bufalin by inhibiting c-MET mediated MEK/ERK Pathways (44). The C-reactive protein/albumin (CRP/Alb) ratio is a novel in ammation-based score in pancreatic cancer (45). Alb is association with IL-12 levels(46).
Our results have clearly shown that miR-155 affects the production and function of autophagy in liver cancer stem cells. Our results indicate that miR-155 activates LC3 and promotes the formation of autophagy. First of all, our study found that miR-155 promoted the binding of IGFII to stemness factors and loaded the stemness factors into the HGF promoter region of hepatocyte growth factor, which enhanced the transcriptional activity of HGF and promoted the expression of HGF. miR-155 signi cantly enhances the transcription and expression ability of HGF denpendent on IGFII. Further research shows that miR-155 promotes HGF-MET-mediated albumin gene (ALB) transcription and translation, and signi cantly promotes Sirt1 transcription and expression through ALB. After Sirt1 expression increases, miR-155 relies on Sirt1 to promote The deacetylation of LC3 in the nucleus enhanced the binding of LC3 to TP53INP2/DOR, resulting in LC3 nucleation. Our results also show that miR-155 promotes the binding of LC3 and ATG4 in the cytoplasm, which causes LC3 to form LC3-I under the processing of ATG4. Immediately thereafter, the binding ability of LC3 to ATG3 and ATG7 is signi cantly enhanced, and the results of western blotting show The level of LC3-II increased signi cantly, indicating that overexpression of miR-155 activated LC3 through the above mechanism. It is meaningful that the level of LC3-II did not increase signi cantly after the addition of Sirt1 inhibitor, suggesting that miR-155 to promote Activation of LC3 dependent on Sirt1. Studies have reported that miR-155 induces the occurrence of autophagy by regulating the expression of autophagy-related genes (47), which is consistent with our research results, suggesting that miR-155 activates LC3 by affecting the expression of Sirt1, thereby inducing the occurrence of autophagy.
Since overexpression of miR-155 activates LC3 in liver cancer stem cells, miR-155 is likely to also promote the assembly of autophagosomes. In fact, our results have shown that excessive miR-155 enhances the interaction between ATG5 and ATG12, ATG16L1 and ATG9. and miR-155 can promote the occurrence of autophagy. Studies have shown that the assembly of autophagosomes mainly includes three steps: the initiation, nucleation and ampli cation of the separation membrane, and involves the participation of three complexes, namely the ULK1 complex (ULK1, FIP200, ATG13 and ATG101), PI3KC3 complex (Beclin-1, VPS34, VPS15 and ATG14L) and ATG16L1 complex (ATG16L1, ATG5 and ATG12) (48).
Although our research shows that miR-155 can promote the assembly of autophagosomes, the exact mechanism of how miR-155 regulates the assembly of autophagosomes is not clear, and further research is needed.
Cellular autophagy can be selectively degraded by autophagy receptors, and then play a role in selectively removing damaged organelles and speci c proteins. p62 (SQSTM1) is a receptor involved in selective autophagy (58), suggesting that autophagy Phagocytosis can play a role in selective degradation.We found that miR-155 may achieve the selectivity for oncoprotein H-Ras by regulating the occurrence of cellular autophagy. Our results show that miR-155 enhances the binding ability of H-Ras and Beclin1, Vps34, Vps15, Uvrage, Bif1, Lamp1, Lamp2, Lamp3, Rubicon, Rab24 in autophagosomes. miR-155 promoted the expression of H-Ras, suggesting that miR-155 enhances the H-Ras expression dependent on cell autophagy. MicroRNA-30a targets BECLIN-1 to inactivate autophagy and sensitizes gastrointestinal stromal tumor cells to imatinib (59) .Accumulating evidence indicates that Vps34 may also contribute to the progression of human cancers and stimulates the p62 phosphorylation (60). UVRAG is one of the key players of autophagy (61). RAB24 has also been connected to several diseases including ataxia, cancer and tuberculosis (62). Our results are likely to lay some foundation for revealing the new function of autophagy, which still needs further study.
In this study, we reveal some of the cellular molecular mechanisms by which miR-155 plays a carcinogenic role in the development of human liver cancer, but the exact molecular mechanisms of many processes need to be further con rmed, including:

Conclusions
We reveal some of the cellular molecular mechanisms by which miR-155 plays a carcinogenic role in the development of human liver cancer, but the exact molecular mechanisms of many processes need to be further con rmed, including:

Declarations
Ethics approval and consent to participate All methods were carried out in "accordance" with the approved guidelines. All experimental protocols "were approved by" a Tongji university institutional committee. Informed consent was obtained from all subjects. The study was reviewed and approved by the China national institutional animal care and use committee.  Construction and identi cation of miR-155 overexpressed and knocked-out human liver cancer stem cell lines. A. Image taken with a uorescence microscope (100x). B. Dot-Blot was used to detect miR-155 in the cells, and U6 was used as the internal reference gene. C. Detect miR-155 precursors in cells using transcription polymerase chain reaction (RT-PCR) with β-actin as an internal reference gene. D. Northern blotting (Northern blotting) was used to detect the precursors, precursors and matures of miR-155 in the cells, and U6 was used as the internal reference gene. E. The quantitative reverse transcription polymerase chain reaction (RT-PCR) was used. The mature body of miR-155 was detected in the cells, and U6 was used as an internal reference gene. F. Detection of circular miR-155 in cells using back-toback RT-PCR.       B. DNA pulldown to analyze the binding ability of CTCF to IGFII DMR probes. C. DNA pulldown to analyze the binding ability of CTCF to IGFII Enhancer probes D. Super-DNA gel migration was used to determine the binding ability of CTCF and H19 ICR DNA probe. The biotin labeled H19 ICR DNA probe, anti-CTCF, anti-Biotin for Super-EMSA. E. Super-DNA gel migration was used to determine the binding ability of CTCF and IGFII DMR DNA probe. F. Super-DNA gel migration was used to determine the binding ability of CTCF and IGFII Enhancer DNA probe. G. Super-DNA gel migration migration was used to determine the binding ability of CTCF to H19 ICR DNA probe. H. Chromosome immunoprecipitation (CHIP) using anti-CTCF. The DNA isolated and puri ed from CHIP precipitate was used as the template, according to H19 ICR, IGFII DMR, The primers designed by IGFII Enhancer DNA sequence were ampli ed by polymerase chain reaction (PCR). IgG CHIP was used as a negative control; using the DNA retained before chromatin immunoprecipitation as a template, and the product ampli ed with primers designed by H19 ICR, IGFII immunoprecipitation (CHIP) using anti-CTCF. J. Analysis of the binding capacity of CTCF and H9 ICR-IGFII Enhancer by chromosomal con guration capture (3C) -chromatin immunoprecipitation (CHIP) method. The cells were cross-linked with formaldehyde and then chromosomal con guration capture (3C) -chromatin immunoprecipitation (CHIP) with anti-CTCF. Using the DNA isolated and puri ed from the CHIP-3C precipitate as a template, polymerase chain reaction (PCR) ampli cation was performed using a pair of mixed primers designed according to H9 ICR and IGFII Enhancer. IgG CHIP-3C was used as a negative control; the DNA retained before chromatin immunoprecipitation was used as a template, and the products ampli ed by independent primers designed by H9 ICR and IGFII Enhancer were used as internal reference (INPUT). K. The binding ability of CTCF and IGFII DMR-IGFII Enhancer in cells by chromosomal con guration capture (3C) -chromatin immunoprecipitation (CHIP). miR-155 enhances the expression of HGF dependent on IGFII t in human liver cancer stem cells A. Coimmunoprecipitation with anti-IGFII and Western blotting with anti-Oct4, anti-Nanog, anti-Sox2, and anti-Cmyc. IgG IP was used as a negative control.B. Chromosome immunoprecipitation (CHIP) using anti-Oct4, anti-Nanog, anti-Sox2, and anti-C-myc. C. Super-DNA-protein complex gel migration (Super-EMSA) using Biotin labeled HGF promoter probe (Biotin-HGF promoter) , anti-RNApolII, and anti-Biotin for. D.
Chromosome immunoprecipitation (CHIP) using anti-RNApolII. E. pEZX-MT-HGF promoter-Luc luciferase reporter gene activity. Each experiment was repeated three times. The values of each group are expressed as mean ± standard deviation (mean ± SEM, n = 3), **, P <0.01, *, P <0.05. F. RT-PCR was used to detect the transcription ability of HGF, and β-actin was used as an internal reference gene. G. Western blotting to detect the translation ability of HGF. β-actin as the internal reference gene. H. Chromosome immunoprecipitation (CHIP) using anti-Oct4, anti-Nanog, anti-Sox2, and anti-C-myc. I. Super-DNA-protein complex gel migration experiment (Super-EMSA). J. Chromosome immunoprecipitation (CHIP) using anti-RNApolII. K. The assay of pEZX-MT-HGF promoter-Luc luciferase reporter gene activity. L. RT-PCR was used to detect the transcription ability of HGF. β-actin was used as an internal reference gene. M. Western blotting was used to detect the translation ability of HGF . β-actin was used as the internal reference gene. Figure 10 miR155 promotes Sirt1 expression dependent on HGF-c-MET in human liver cancer stem cells. A. Coimmunoprecipitation with anti-HGF and the precipitates were analyzed by Western blotting with anti-c-MET. B. RT-PCR to detect the transcriptional capacity of ALB in cells.β-actin as an internal reference gene.C. Western blotting was used to detect the translation ability of ALB . β-actin was used as the internal reference gene. D. RT-PCR was used to detect the transcription ability of ALB . β-actin was used Figure 12 miR155 enhances the expression levels of pRaf1, pMEK1 / 2, pERK1 / 2, pElK, pJak, Jun, pAKT, pmTOR, P70S6K, 4E-BP1, SGK1, C-myc dependent on H-Ras in human human liver cancer stem cells. A. Coimmunoprecipitation with anti-H-Ras and Western blotting with anti-ATG3, anti-Beclin1, anti-Vps34, anti-Vps5, anti-Uvrage, anti-Bif1, anti-Lamp1, -Lamp2, anti-lamp3, anti-rubicon, anti-Rab24. B. Western blotting was used to detect the translation ability of H-Ras .β-actin was used as the internal reference gene. C.
Western blotting was used to detect the translation ability of H-Ras.β-actin was used as the internal reference gene. D. Western blotting was used to detect the translation ability of H-Ras .β-actin was used as the internal reference gene. E. GST pulldown analysis with anti-GST. The precipitate was analyzed by Western blotting with anti-GTP-Ras. F. GST pulldown analysis with anti-GST. The precipitate was analyzed by Western blotting with anti-GTP-Ras. G&H. Western blotting to detect pRaf1, pMEK1 / 2, pERK1 / 2, pElK, pJak, Jun, pAKT, pmTOR, P70S6K, 4E-BP1, SGK1, C-myc .β-actin as an internal reference gene. U6 as an internal reference gene. C. Real-time RT-PCR for mature miR-15-5p. U6 as an internal reference