| Objectives: To validate the activation effect of the FGFR2 on the PI3k-AKT pathway,we further explore the mechanism of sno RD126 to increase FGFR2,explore the key structure area of sno RD126,and further cover the role of snord126 in the development of liver cancer.Explore the possibility of aso in vitro,which inhibits sno RD126,and discusses the possibility of aso as a new liver cancer treatment drug.Material and Methods: Molecular cloning technology was used to construct a stable line with overexpression of sno RD126,q RT-PCR was used to detect the expression of sno RD126,WB experiment was used to detect whether PI3K-AKT pathway was activated or inhibited,and RNA-pull down technology was used for mass spectrometry analysis.Representative possible binding proteins were selected from the mass spectrometric results and combined with RNA-pull down and Western blot(WB)techniques were used to verify the mass spectrometric results.By point mutating the C box,D box,C’ box and D’ box of sno RD126,the mutated sno RD126 was used to make the probe,and then the pulldown protein was carried out,and then the WB was verified.Finally,the protein hn RNPK was targeted,and then the RIP experiment was used to reverse verify the binding of hn RNPK to sno RD126 and the domain mutated sno RD126,to see whether the binding of hn RNPK to the protein after domain mutation would be affected.Cell lines with single and co-mutated C’ and D domains of sno RD126 were constructed.The proliferation effect of the treated cells was detected by CCK8,plate cloning and EDU,and the transcription level of FGFR2 was detected by q RT-PCR assay.Western blot assay was used to detect whether PI3K-AKT pathway was activated after cell treatment,and Chchip assay was used to detect whether the regulation of sno RD126 and hn RNPK protein complex on the transcription level of FGFR2 was transcriptional regulation.The dual luciferase reporter assay further illustrates this point.After transfection of ASO targeting sno RD126 on liver cancer cells,CCK8 and plate cloning experiments were used to evaluate the effect of ASO on liver cancer cell proliferation.This result was further verified in vivo,namely,human hepatoma cells were first injected subcutanically into nude mice,and a certain concentration of ASO was injected into the tumor after tumor formation.Finally,tumor weighing,histochemistry or Western blot tests were performed to detect the phosphorylation level of cell proliferation markers Ki67 and PI3K-AKT pathway.Sixty-eight cases of hepatoma carcinoma tissues that were surgically resected and matched adjacent cancer-free liver tissues in the Liver Surgery Center of Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology from May 2016 to August 2017 were selected for analysis in combination with clinicopathological factors and prognosis of liver cancer.Results:After overexpression of sno RD126 in Huh7 cell line,the transcription level of FGFR2 was increased and the PI3K-AKT pathway was activated;after knockdown of sno RD126 in Hep G2 cell line,the transcription level of FGFR2 was decreased and the PI3K-AKT pathway was inhibited.After the mutation,the sno RD126 probe pulldown protein was detected by WB assay,and the results showed that the protein bands of hn RNPK and PRPF8 were significantly weakened in the C’ box mutation and D box mutation groups,and the changes of hn RNPK were more obvious.RIP assay was performed in wild-type Hep G2 and sno RD126 Huh7 cell lines that overexpressed wild-type and mutant.The binding of hn RNPK and sno RD126 was further confirmed by the experimental results.Through CCK8,plate cloning and EDU experiments,we found that the proliferation promotion effect of wild-type sno RD126 was obvious.However,when the C’ and D domains of sno RD126 were mutated,the proliferation promotion effect of sno RD126 disappeared.After the protein hn RNPK was knocked down,we found that the proliferation effect of liver cancer was inhibited.When sno RD126 was upregulated,the proliferation effect of sno RD126 disappeared when hn RNPK was knocked down.When hn RNPK was knocked down in wild-type Hep G2 cells,the transcription level of FGFR2 was reduced.Moreover,when hn RNPK was knocked down in sno RD126 upregulated Huh7 cells,the FGFR2 that should have been upregulated was also inhibited.Western blot experiments also showed that the PI3K-AKT pathway was significantly inhibited when sno RD126 was knocked down,and the PI3K-AKT pathway was significantly inhibited when hn RNPK protein was knocked down.In Huh7 cells upregulated by sno RD126,hn RNPK was knocked down.The PI3K-Akt pathway,which should have been activated,is also inhibited.Ch IP assay showed that hn RNPK could bind to the 0-500 bp region of the FGFR2 promoter,which was verified by our subsequent dual luciferase reporter gene assay.After mutations in the C’ and D domains of sno RD126,its ability to promote tumor cell proliferation in vivo was inhibited.Through CCK8 and plate cloning experiments,we found that ASO inhibited the proliferation promoting effect of sno RD126 significantly,and in vivo experiments showed that ASO inhibited the cancer-promoting effect of sno RD126.The expression level of sno RD126 was detected by q RT-PCR in patients with liver cancer compared with their adjacent tissues.The expression level of sno RD126 was mainly high in liver cancer tissues,and patients with high expression of sno RD126 tended to have a lower overall survival rate.Conclusion: sno RD126 binds to hn RNPK through its domain C’ and D and plays a role in promoting cancer.Transcriptional up-regulation of FGFR2 and activation of PI3K-AKT pathway promotes liver cancer proliferation.ASO inhibited the cancer-promoting effects of sno RD126 in vivo and in vitro,suggesting that ASO could be used as a potential therapeutic drug for the treatment of liver cancer.sno RD126 is mostly highly expressed in liver cancer,and liver cancer patients with high expression of sno RD126 tend to have poor prognosis. |
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