| Background:Studies have shown that KRAS mutations are present in nearly 90% of pancreatic cancer patients’ cancer tissues,and mutated KRAS can maintain the stemness of pancreatic cancer stem cells and drive their self-renewal.In addition,it has been demonstrated that the pluripotency gene Lin28 B is associated with the development of various tumors and is involved in regulating the biological behavior of various tumor stem cells,and the Lin28B/let-7 pathway has been found to regulate the translation process of KRAS.Accordingly,we speculate that KRAS and Lin28B/let-7 pathway may be involved in regulating the growth of pancreatic cancer stem cells,but the specific regulatory mechanism is not yet clear.Objective:In this study,we analyzed the expression level of RNA-binding protein Lin28 B in pancreatic cancer tissues and cells,and explored the effect of Lin28 B on the proliferation ability,migration ability and stemness of pancreatic cancer cells,and further explored the nuclear translocation mechanism of Lin28 B and its potential molecular biological mechanism to promote the stemness of pancreatic cancer cells,which provided a theoretical basis and new ideas for the treatment of pancreatic cancer.Methods:1.The m RNA expression level of Lin28 B in pancreatic cancer tissues and paired adjacent normal tissues were detected by q RT-PCR.2.To detect the m RNA and protein expression levels of Lin28 B in three human pancreatic cancer cell lines,PANC1,SW1990 and Pa Tu8988,by Western blot and q RT-PCR.3.We transfected sh-Lin28 B plasmid into pancreatic cancer cell lines with relatively high Lin28 B expression,and detect the effects of Lin28 B on the proliferation ability and migration ability of pancreatic cancer cell lines by Western blot and CCK-8 assay and wound healing assay,respectively.4.We analyzed Lin28 B sequence by bioinformatics software to predict the function of Lin28 B,and to explore the interaction between PKCβ and Lin28 B via point mutation techniques and immunoprecipitation(IP)assays,and to clarify the interaction sites of PKCβ and Lin28 B.5.We investigated the interaction of PKCβ with Lin28 B on the regulation of phosphorylation level and nuclear translocation of Lin28 B by Phos-tag system technique,nuclear /cytoplasmic extraction and immunofluorescence assay.6.We investigated whether KRAS can regulate the nuclear translocation mechanism of Lin28 B through PKCβ using Western blot,co-transfection,q RT-PCR,nuclear / cytoplasmic extraction and immunofluorescence double-staining assay.7.We predicted the genes targeted by Lin28B/let-7 axis by bioinformatics and analyzed the correlation between Lin28 B and TET3 expression in pancreatic cancer tissues and cells by immunohistochemistry,Western blot and q RT-PCR.8.We analyzed the expression levels of TET3 protein and m RNA by Western blot and q RTPCR after overexpression or knockdown of Lin28 B in pancreatic cancer cells.9.We analyzed the protein and m RNA expression levels of Lin28 B and TET3 by overexpression of let-7i in pancreatic cancer cells using Western blot and q RT-PCR.And we also detected the expression levels of TET3 after co-transfected Flag-Lin28 B and let-7i in pancreatic cancer to analyze the regulatory effect of Lin28B/let-7i axis on TET3 expression.10.We verified the interaction of let-7i with Lin28 B and TET3 by using a dual luciferase reporter gene assay and mutating the binding sites of let-7i interacting with Lin28 B and TET3 respectively.11.We investigated the effect of Lin28B/let-7i/TET3 axis on the proliferation and invasion ability of pancreatic cancer cells through CCK-8 assay,colony formation assay and transwell assay,and analyzed the effect of Lin28B/let-7i/TET3 axis on the stemness of pancreatic cancer cells by using Western blot assay to detect cancer cell stemness markers.Results:1.The results of q RT-PCR showed that the m RNA expression level of Lin28 B was relatively high in pancreatic cancer tissues compared with paired adjacent normal tissues.2.Western blot and q RT-PCR were performed to detect Lin28 B protein expression levels and m RNA expression in three human pancreatic cancer cell lines,PANC1,SW1990 and Pa Tu8988,respectively,and we found that Lin28 B expression was highest in PANC1 cells,followed by SW1990,and lowest in Pa Tu8988.3.By down regulating Lin28 B in PANC1 cell lines,CCK-8 assay and wound healing assay showed that the proliferation and migration ability of pancreatic cancer cells were suppressed after Lin28 B knockdown,and the expression of Cyclin B1 and CDC25 B,which are related to cell proliferation,were also significantly down-regulated.4.Bioinformatics software analysis of Lin28 B sequence revealed two conserved sequences that may interact with PKC,and the two conserved sequences are the key sequences that determine the nuclear localization of Lin28 B.Immunoprecipitation further confirmed the interaction between PKCβ and Lin28 B,and point mutation experiments also clarified the interaction sites between PKCβ and Lin28 B.5.Phos-tag system technique and Western blot showed that PKCβ could directly phosphorylate the S243 site of Lin28 B in pancreatic cancer cells,and the results of nuclear / cytoplasmic extraction and immunofluorescence assay showed that PKCβ interacted with Lin28 B promoted the nuclear translocation of Lin28 B.6.Compared with the control group,nuclear localization of Lin28 B was significantly increased when KRAS overexpressed in pancreatic cancer cells,while we observed nuclear Lin28 B showed significantly decreased after co-transfection with Flag-KRAS and sh-PKCβ.Besides,upregulation of KRAS promoted the co-localization of PKCβ and Lin28 B.7.Bioinformatics predicted that let-7i has potential complementary binding sites with the 3’UTR region of Lin28 B and TET3,and immunohistochemistry,Western blot and q RT-PCR results showed that Lin28 B and TET3 had correlation in protein and m RNA expression levels in pancreatic cancer tissues and cell lines.8.Western blot and q RT-PCR results showed that the protein and m RNA levels of TET3 in pancreatic cancer cells were significantly reduced after down-regulation of Lin28 B expression,while up-regulation of Lin28 B expression showed that the protein and m RNA levels of TET3 were significantly increased.9.When let-7i was overexpressed in PANC1 cells,Western blot and q RT-PCR results showed that m RNA levels and protein expression of Lin28 B and TET3 were down-regulated.It showed TET3 protein and m RNA levels increased in Flag-Lin28 B overexpressing cells,whereas no significant changes were observed when co-transfected with Flag-Lin28 B and let-7i compared to the control group.10.Dual luciferase reporter gene assay and point mutation assay showed that the fluorescence intensity produced by Lin28 B 3’UTR and TET3 3’UTR luciferase plasmids was decreased when up-regulating let-7i compared with the control,while both of the Lin28 B 3’UTR and TET3 3’UTR mutant plasmids did not show a significant decrease in fluorescence intensity.11.The results of CCK-8 assay,colony formation assay and transwell assay showed that Lin28B/let-7i/TET3 axis could promote the proliferation,colony formation and invasive ability of pancreatic cancer cells,and it could promote the protein expression of stemness markers,OCT4,NANOG and SOX2.Conclusion:Lin28B expression was up-regulated in pancreatic cancer tissues and promoted the proliferative capacity,invasive ability and stemness maintenance of pancreatic cancer cells.In addition,KRAS could promote the phosphorylation and nuclear translocation of Lin28 B in pancreatic cancer cells through PKCβ,and the main site of interaction between PKCβ and Lin28 B was the S243 site of Lin28 B.The nuclear translocation of Lin28 B could competitively bind let-7i to upregulate the expression of TET3,thus promoting the proliferation,invasion and stemness maintenance of pancreatic cancer cells.Therefore,Lin28 B is expected to be a potential therapeutic target for pancreatic cancer. |
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