Study On The Mechanism Of Lovastatin Through KLF 2-mediated MAPK/ERK Pathway And Cholesterol Synt Hesis Pathway To Exhibit Anti-leukemic Activity

Objective:This project will focus on MAPK/ERK signaling pathway and cholesterol biosynthesis pathway,to elucidate the anti-leukemia mechanism of Lovastatin,and provide scientific theoretical basis for the use of Lovastatin in the treatment of leukemia.Methods:(1)The effects of Lovastatin on HEL cell cycle and apoptosis were detected by flow cytometry.(2)Western-Blotting assay was used to detect the effect of Lovastatin on Fli-1 expression.(3)Q-Real time-PCR was used to analyze the effect of Lovastatin on FAM83 A and DDIT4 expression in HEL cells.(4)The effect of Lovastatin on the expression of FAM83 A,DDIT4,ERK and phosphorylated ERK was examined by Western-Blotting assay.(5)Stable HEL cell lines with silencing of KLF2 and DDIT4 were constructed using lentivirus.(6)The effects of sh KLF2 and sh DDIT4 on the proliferation of HEL cells were examined by MTT assay.(7)The effect of sh DDIT4 on ERK and phosphorylated ERK expression was detected by Western-Blotting assay.(8)The effect of Lovastatin on the expression of KLF2 in HEL cells was analyzed by Q-Real time-PCR.(9)The effect of Lovastatin on KLF2 in HEL cells and KLF2 protein expression in spleen of leukemic mice was detected by Western-Blotting assay.(10)The effect of sh KLF2 on the expression of FAM83 A and DDIT4 was detected by QReal time-PCR,and the effect of sh KLF2 on the expression of FAM83 A,DDIT4,ERK and phosphorylated ERK was detected by Western-Blotting assay.(11)The effect of Lovastatin on the expression of 18 genes of cholesterol synthesis pathway and related genes SREBP1,SREBP2,APOA1 was detected by Q-Real time-PCR,and HMGCR,LSS,DHCR7 in 18 genes of cholesterol synthesis pathway were detected by WesternBlotting assay.protein expression.(12)Q-Real time-PCR was used to detect the effect of sh KLF2 on the expression of 12 of the 18 genes of cholesterol synthesis pathway and SREBP1 and SREBP2,and the expression of HMGCR and LSS of cholesterol pathway was further detected by Western-Blotting assay.(13)A leukemia mouse model was constructed using F-Mu LV virus to detect the effect of Lovastatin on the survival time of leukemia mice.Results:(1)Flow cytometry results showed that Lovastatin had an apoptosis-inducing effect on HEL cells,and Lovastatin blocked HEL cells at G1 phase(P<0.05).(2)Fli-1is a classical pro-oncogene in HEL cells,but Western-Blotting results showed that Lovastatin had no effect on Fli-1 expression.(3)Q-Real time-PCR results showed that Lovastatin inhibited the expression of FAM83 A and DDIT4.(4)Western-Blotting results showed that Lovastatin inhibited the protein levels of FAM83 A,DDIT4 and phosphorylated ERK.(5)After silencing KLF2 in HEL cells using lentivirus,the results of cell growth curve plotted by MTT assay showed that sh KLF2 promoted cell proliferation after silencing KLF2 compared with control Scrambled.After treatment with Lovastatin(sh KLF2+Lovastatin),the inhibitory effect on proliferation was diminished compared to Scrambled+Lovastatin.After silencing DDIT4,sh DDIT4 inhibited cell proliferation compared to control Scrambled.After treatment with Lovastatin(sh DDIT4+Lovastatin),the inhibition of proliferation was enhanced compared to Scrambled+Lovastatin.(6)Western-Blotting results showed that sh DDIT4 downregulated the expression of phosphorylated ERK.(7)Q-Real time-PCR results showed that Lovastatin significantly induced KLF2 expression.(8)Western-Blotting results showed that Lovastatin promoted KLF2 in HEL cells and KLF2 protein expression in the spleen of leukemic mice.(9)Western-Blotting results showed silencing of KLF2 in HEL cells with elevated levels of DDIT4,FAM83 A and phosphorylated ERK protein compared to Scrambled.Combined with the results of QReal time-PCR,it showed that after treatment with Lovastatin(sh KLF2+Lovastatin),the m RNA and protein levels of DDIT4 and FAM83 A were elevated and phosphorylated ERK expression was upregulated compared to Scrambled+Lovastatin.(10)Q-Real time-PCR results showed that Lovastatin promoted the expression of 16 of18 genes in the cholesterol synthesis pathway(no effect on the expression of FDFT1 and IDI1);also,Lovastatin induced the expression of SREBP2 and APOA1,but had no effect on the expression of SREBP1.In addition,Western-Blotting results showed that Lovastatin promoted the protein expression of HMGCR,LSS,and DHCR7 in the cholesterol pathway.(11)Q-Real time-PCR results showed that sh KLF2 promoted the expression of SQLE,decreased the expression of MSMO1,MVD,HMGCR,MVK,TM7SF2 and HMGCS1,and had no effect on the expression of CYP51A1,LSS,EBP,DHCR24,DHCR7,SREBP1 and SREBP2.Meanwhile,Western-Blotting results showed that sh KLF2 decreased the protein level of HMGCR and did not affect the protein level of LSS.(12)The results of leukemic mice experiments showed that Lovastatin significantly prolonged the survival time of leukemic mice compared with the control group(P<0.05).Conclusions:(1)Lovastatin exerts anti-leukemic effects through cycle block and induction of apoptosis.(2)Lovastatin blocks the proliferation of leukemic cells through the KLF2-mediated DDIT4/MAPK/ERK signaling pathway.(3)Lovastatin exerts anti-leukemic effects through some genes in the KLF2-regulated cholesterol synthesis pathway.