Research On Proliferation And Mechanism Of Laptm5 3’UTR On Mouse B Lymphoma Cell

Objective:This study explored whether Laptm5 3’UTR affect proliferation of mouse B lymphoma cell. This study also provides foundation to explore the function of competing endogenous RNA (ceRNA).Methods:1. The expression level of Laptm5 mRNA was detected by Real-time PCR. Western blot was used to detect the expression level of Laptm5 protein.2. In order to establish the over-expression cell lines, we first established plasmids including Laptm5 3’UTR wild type (wt) and mutation type (mut). Laptm5 3’UTR sequence with miR-17-3p complementary sequence was mutated in mutation type. Secondly, in order to pack retrovirus, we transfect the plasmids to 293T cell line by lipofection transfection. Thirdly, we chose the 38B9 cell line, adding an auxiliary reagent which named polybreen and retrovirus to enhance infection, they were replaced with fresh medium and added puromycin to enhance the efficiency of infection after 48 hours. Real-time PCR was used to detect the infection efficiency and evaluate the over-expression of Laptm5 3’UTR wt and mut.3. Cell count and cck-8 were used to detect the proliferation of over-expression of Laptm5 3’UTR wt and mut.4. MiR-17-3p was one of miRNAs predicted by bioinformatics. Real-time PCR was used to detect the expression of miR-17-3p in both 38B9 and B cell. We established miR-17-3p plasmid and containing Laptm5 3’UTR wt and mut sequence of luciferase gene fusion plasmid. We used Luciferase to verify that the Laptm5 3’UTR was regulated by miR-17-3p.Results:1. The expression of Laptm5 mRNA and protein in 38B9 were lower than B cell, suggesting that the Laptm5 probably suppress tumor in B cell lymphoma development.2. We established Laptm5 3’UTR wt and mut plasmids successfully. Real-time PCR results showed that the expression of Laptm5 3’UTR in wt and mut were higher than control after retrovirus infection and puromycin selection. The differences between the over-expression group and the negative control group were statistically significant (P<0.05).3. Cell count results showed that by increasing the expression of Laptm5 3’UTR, the cell counts of Laptm5 3’UTR wt was obviously less than Laptm5 3’UTR mut and control. Similarly, CCK-8 results showed the over-expression of LaptmS 3’UTR group cells obviously inhibit the proliferation (P<0.01).4. Real-time PCR results showed that the expression of miR-17-3p in 38B9 was higher than B cell. Luciferase results showed that Laptm5 3’UTR wt sequence of luciferase gene fusion plasmid activity was obviously lower than Laptm5 3’UTR mut.Conclusions:The expression of Laptm5 declined in mouse B lymphoma cell. B lymphoma cell obviously inhibit the proliferation after increasing the expression of Laptm5 3’UTR. The expression of miR-17-3p was declined in B lymphoma cell. MiR-17-3p target negatively regulated the Laptm5 3’UTR, suggesting that Laptm5 3’UTR probably competing combine miR-17-3p and thus affect the expression of miR-17-3p other target protein. This is the possible mechanism which Laptm5 3’UTR can affect the function of B cell lymphoma cells in mice.