The Effect Of Long Non-coding RNA On The PTEN Gene Expression

BackgroundAt present, the top five causes of Chinese population are follows : malignant tumor,cerebrovascular disease,respiratory diseases,heart disease and injury.With increasing incidence and mortality,cancer is the leading cause of death in China.And it is the major public health problem.The results from The National Central Registry of China indicated that an estimated4292,000 new cancer cases and 2814,000 cancer deaths would occur in China in 2015.So,it is important to study the pathogenesis of tumor,which would supply chances to seek candidate diagnostic biomarkers and therapeutic targets.Tumorigenesis is a well-known multi-step process involving inactivation of tumor suppressor genes and activation of oncogenes.Studies have confirmed that some tumor-suppressor gene mutation is one of the most common molecular changes.Whereas it is thought that the long non-coding RNAs could not be translated in to protein.Emerging evidence indicates that lncRNAs play a critical role in the initiation and progression of tumor disease.PTEN has the protein phosphatase activity as well as the lipid phosphatase activity.It has been confirmed that the mutation of PTEN was found in many kinds of malignant tumors.PTEN plays an important role in the PI3K/AKT signal transduction pathway.PTEN is highly homologous to PTENP1.The high homology region is located in the 3 ’non encoding region of PTENP1 and PTEN.Within the high homology region, we found perfectly conserved seed matches for the PTEN-targeting mic RNA.Related studies have shown that in some malignant tumor cell lines such as breast and prostate, some micRNA can not only target the combination PTENP1 but alsoPTEN.But the specific mechanism is not clear.In this study,HEK293 cells were selected as the experimental subject, These cells were derived from 293 cells.The cell has the characteristics of immortalization.While immortalization is a necessary stage of cell malignant transformation.ObjectiveIn this study, we designed specific PCR primer sets in the non-homologous 3’UTR regions to overexxpress the high homology region. We detected the expression of PTEN gene and the expression of the important molecules in the PI3K/AKT signaling pathway.Aims to explore the mechanism of the effect of PTENP1 on the expression of PTEN gene.Materials and methods1.HEK293 T cells were used as the experimental objects, and the cells were cultured in DMEM culture medium.2. Amplification of cDNA PTENP1 plasmid into Escherichia coli Selected by ampicillin screening, select positive clones, shake bacteria, plasmid extraction, enzyme digestion and sequenc.After the correct sequencing results, the enzyme fragment is linked with pcDNA3.1(+).Extract plasmids using endotoxin-free plasmid extraction kit, obtaine a large number of plasmid,then reserve under-80℃.3. The experimental group: the well condition of the 293 T cells are divided into 3 groups:normal control group(A group), empty plasmid group(B group), transfection of pcDNA3.1(+)-PTENP1 plasmid group(C group).Cells are transfected using Lipofectamin2000 transfection kit according operation steps. The cells were observed by inverted microscope, and the effect of transfection was evaluated.4. Extract total RNA using Trizo reagent, the expression of PTEN gene is detected by RT-PCR.5. Blot Western method is used to detect the expression of PTEN gene, and the expression of PI3K、p-PI3K、AKT、p-AKT in the PI3K/AKT signaling pathway.6. All the original data are represented as mean + SEM.Statistical analysis was performed using Statistics SPSS software. If statistical results is set as P<0.05, the difference is statistical significant.Results1. Obtain the target fragment through the PCR.. The sequencing results are fully consistent with Genebank.2. The pc DNA3.1(+)-PTENP1 plasmid is constructed successfully.3. Three groups of cells are successfully transfected with pcDNA3.1(+)-PTENP1 plasmid or pcDNA3.1(+) empty plasmid.RT-PCR reults show that C group is higher than A and B about the expression of PTEN.4. After transfection, Western Blot results show that the content of PTEN protein in C group is higher than A and B group.7. After successful transfection, Western Blot results show that the expression of PI3 K and AKT in C group is lower than A and B group.and the difference is statistical significant(P<0.05).Conclusion1. Overexpression of PTENP1 and PTEN high homologous sequences can positively regulate the expression of PTEN gene, which may play a role through competitive endogenous RNA mechanism.2. PTEN has a negative regulatory effect on the PI3K/AKT signaling pathway, which is realized by inhibiting the phosphorylation of downstream molecules.