The Role Of PRPS2 And Its Molecular Mechanism In The Carcinogenesis Of Cervical Cancer

BackgroundCervical cancer is one of the most frequent cancer in female genital system worldwide.The persistent infection of high risk human papillomavirus(HPV)and the constant expressions of oncogene HPV E6/E7 in host cells are the predisposing factors.The long-term infection of HPV and the stable expressions of two oncoproteins cause the chromosomal instability,mutation accumulation of gene related to growth metabolism,intracellular key signal pathways changes in host cells and eventually lead to malignant transformation and carcinogenesis.Phosphoribosyl-pyrophosphate synthetase(PRPS2)is one key rate-limiting enzyme in the nucleotide biosynthesis pathway.It can catalyze the synthesis of phosphoribosyl pyrophosphate(PRPP)from ribose-5-phophate(R5P).And PRPP is an important substrate in the synthesis of purine and pyrimidine nucleosides,so PRPS is essential during nucleotide synthesis.PRPS includes three isozymes of PRPS1,PRPS2 and PRPS3(PRPS1L1),Among them,PRPS1 plays a major role in the synthesis of nucleic acids,and there are few studies on other two isozymes.It has been reported that PRPS2 was involved in the occurrence of Burkitt's lymphoma in mice induced by Myc overexpression,and its expression was regulated by Myc,suggesting that PRPS2 may contribute to the development of tumors.But so far there have been few reports on the effect of PRPS2 in tumorigenesis and development,and the mechanism is unclear.ObjectiveIn this study,we mainly used cervical cancer cells experiments to explore the role of PRPS2 in the development of carcinoma of uterine cervix and the molecular pathways involved.Methods and Results1.The expression of PRPS2 in cervical cancer tissues is higher than normal tissuesWe collected 70 cervical cancer samples and 45 normal tissues and made into cervical tissue sections.The protein expression of PRPS2 in cervical tissues was detected by immunohistochemistry(IHC)to compare the expression differences between normal cervical tissues and cervical cancer tissues.The results shown that PRPS2 is mainly expressed in the cell membrane in normal cervical tissues,and is mainly expressed in cytoplasm in cervical cancer tissues,and its expression in cervical cancer tissues is significantly higher than that in normal cervical tissues.2.PRPS2 knockdown in cervical cancer cells significantly inhibited cell proliferationTo begin with,according to the detection of PRPS2 basic protein expression by Western blot in cervical cancer cells,we conduct cells experiments with HeLa,SiHa and CaSki cells.Lipofectamine 2000 liposomes were used to transfect with PRPS2-specific siRNA siPRPS2 and siNC into HeLa,SiHa and CaSki cervical cancer cells.Western blot was used to detect the knockdown efficiency.Next;we employed Cell Counting Kit8(CCK-8),cell colony formation,cell cycle and cell apoptosis assays to determine the effect of PRPS2 on cell proliferation.The results indicated that PRPS2 knockdown restrained the viability and proliferation of cervical cancer cells,meanwhile the cell cycle G2 was arrested and the proportion of apoptosis was distinctly increased.3.PRPS2 knockdown in cervical cancer cells significantly impaired cell migration and invasionsiPRPS2 and siNC were transfected into HeLa SiHa and CaSki cervical cancer cells.Transwell chambers and Matrigel were applied to examine the migration and invasion of cervical cancer cells.The results demonstrated that compared with transfected with siNC,the migration and invasion ability of cervical cancer cells with siPRPS2 were obviously decreased.Western blot was used to detect the intracellular epithelial-mesenchymal transition(EMT)-related marker proteins in the siPRPS2 experimental group and the siNC control group.It was found that PRPS2 did not affect the migration and invasion of cervical cancer cells through the EMT pathway.4.PRPS2 knockdown in cervical cancer cells promoted cell autophagyWe further studied the effect of PRPS2 on autophagy by Western blot in HeLa cells.The results illustrated that after PRPS2 knockdown the protein expressions of LC3? and Beclinl were obviously risen.It indicated that PRPS2 knockdown could promote cell autophagy.5.Effect of PRPS2 on cervical cancer cell signaling pathwaysTo investigate the signaling pathways PRPS2 involved in,we detected the protein expression levels of the molecules closely related to the growth and proliferation by Western blot in the siPRPS2 experimental group and the siNC control group.Compared with the siNC control group,after interfering with PRPS2,the phosphorylation levels of AMPK a and its downstream molecules were increased;meanwhile the expression of RB was significantly increased and the expression of E2F1 was decreased;however,there was no change in p-AKT level.This result indicated that PRPS2 regulated various biological characteristics of cervical cancer cells through the AMPK pathway and the RB pathway.Conclusion and innovationThrough the above experiments,we found that PRPS2 was overexpressed in cervical cancer compared with normal cervical tissue;in cytological experiments,PRPS2 could promote the growth,proliferation,migration,invasion and cell cycle progression of cervical cancer cells.PRPS2 inhibited AMPK activity and decreased the expression level of RB protein and increased its downstream molecule E2F1.In addition,PRPS2 was also involved in regulating the autophagy process of cervical cancer cells.These results indicated that PRPS2 was involved in the regulation of the growth,proliferation,cell apoptosis and cell cycle progression of cervical cancer by regulating the AMPK and RB pathways,and has an important role in promoting the development of cervical cancer.This study firstly explored the role of PRPS2 in the development of cervical cancer through in vitro and in vivo experiments,and conducted a preliminary study on the molecular pathways involved in regulation.As a new tumor-associated protein,PRPS2 has the potential to play a role in the diagnosis and adjuvant treatment of cervical cancer.This study provides an important theoretical basis for guiding the design of PRPS2 as a target for the adjuvant treatment of cervical cancer.