The Function Of LRP5 In Cervical Cancer

BackgroundCervical cancer(CC)is one of the common gynecologic malignancies and the fourth leading cause of cancer death among women worldwide.Abnormal activation of the classical Wnt/?-catenin signaling pathway enhances the proliferation,migration and invasion ability of CC cells,which is one of the important causes of CC.However,the exact role of LRP5,as an auxiliary receptor of Wnt ligand,in the occurrence of CC has not been fully confirmed,which needs to be further explored.ObjectiveTo explore the effect of activation or knockout of LRP5 gene on CC cells,and to reveal the role and molecular mechanism of LRP5 gene in the pathogenesis of CC.Methods1.IHC: The paraffin fixed microarray of CC tissues was stained by immunohistochemical technique to detect the the expression level of LRP5 protein in adjacent normal tissues to cancer and CC tissues.2.Construction of CC stable cell lines knocked out or activated by LRP5 genea)Plasmid preparation: CRISPR/Cas9 KO plasmid that can knock out LRP5 gene,CRISPR Act plasmid that can activate LRP5 gene and their control plasmids were purchased.b)Screening cells: HeLa cells were seeded into 6-well plates,and cells were transfected with LRP5 CRISPR/Cas9 KO plasmid(NC-KO as control plasmid)or LRP5 CRISPR Act plasmid(NC-Act as control plasmid),and then continuously screened with puromycin.Extraction of cellular RNA,and the up-regulation or down-regulation of the LRP5 gene was analyzed by qRT-PCR.c)Screening monoclonal stable cell lines: Single cells selected for puromycin were separated into 96-well plates by flow cytometry and expanded.The changes of LRP5 gene were identified by qRT-PCR and Western Blot analysis,and LRP5-KO Hela monoclonal stable cells with the most obvious LRP5 gene down-regulation and LRP5-Act Hela monoclonal stable cells with the most obvious LRP5 gene up-regulation were selected for subsequent experiments compared with the control group.d)In vitro experiments: cell proliferation experiment,cell colony formation experiment,cell scratch healing experiment,flow cytometry cell cycle experiment,and cisplatin sensitivity experiment were performed on the selected stably transfected cells.3.To identify the effect of activation or knockout of LRP5 gene on the tumorigenic ability of Hela cells in vivoa)Cell preparation: The stably cultured LRP5-KO or LRP5-Act HeLa cells and their corresponding control cells were separately expanded.b)Injection into nude mice: HeLa cell suspension was injected subcutaneously in the bilateral inguinal and axillary regions of nude mice.Tumor size was measured every two days from the day of injection,after which the mice were sacrificed by anesthesia and the tumors were excised and weighed.c)Analytical verification: RNA was extracted from tumor tissue for qRT-PCR analysis to confirm the expression of LRP5 gene in the tumor.Results1.Immunohistochemistry results showed that the expression level of LRP5 protein in cervical cancer tissues was significantly increased compared with normal cervical tissues.2.qRT-PCR results showed that after the activation of LRP5 gene in HeLa cells,its mRNA level was increased,and the mRNA levels of downstream genes such as CTNNB1,CyclinD1,CD44,COX2 and c-Myc in the canonical Wnt/?-catenin signaling pathway were up-regulated.In addition,activation of LRP5 up-regulates IL-6 and STAT3 mRNA levels.On the contrary,knockout of the LRP5 gene in HeLa cells significantly reduced mRNA levels and down-regulated mRNA levels of downstream genes,which also reduced the mRNA levels of IL-6 and STAT3.3.Western Blot results showed that activation of LRP5 gene in HeLa cells up-regulated its protein expression level and the protein levels of downstream genes CyclinD1 and c-Myc were also increased.In addition,activation of LRP5 gene promotes IL-6-induced phosphorylation level of STAT3 protein(p-STAT3).On the contrary,knockout of the LRP5 gene down-regulated the protein expression levels of LRP5,CyclinD1,and c-Myc,and inhibited the IL-6-induced p-STAT3 protein expression level.4.Cell proliferation experiment showed that activation of LRP5 gene accelerated the proliferation of HeLa cells.On the contrary,knocking out LRP5 gene significantly inhibited the proliferation of HeLa5.Cell colony formation experiment showed that activation of LRP5 gene significantly enhanced the ability of HeLa cells to form clones in vitro.On the contrary,knocking out the LRP5 gene reduced the ability of HeLa cells to clone in vitro.6.Cell scratch healing experiment showed that activation of LRP5 gene enhanced the migration ability of HeLa cells.On the contrary,knocking out the LRP5 gene inhibited the migration ability of HeLa cells.7.Flow cytometry detection of cell cycle results showed that after knockout of LRP5 gene,the number of HeLa cells in S phase decreased,whereas in G2/M phase increased,HeLa cells were blocked in G2/M phase.8.Drug sensitivity experiment results showed that activation or knockout of LRP5 gene did not affect the sensitivity of HeLa cells to cisplatin.9.The results of in vitro tumorigenesis in nude mice showed that activation of LRP5 gene enhanced the tumorigenic ability of HeLa cells in nude mice.On the contrary,knocking out LRP5 gene significantly reduced the tumorigenic ability of HeLa cells in vivo.Conclusions1.The expression of LRP5 protein is increased in CC tissues,and its expression level of LRP5 is negatively correlated with the survival time of CC patients;2.Activation of the LRP5 gene may enhances the proliferation,migration,and tumorigenic ability of CC cells by activating the canonical Wnt/?-Catenin signaling pathway and IL-6/STAT3 signaling pathway;3.Knockout of the LRP5 gene may inhibits the proliferation,migration,and tumorigenic ability of CC cells by inhibiting the canonical Wnt/?-Catenin pathway and IL-6/STAT3 signaling pathway;4.LRP5 gene may be a potential target for CC diagnosis and treatment.